Novel interleukin-15 (IL-15) fusion proteins and uses thereof

Abstract

The present disclosure provides novel and improved IL-15 fusion proteins for use in the treatment of cancer and other disorders. In various embodiments, the fusion proteins of the invention comprise two functional domains: an IL-15 / IL-15RαSushi domain (also referred to as an "IL-15 / IL-15RαSushi complex") and an antibody domain, each of which can take various forms. IL-15 is fused to the C-terminus of the antibody domain, allowing for coexpression and non-covalent complex formation with IL-15RαSushi. Importantly, the fusion proteins of the invention address some of the limitations identified in IL-15 therapies evaluated to date. Specifically, the fusion proteins demonstrate an extended in vivo half-life of IL-15 and optimized preclinical activity compared to rIL-15 and related cytokine therapies.

Description

[Background technology]

[0001] Cancer has traditionally been treated with chemotherapy, radiation, targeted therapy, and surgery. However, over the past decade, immunotherapy has emerged as a fifth pillar of cancer treatment, revolutionizing the fight against cancer. The development of T cell checkpoint (CTLA-4 and PD-1 / PD-L1) inhibitors established the benchmark for immunotherapy drugs. These treatments have been shown to induce response rates approaching 50% in certain cancer patients through the efficient expansion and reactivation of tumor-specific T cell pools.

[0002] Recently, interleukin-15 (IL-15), a cytokine member of the four-alpha helix bundle family, has emerged as a potential immunomodulatory agent for cancer therapy. IL-15 binds to its specific receptor, IL-15Rα, which is expressed on antigen-presenting dendritic cells, monocytes, and macrophages, and to IL-15Rβ and the common cytokine receptor γ chain (γ) on reactive cells, including T cells and natural killer (NK) cells. c IL-15 transactivates the heterodimeric receptor complex composed of IL-15 and IL-15-dependent IL-15 receptors, inducing signal transduction. IL-15 exhibits broad activity, inducing differentiation and proliferation of T cells, B cells, and natural killer (NK) cells. IL-15 also activates CD8 + Enhances the cytolytic activity of T cells and antigen-experienced CD8 + CD44 hi IL-15 induces long-term 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 (Tregs). Therefore, it is hypothesized that enhancing IL-15 activity may be a promising anti-cancer therapeutic agent by enhancing innate and adaptive immunity and triggering tumor attack (Steel et al., Trends in Pharmacological Sciences, Vol. 33(1): pp. 35-41, 2012).

[0003] A first-in-human phase I clinical trial of intravenous infusion of recombinant human IL-15 in patients with metastatic melanoma reported that IL-15 can be safely administered to patients with metastatic malignancies and that IL-15 administration significantly altered the homeostasis of lymphocyte subsets in the blood, with NK cells and gamma-delta cells being most dramatically affected, followed by CD8 memory T cells (Conlon et al., J Clin Oncol., 33(1), pp. 74-82).

[0004] Despite these new advances in using IL-15 as a cancer immunotherapy to enhance immune responses, there are still limitations to its effective use as a therapeutic agent. For example, IL-15 has a short half-life (<40 minutes), which 1) results in low bioavailability that hinders its in vivo antitumor efficacy, and 2) requires high doses to achieve therapeutically relevant exposure, resulting in toxicity. Furthermore, IL-15 is understood to be expressed at low levels in standard mammalian cell systems.

[0005] There is still a strong demand for new, highly effective and safe therapeutic agents in cancer treatment. Summary of the Invention

[0006] In one aspect, the present invention provides new and improved IL-15 fusion proteins for use in the treatment of cancer. In various embodiments, the fusion proteins of the invention have two functional domains: an IL-15 / IL-15 receptor alpha (IL-15Rα) component (also referred to as an "IL-15 / IL-15Rα complex") and a heterologous protein, each of which can take a variety of forms. In various embodiments, the fusion proteins are configured such that IL-15 is fused to either the C-terminus of the heterologous protein or the N-terminus of the heterologous protein and is coexpressed with the IL-15Rα domain to form a noncovalent complex (see Figures 1B and 1C, where the heterologous protein is an Fc domain, and Figures 30A and 30B, where the heterologous protein is an antibody).

[0007] In various embodiments, the IL-15 fusion proteins of the invention comprise an IL-15 / IL-15Rα complex in which the IL-15 domain comprises the sequence of the mature human IL-15 polypeptide set forth in SEQ ID NO:2 (also referred to as huIL-15 or IL-15 wild-type (wt)). In various embodiments, the IL-15 domain is an IL-15 variant (or mutant) that comprises a sequence derived from the sequence of the mature human IL-15 polypeptide set forth in SEQ ID NO:2. IL-15 variants (or mutants) are designated by the original amino acid, its position within the mature sequence, and the variant amino acid. For example, huIL-15 "S58D" refers to human IL-15 containing an S to D substitution at position 58 of SEQ ID NO:2. In various embodiments, the IL-15 variant functions as an IL-15 superagonist, as evidenced by increased binding activity to IL-15Rβ or increased functional activity compared to the native IL-15 polypeptide. In various embodiments, the IL-15 variant functions as an IL-15 antagonist, e.g., as evidenced by reduced or absent binding or functional activity to IL-15Rβ compared to the 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 the 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. The amino acid change may include IL-15Rβ and / or IL-15Rβγ. cThe domain of IL-15 that interacts with can include one or more of an amino acid substitution, deletion, or insertion. In various embodiments, the amino acid changes are one or more amino acid substitutions at positions 30, 31, 32, 58, 62, 63, 67, 68, or 108 of SEQ ID NO: 2. In various embodiments, the amino acid changes are a D to T substitution at position 30, a V to Y substitution at position 31, an H to E substitution at position 32, an S to D or H or R or Q or I or P substitution at position 58, a T to D substitution at position 62, a V to F or A or R or K substitution at position 63, an I to V substitution at position 67, an I to F or H or D or K or Q or G substitution at position 68, or a Q to A or M or S or E or K substitution at position 108 of the mature human IL-15 sequence, or any combination of these substitutions. In various embodiments, the IL-15 variant comprises one, two, three, four, five, or six amino acid deletions at the N-terminus of SEQ ID NO:2. In various embodiments, the IL-15 variant comprises one, two, three, four, five, or six, seven, eight, nine, or ten amino acid deletions at the C-terminus of SEQ ID NO:2. In various embodiments, the IL-2 variant comprises an amino acid insertion of "GS" (SEQ ID NO:12), or "GGSGG" (SEQ ID NO:153), or "GSSGGSGGS" (SEQ ID NO:154) after position N95 of SEQ ID NO:2. In various embodiments, the IL-15 variant comprises a combination of amino acid substitutions, deletions, or insertions. In various embodiments, the IL-15 variant polypeptide comprises the sequence set forth in SEQ ID NOs:24-45, 56-63, and 66-81.

[0008] In various embodiments, the IL-15 fusion proteins of the invention comprise an IL-15 / IL-15Rα complex in which the IL-15Rα comprises either the IL-15Rα sushi domain (SEQ ID NO:5) or the IL-15Rα extracellular domain (SEQ ID NO:4), or any functional domain thereof. In various embodiments, the IL-15Rα domain comprises a sequence that is at least 90% similar 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% similar 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% similar 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% similar to the sequence set forth in SEQ ID NO:5.

[0009] In various embodiments, the IL-15 fusion proteins of the invention comprise an IL-15 / IL-15RαSushi complex and at least one heterologous protein.

[0010] In various embodiments, the IL-15 fusion proteins of the present invention comprise an IL-15 / IL-15Rα complex in which IL-15 is fused to either the C-terminus or the N-terminus of the heterologous protein.

[0011] In various embodiments, the IL-15 fusion proteins of the present invention contain dimeric or monomeric IL-15 / IL-15Rα-heterologous protein complexes.

[0012] 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 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, or any combination thereof. In various embodiments, the Fc domain comprises amino acid changes that result in an Fc domain with altered complement binding or Fc receptor binding properties. Amino acid changes that produce Fc domains with altered complement binding or Fc receptor binding properties are known in the art. In various embodiments, the Fc domain sequence used to generate the dimeric IL-15 / IL-15Rα complex-Fc fusion protein is the human IgG1-Fc domain sequence set forth in SEQ ID NO: 6. SEQ ID NO: 6 comprises amino acid substitutions that abolish FcγR binding and C1q binding. In various embodiments, the heterodimeric Fc domain sequence used to generate the 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 contains amino acid substitutions that abolish FcγR binding and C1q binding. In various embodiments, the heterodimeric Fc domain sequence used to generate the 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 contains amino acid substitutions that abolish FcγR binding and C1q binding.

[0013] In various embodiments, the IL-15 fusion proteins of the invention comprise an IL-15 / IL-15Rα complex and the heterologous protein is a full-length non-binding antibody for half-life extension or a specific antibody or fragment used for targeting, multifunctionality, and half-life extension.

[0014] 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 antibody (monospecific or bispecific) of the full-length IgG type or antibody fragment type, which exhibits an additive or synergistic effect with the IL-15 / IL-15RαSushi complex.

[0015] In various embodiments, the IL-15 fusion proteins of the invention comprise an IL-15 / IL-15Rα complex, where the heterologous protein allows for tissue- or tumor-specific targeting, increasing the local concentration and penetration of IL-15 into the tumor microenvironment, thereby increasing tumor cell killing efficacy and reducing systemic toxicity.

[0016] In various embodiments, the heterologous protein is an antibody, or antibody fragment, or ligand or variant thereof, or receptor or variant thereof, capable of binding to a tumor-associated antigen (TAA) or tissue-specific antigen or target, a cell surface molecule or extracellular matrix protein, a protease(s), and any post-translationally modified residue(s). In various embodiments, the antibody is an anti-fibroblast activation protein (FAP) antibody or antibody fragment. In various embodiments, the antibody is a humanized anti-FAP antibody comprising the heavy and light chain sequences set forth in SEQ ID NOs: 109-110. In various embodiments, the heterologous protein is an antibody or antibody fragment against an immune checkpoint regulator. In various embodiments, the antibody is an antagonistic Programmed Death-1 (PD-1) antibody or antibody fragment. In various embodiments, the antibody is an antagonistic humanized PD-1 antibody comprising the heavy and light chain amino acid sequences set forth in SEQ ID NOs: 111-112. In various embodiments, the antibody is an antagonistic human PD-1 antibody comprising the heavy and light chain amino acid sequences set forth in SEQ ID NOs: 113-114. In various embodiments, the antibody is an antagonistic Programmed Death Ligand-1 (PD-L1) antibody or antibody fragment.

[0017] In various embodiments, the heterologous protein is covalently linked to the IL-15 polypeptide of the IL-15 / IL-15RαSushi complex by 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 (or any number therebetween) that is relatively free of secondary structure. In various embodiments, the linker is enriched in G / S content (e.g., at least about 60%, at least about 70%, at least about 80%, or at least about 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 set forth in SEQ ID NOs: 9-12, 47, and 153-154. Each peptide linker sequence can be independently selected.

[0018] In another aspect, the present disclosure provides a pharmaceutical composition comprising an isolated IL-15 fusion protein of the invention in admixture with a pharmaceutically acceptable carrier.

[0019] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a subject in need thereof.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, colon cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, and rhabdomyosarcoma.

[0020] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the 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, radiation therapy, 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 a vaccine such as Bacillus Calmette-Guerin (BCG).In various embodiments, the combination therapy can include administering to a subject a therapeutically effective amount of immunotherapy, including depleting antibodies against specific tumor antigens. treatment with antibody-drug conjugates; treatment with agonist, antagonist, or blocking antibodies against costimulatory or co-inhibitory molecules (immune checkpoints), such as CD276, CD272, CTLA-4, PD-1, PD-L1, CD40, SIRPα, 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 (BiTE®), such as blinatumomab; treatments involving the administration of biological response modifiers, such as IL-2, IL-7, IL-10, IL-12, IL-21, G-CSF, GM-CSF, IFN-α, IFN-β, and IFN-γ; treatment with therapeutic vaccines, such as sipuleucel-T; dendritic cell vaccines or treatment with a tumor antigen peptide vaccine; treatment with chimeric antigen receptor (CAR)-T cells; treatment with CAR-NK cells; treatment with tumor-infiltrating lymphocytes (TILs); treatment with adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR-transgenic T cells); treatment with TALL-104 cells; treatment with immunostimulants such as Toll-like receptor (TLR) agonists such as TLR4 agonist CpG, TLR7 agonist CpG, TLR8 agonist CpG, TLR9 agonist CpG, and imiquimod; and treatment with vaccines such as Bacillus Calmette-Guerin (BCG); the above combination therapies enhance tumor cell killing by effector cells, i.e., there is synergy between the IL-15 / IL-15RαSushi-Fc fusion protein and immunotherapy when administered simultaneously.

[0021] In another aspect, the present disclosure provides methods for expanding and renewing NK and T cells in vitro and in vivo, in combination with optional adoptive cell transfer NK cell-T cell therapy or CAR-NK-CAR-T therapy to maintain cell survival and half-life.

[0022] In another aspect, the present disclosure provides a method for treating a viral infection in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of the present invention. In one embodiment, the subject is a human subject.

[0023] In another aspect, the present disclosure provides a use of an IL-15 fusion protein for preparing a medicament for treating cancer.

[0024] In another aspect, the disclosure provides a use of an IL-15 fusion protein for the preparation of a medicament for treating a viral infection.

[0025] 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.

[0026] In another aspect, the present disclosure provides a vector comprising a nucleic acid described herein. In various embodiments, the vector is an expression vector. In another aspect, the present disclosure provides an isolated cell comprising a nucleic acid of the present disclosure. In various embodiments, the cell is a host cell comprising an expression vector of the present disclosure. In another aspect, a method of producing an IL-15 fusion protein by culturing the host cell described above under conditions that promote expression of the protein or polypeptide is provided. [Brief explanation of the drawings]

[0027] [Figure 1] Figure 1 shows several forms of IL-15 / IL-15Rα-Fc fusion proteins of the present invention: (A) IL-15 / IL-15Rα heterodimeric Fc fusion form; (B) monovalent IL-15 / IL-15Rα (non-covalent)-Fc fusion form; (C) bivalent IL-15 / IL-15Rα (non-covalent)-Fc fusion form; (D) monovalent IL-15 (non-covalent) / IL-15Rα-Fc fusion form; and (E) bivalent IL-15 (non-covalent) / IL-15Rα-Fc fusion protein form. In each fusion protein form, the IL-15 / IL-15Rα complex can be located at either the C-terminus or N-terminus of the Fc domain; IL-15Rα can be either the IL-15Rα Sushi domain or the IL-15Rα ECD. [Figure 2] Figure 2 shows the A) purity and B) percentage of monomer of exemplary IL-15 / IL-15Rα (non-covalent)-Fc fusion proteins P-0217, P-0234, and P-0313, as measured by SDS-PAGE and SEC-HPLC, respectively. All three fusion proteins contain 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 by an S58D substitution in the IL-15 domain. [Figure 3]Figure 3 shows SEC (size exclusion chromatography) chromatograms of several IL-15 / IL-15Rα-Fc fusion proteins of different configurations. Unless otherwise noted, all of these exemplary fusion proteins contain the IL-15 / IL-15Rα complex at the C-terminus. P-0162 is a monomeric IL-15-only Fc fusion protein. P-0197 is a monovalent IL-15 / IL-15Rα (non-covalent)-Fc fusion, the schematic of which is shown in Figure 1B. P-0153 is a monomeric IL-15 / IL-15Rα fusion in the form of a heterodimeric Fc fusion (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 bivalent IL-15 / IL-15Rα (noncovalent)-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. [Figure 4] Figure 4 shows the effect of different IL-15 / IL-15Rα-Fc fusion forms on IL-15Rβ-binding activity in an ELISA assay. IL-15Rα increases the IL-15Rβ-binding activity of IL-15-Fc fusion proteins. 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; and P-0162 is a C-terminal monovalent IL-15-Fc fusion protein without IL-15RαSushi complexed thereto. [Figure 5]Figure 5 shows the effect of IL-15Rα on the biological activity of IL-15-Fc fusion proteins. IL-15Rα enhances the biological activity of IL-15-Fc fusion proteins. The induction of CD69-positive NK cells (Figure 5A) and CD8+ T cells (Figure 5B) was measured in an ex vivo human PBMC FACS-based assay. P-0197 is a C-terminal monovalent IL-15 / IL-15RαSushi (non-covalent)-Fc fusion protein; P-0162 is a fusion protein with the same structure as P-0197 but without IL-15RαSushi complexed. [Figure 6] Figure 6 shows the effect of different IL-15 / IL-15Rα complex formation configurations on the biological activity of IL-15-Fc fusion proteins. The induction of CD69+ NK cells (Figure 6A) and CD8+ T cells (Figure 6B) was measured in an ex vivo human PBMC FACS-based assay. P-0165 is a C-terminal monovalent IL-15 (non-covalent) / IL-15Rα-Fc fusion protein; P-0197 is a C-terminal monovalent IL-15 / IL-15Rα (non-covalent)-Fc fusion protein; and P-0153 is a C-terminal IL-15 / IL-15Rα heterodimeric Fc fusion protein. [Figure 7] Figure 7 shows the effect of the linker on the biological activity of different types of IL-15 / IL-15Rα-Fc fusion proteins. The induction of CD69-positive NK cells (Figure 7A) and CD8 T cells (Figure 7B) was measured in an ex vivo human PBMC FACS-based assay. P-0165 and P-0166 are monovalent IL-15 (non-covalent) / IL-15Rα-Fc fusion proteins 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 15-amino acid rigid linker, a 10-amino acid flexible linker, and a 15-amino acid flexible linker, respectively. [Figure 8]Figure 8 shows the effect of N- or C-terminal fusion on the activity of IL-15 / IL-15Rα-Fc 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 and the standard are C-terminal bivalent IL-15 (non-covalent) / IL-15Rα-Fc fusion proteins and N-terminal bivalent IL-15 (non-covalent) / IL-15Rα-Fc fusion proteins, respectively. (B) P-0234 and P-0223 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. [Figure 9] Figure 9 shows the effect of the IL-15Rα complete ECD domain or the IL-15Rα sushi domain on the biological activity of IL-15 / IL-15Rα-Fc fusion proteins. The induction of CD69-positive NK cells was measured in an ex vivo human PBMC FACS-based assay. (A) P-0234 and P-0220 are C-terminal bivalent IL-15 / IL-15Rα (non-covalent)-Fc fusion proteins with IL-15Rα sushi and the complete ECD, respectively. (B) P-0223 and P-0224 are N-terminal bivalent IL-15 / IL-15Rα (non-covalent)-Fc fusion proteins with IL-15Rα sushi and the complete ECD, respectively. (C) P-0221 and P-0222 are N-terminal monovalent IL-15 / IL-15Rα (non-covalent)-Fc fusion proteins carrying IL-15Rα sushi and the complete ECD, respectively. [Figure 10]Figure 10 shows that the S58D substitution in the IL-15 polypeptide enhanced 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 and P-0314 are bivalent IL-15 (non-covalent) / IL-15Rα-Fc fusion proteins containing wild-type IL-15 and the S58D variant of IL-15, respectively. P-0234 and P-0313 are bivalent IL-15 / IL-15Rα (non-covalent)-Fc fusion proteins containing wild-type IL-15 and the S58D variant of IL-15, respectively. [Figure 11] Figure 11 shows that fusion proteins containing the IL-15S58D variant exhibited enhanced ability to induce Ki67 expression on CD8+ T cells (A), CD4+ T cells (B), and NK cells (C). P-0218 and P-0314 are bivalent IL-15 (non-covalent) / IL-15Rα-Fc fusion proteins containing wild-type IL-15 and the S58D variant of IL-15, respectively. P-0234 and P-0313 are bivalent IL-15 / IL-15Rα (non-covalent)-Fc fusion proteins containing wild-type IL-15 and the S58D variant of IL-15, respectively. [Figure 12] Figure 12 shows serum IL-15 concentrations in mice treated with rhIL-15, standard, and P-0234 in a 4-day repeated-dose study. Female B Balb / C mice were intraperitoneally injected daily with vehicle, rhIL-15 (0.03 mg / kg), standard (0.1 mg / kg and 0.5 mg / kg), or P-0234 (0.1 mg / kg and 0.5 mg / kg). Terminal blood was collected 1 hour after the final injection on day 4, and serum IL-15 levels were measured using an ELISA assay. [Figure 13]Figure 13 shows (A) body weight and (B) percent change in body weight from day 0 in Balb / C mice treated with rhIL-15, standard, and P-0234 in a 4-day repeated-dose 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. [Figure 14] Figure 14 shows the effect of IL-15 compounds on the proliferation and proliferation of NK cells in the peripheral blood of Balb / C mice in a 4-day repeated-dose study. After four daily doses, blood was collected for Ki67 measurement and NK cell phenotyping by FACS. (A) Percentage of NK cells positive for the Ki67 proliferation marker; (B) Percentage of NK cells in the CD3-negative lymphocyte population. 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 vehicle group, #p<0.001 compared to equivalent dose control, ##p<0.01 compared to equivalent dose control. [Figure 15] Figure 15 shows the effects of IL-15 compounds on the proliferation, proliferation, and activation of splenic NK cells in Balb / C mice in a 4-day repeated-dose study. (A) Percentage of splenic NK cells positive for the Ki67 proliferation marker; (B) Total NK cells in the spleen; (C) Percentage of CD69-positive splenic NK cells. 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 vehicle group, **p<0.01 compared to vehicle group, *p<0.05 compared to vehicle group. [Figure 16] Figure 16 shows serum concentrations of P-0313 and standard after a single intraperitoneal injection in Balb / C mice. Blood was collected from mice treated with 0.3 mg / kg of P-0313 or standard at -24 hours (pre-dose) and 1, 4, 24, 72, 144, and 192 hours after administration. (A) Detection of human Fc-IL-15 complexes by an in-house ELISA assay; (B) Detection of human IL-15 by a commercial ELISA assay. [Figure 17] FIG. 17 shows the body weight of Balb / C mice after a single injection of P-0313 and standard over an 8 day period. [Figure 18] Figure 18 shows the dose- and time-dependent effects of IL-15 / IL-15Rα-Fc fusion protein on Ki67 expression on NK cells (A) and CD8+ T cells (B) after a single injection in Balb / C mice. Blood was collected at -24 hours (pre-dose) and at 1, 4, 24, 72, 144, and 192 hours for lymphocyte phenotyping and Ki67 measurement by FACS analysis. Data are presented as mean ± SEM. Statistical analysis was performed by two-way ANOVA followed by Tukey's post-hoc test. ****p<0.0001, **p<0.01, *p<0.05 compared to the PBS group at each time point. [Figure 19] Figure 19 shows the dose- and time-dependent effects of IL-15 / IL-15Rα-Fc fusion protein on the proliferation 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 (pre-dose) and at 1, 4, 24, 72, 144, and 192 hours, and lymphocyte phenotyping was performed by FACS analysis. Data are presented as mean ± SEM. Statistical analysis was performed by two-way ANOVA followed by Tukey's post-hoc test. ****p<0.0001, ***p<0.001, *p<0.05 compared to the PBS group at each time point. [Figure 20]Figure 20 shows the inhibition of lung metastasis by P-0313 and standard in a mouse CT26 lung metastasis model. Vehicle, standard (0.3 mg / kg), or P-0313 (0.03 mg / kg and 0.1 mg / kg) was administered every 5 days (Q5D) for three doses, starting 1 day after CT26 cell injection. On day 16, mice were sacrificed, and the number of metastatic nodules in the lungs was counted under a microscope. (A) Representative lung photographs taken under a light microscope show metastatic nodules in each treatment group. (B) Number of lung nodules for all animals. 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<0.05 compared to the PBS group. [Figure 21] Figure 21 shows immunopharmacodynamic profiling in the CT26 lung metastasis model after treatment with P-0313 or standard. Three days after three intraperitoneal injections of P-0313, standard, or PBS every five days, flow cytometry confirmed increased numbers of circulating A) NK cells and B) CD8+ T cells per μL of whole blood in CT26 metastatic mice. 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<0.001, **p<0.01 compared to the PBS group. [Figure 22] Figure 22 shows spleen weights in mice treated with P-0313 or standard in the CT26 lung metastasis model. Spleens were harvested 3 days after three intraperitoneal injections of IL-15 / IL-15Rα-Fc fusion protein every 5 days. 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. [Figure 23]Figure 23 shows hepatotoxicity assessment in CT26 lung metastasis mice treated with P-0313 or standard. Livers were harvested 3 days after treatment, 3 times every 5 days. A) Liver weight; B) Serum ALT level; and C) Serum AST level. Serum ALT and AST levels were measured using commercially available ELISA kits. Data are presented as mean ± SEM. [Figure 24] Figure 24 shows the antitumor activity of P-0313 in a subcutaneously established CT26 mouse colon tumor model. On day 0, 1 x 10 CT26 cells were injected subcutaneously. Starting when the mean tumor volume reached approximately 70 mm (day 11), vehicle (PBS) or P-0313 (0.1 mg / kg or 0.05 mg / kg) injections were administered twice every 5 days. (A) Growth curve of CT26 subcutaneous tumors. (B) Changes in body weight from baseline. Data are presented 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. [Figure 25] Figure 25 shows the growth curves of subcutaneous CT26 tumors in individual mice administered (A) PBS vehicle, (B) 0.05 mg / kg P-0313, or (C) 0.01 mg / kg P-0313. n=10 / group. [Figure 26] Figure 26 shows the proliferation and proliferation of NK cells and CD8+ T cells in P-0313-treated mice in the CT26 mouse colon carcinoma tumor model. Treatment began 11 days after tumor implantation and was administered twice every 5 days. Flow cytometry analysis on day 19 confirmed increased Ki67 expression (A-B) and increased circulating cell counts (per μL of whole blood) (C-D) in NK cells and CD8+ T cells. 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<0.05 compared to the PBS group. [Figure 27]Figure 27 shows immunophenotyping of splenic NK cells and splenic CD8+ T cells in CT26 colon tumor-bearing mice treated with P-0313. Treatment was administered twice every 5 days, starting 11 days after tumor implantation, and flow cytometry on day 21 confirmed an increase in the number of splenic NK cells (A) and CD8+ T cells (B). 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. [Figure 28] Figure 28 shows the antitumor activity of P-0313 in an unestablished CT26 colon tumor model. Three days after subcutaneous implantation of 1 x 10 CT26 cells, mice were injected with vehicle (PBS) or P-0313 (0.1 mg / kg) every 5 days for 5 doses. (A) Growth curve of CT26 subcutaneous tumors after tumor cell implantation on day 0. (B) Tumor weight on day 25. Data are presented 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 to the PBS group. [Figure 29] Figure 29 shows spleen weight and body weight gain / loss in CT26 tumor-bearing mice treated with P-0313. Three days after CT26 tumor cell implantation, mice were injected with vehicle (PBS) or P-0313 (0.1 mg / kg) five times every five days. (A) Spleen weight on day 25. (B) Body weight gain / loss over 25 days. Data are presented 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. [Figure 30] FIG. 30 shows exemplary IL-15 / IL-15Rα (non-covalent) antibody fusion proteins in which the IL-15 / IL-15Rα complex is fused to the C-terminus of A) a bivalent homodimeric heavy chain, B) a monovalent heterodimeric heavy chain, and C) a light chain. [Figure 31]Figure 31 shows the effect of various IL-15 single amino acid substitutions at positions A) S58, B) V63, C) I68, or D) Q108 on the cytokine's ability to induce Ki67 expression on CD8+ T cells. All exemplary IL-15 variant fusion proteins are bivalent IL-15 / IL-15Rα (non-covalent) Fc fusions, except for P-0764 and P-0793 (Figure 31D), which are monovalent IL-15 / IL-15Rα (non-covalent) Fc fusions. P-0313, a well-characterized bivalent IL-15S58D / IL-15Rα (non-covalent) Fc fusion protein, was included as a control. [Figure 32] Figure 32 shows the dose-dependent induction of Ki67 expression on CD8+ T cells after treatment with IL-15 variant fusion proteins in fresh human PBMCs. P-0866, P-0867, and P-0868 are all bivalent IL-15 / IL-15Rα (non-covalent) Fc fusion proteins containing one, two, and three amino acid deletions at the N-terminus of IL-15, respectively. P-0234 is a homogenate containing wild-type IL-15. [Figure 33] Figure 33 shows the activity assessment of various IL-15 fusion proteins carrying combination mutations by analyzing the dose-dependent induction of Ki67 expression on CD8+ T cells in fresh human PBMCs. A) P-0773, P-0772, and P-0768 contain an additional V63A substitution relative to their respective single amino acid change-carrying counterparts, P-0358 (I68H), P-0736 (I68Q), and P-0737 (I68G). P-0771 contains a single V63A mutation. All exemplary IL-15 variant fusion proteins are bivalent IL-15 / IL-15Rα (non-covalent) Fc fusions. B) P-0886 and P-0888 are bivalent IL-15 / IL-15Rα (non-covalent) antibody fusion proteins. P-0886 contains two amino acid deletions at the IL-15 N-terminus, while P-0888 contains one additional V63A single amino acid substitution. P-0313 and P-0234 are highly potent bivalent IL-15 / IL-15Rα (non-covalent) Fc fusion proteins and were included as controls. [Figure 34] Figure 34 shows the dose-dependent induction of Ki67 expression on A) CD8+ T cells and B) NK cells after treatment with various IL-15 variant fusion proteins in fresh human PBMCs. Compared to the highly potent control P-0313, all test compounds showed varying levels of attenuation of activity. [Figure 35] Figure 35 shows the dose-dependent induction of Ki67 expression on CD8+ T cells after treatment with IL-15 variant fusion proteins in fresh human PBMCs. A) P-773 is a bivalent IL-15 / IL-15Rα (non-covalent) Fc fusion, while P-0870 is its antibody fusion counterpart containing the same amino acid substitutions, V63A / I68H, to attenuate IL-15 potency. B) P-867 is a bivalent IL-15 / IL-15Rα (non-covalent) Fc fusion containing two amino acid deletions at the N-terminus of IL-15, and P-0886 is the antibody fusion counterpart of P-0867. DETAILED DESCRIPTION OF THE INVENTION

[0028] The present disclosure provides novel and improved IL-15 fusion proteins for use in 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 as an "IL-15 / IL-15RαSushi complex") and an Fc domain, each of which can take various forms, and IL-15 is fused to the C-terminus or N-terminus of the Fc domain, configured for coexpression and non-covalent complex formation with IL-15Rα, IL-15RαSushi, or IL-15RαECD, or a functional fragment thereof (see FIG. 1). In various embodiments, the fusion proteins of the present invention have two functional domains, an IL-15 / IL-15RαSushi domain and an antibody or antibody fragment, each of which can take a variety of forms, and IL-15 is fused to the C-terminus or N-terminus of an antibody heavy or light chain, and are configured to co-express and form a non-covalent complex with IL-15Rα, IL-15RαSushi, or IL-15RαECD, or a functional fragment thereof (illustrated in Figure 30).

[0029] The present disclosure provides IL-15 variants that have amino acid substitutions, deletions, or insertions and function as IL-15 superagonists, IL-15 agonists, or IL-15 antagonists for use in the treatment of cancer and other disorders.

[0030] The inventors have realized that, in order to extend the circulating half-life of IL-15 or IL-15 fusion proteins and / or increase the biological activity of IL-15 or IL-15 fusion proteins, it is highly desirable to covalently link IL-15 to a human Fc or antibody / antibody fragment at either the N- or C-terminus to enhance the presentation of IL-15 to its signaling receptor and prevent dissociation of IL-15 from the fusion protein. Furthermore, the inventors have determined that it would be highly desirable to create a fusion protein complex containing the IL-15Rα domain noncovalently linked to IL-15 in order to more naturally present IL-15 to the IL-15 signaling receptor. Using the formats and strategies described herein, the inventors demonstrate that increased protein expression, potentially reduced immunogenicity, and protection of IL-15 from degradation can be achieved. In various embodiments described herein, the C-terminal location of the IL-15-IL-15Ra complex is preferred to achieve enhanced biological activity and developability.

[0031] Thus, the present disclosure provides IL-15 variants that have one or more amino acid substitutions, deletions, or insertions and function as IL-15 antagonists with optimally attenuated potency to prevent pathway overactivation, reduce undesirable target sinks to reduce systemic toxicity, and improve pharmacokinetics.

[0032] definition As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably and refer to a polymer of amino acid residues. In various embodiments, a "peptide," "polypeptide," and "protein" are amino acid chains in which the alpha carbons are linked through peptide bonds. The terminal amino acid at one end of the chain (the amino terminus) has a free amino group, while the terminal amino acid at the other end of the chain (the carboxy terminus) has a free carboxyl group. As used herein, the term "amino terminus" (abbreviated N-terminus) refers to the free alpha-amino group on the amino terminal amino acid of a peptide, or to the alpha-amino group of an amino acid anywhere else within the peptide (when participating in a peptide bond). Similarly, the term "carboxy terminus" refers to the free carboxyl group at the carboxy terminus of a peptide, or to the carboxyl group of an amino acid anywhere else within the peptide. Peptides also encompass virtually any polyamino acid, including, but not limited to, peptidomimetics, such as those in which amino acids are linked by ethers rather than amide bonds.

[0033] The polypeptides of the present disclosure encompass polypeptides that have been modified in any way and for any reason, such as (1) reducing susceptibility to proteolysis, (2) reducing susceptibility to oxidation, (3) changing binding affinity for the purpose of protein complex formation, (4) changing binding affinity, and (5) imparting or modifying other physicochemical or functional properties. For example, a single amino acid substitution (e.g., conservative amino acid substitution) or multiple amino acid substitutions may be made in the native sequence (e.g., in the portion of the polypeptide outside the domain that forms intermolecular contacts). A "conservative amino acid substitution" refers to the substitution of an amino acid in a polypeptide with a functionally similar amino acid. Each of the following six groups contains amino acids that are conservative substitutions for each other: 1) Alanine (A), Serine (S), and Threonine (T) 2) Aspartic acid (D) and glutamic acid (E) 3) Asparagine (N) and Glutamine (Q) 4) Arginine (R) and Lysine (K) 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V) 6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W)

[0034] "Non-conservative amino acid substitutions" refer to the substitution of a member of one of these classes for a member of another class. In making such changes, various embodiments may consider the hydropathic index of amino acids. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. The respective hydropathic indices are as follows: 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); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0035] The importance of the hydropathic amino acid index in conferring interactive biological function to a protein is understood in the art (see, e.g., Kyte et al., 1982, J. Mol. Biol., 157:105-131). It is known that substitution of a particular amino acid with another amino acid having a similar hydropathic index or hydropathic score can retain similar biological activity. When making changes based on the hydropathic index, various embodiments include substitutions of amino acids whose hydropathic indices are within ±2 of each other, in various embodiments within ±1, and in various embodiments within ±0.5.

[0036] It is also understood in the art that substitutions of similar amino acids can be made efficiently on the basis of hydrophilicity, particularly when the biologically functional proteins or peptides thus generated are intended for use in the immunological embodiments disclosed herein. In various embodiments, the maximum local average hydrophilicity of a protein, as controlled by the hydrophilicity of adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., correlates with the biological properties of the protein.

[0037] The following hydrophilicity values ​​have been 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, various embodiments include substitutions of amino acids with hydrophilicity values ​​within ±2, in various embodiments within ±1, and in various embodiments within ±0.5.

[0038] Exemplary amino acid substitutions are listed in Table 1. TIFF0007807076000001.tif187170

[0039] Those skilled in the art can use well-known techniques to determine suitable variants of the polypeptides described herein. In various embodiments, those skilled in the art can identify suitable regions of the molecule where changes can be made without destroying activity by targeting regions not believed to be important for activity. In other embodiments, those skilled in the art can identify residues and portions of the molecule that are conserved among similar polypeptides. In further embodiments, even regions that may be important for biological activity or structure can be subject to conservative amino acid substitutions without destroying biological activity or adversely affecting polypeptide structure.

[0040] Additionally, one skilled in the art can investigate structure-function studies that identify residues in similar polypeptides that are important for activity or structure. Taking such comparisons into account, one skilled in the art can predict the importance of amino acid residues in a polypeptide that correspond to amino acid residues important for the activity or structure of the similar polypeptide. One skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0041] Furthermore, one skilled in the art can analyze the three-dimensional structure and amino acid sequence in relation to the structures of similar polypeptides. In light of such information, one skilled in the art can predict the arrangement of amino acid residues of a polypeptide relative to its three-dimensional structure. In various embodiments, one skilled in the art can select amino acid residues predicted to be present on the surface of the polypeptide so as not to make fundamental changes to these residues, as these residues may be involved in important interactions with other molecules. Furthermore, one skilled in the art can generate test variants containing single amino acid substitutions at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about suitable variants. For example, if a change to a particular amino acid residue is found to destroy activity, undesirably reduce activity, or result in inappropriate activity, variants containing such changes can be avoided. In other words, based on the information gathered from such routine experiments, one skilled in the art can easily identify amino acids for which further substitutions, alone or in combination with other mutations, should be avoided.

[0042] The terms "polypeptide fragment" and "truncated polypeptide," as used herein, refer to a polypeptide having a deletion at the amino terminus and / or carboxy terminus compared to the corresponding full-length protein. In certain embodiments, a fragment can be, for example, 5 or more, 10 or more, 25 or more, 50 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1000 or more amino acids in length. In certain embodiments, a fragment can be, for example, 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 450 or less, 400 or less, 350 or less, 300 or less, 250 or less, 200 or less, 150 or less, 100 or less, 50 or less, 25 or less, 10 or less, or 5 or less amino acids in length. The fragment may further comprise one or more additional amino acids at one or both termini, for example, an amino acid sequence derived 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).

[0043] The terms "polypeptide variant," "hybrid polypeptide," and "polypeptide variant," as used herein, refer to a polypeptide comprising an amino acid sequence in which one or more amino acid residues have been inserted, deleted, and / or substituted into the amino acid sequence compared to another polypeptide sequence. In certain embodiments, the number of inserted, deleted, or substituted amino acid residues 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 encompass fusion proteins.

[0044] A "derivative" of a polypeptide is a polypeptide that has been chemically modified, for example, by conjugation to another chemical moiety, such as polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.

[0045] As used herein, the term "% sequence identity" is used interchangeably with the term "% identity" and refers to the amino acid sequence identity between two or more peptide sequences or the nucleotide sequence identity between two or more nucleotide sequences when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same as 80% sequence identity measured by a specified algorithm, meaning that a given sequence has at least 80% identity to another sequence of a different length. In some embodiments, the % 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 more sequence identity to the given sequence. In certain embodiments, the percent identity is within the range of, for example, 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%.

[0046] As used herein, the term "% sequence identity" is used interchangeably with the term "% homology" and refers to the amino acid sequence identity between two or more peptide sequences or the nucleotide sequence identity between two or more nucleotide sequences when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same as 80% sequence identity as measured by a defined algorithm, i.e., a homolog of a given sequence has greater than 80% sequence identity over a certain length of the given sequence. In certain embodiments, the % 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 more sequence identity to the given sequence. In certain embodiments, the percent homology is within the range of, for example, 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%.

[0047] Exemplary computer programs that can be used to determine identity between two sequences include, but are not limited to, the suite 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 compared to amino acid sequences in GenBank Protein Sequences or other public databases, sequence searches are typically performed using the BLASTP program. The BLASTX program is preferred for searching nucleic acid sequences translated in all reading frames against amino acid sequences in GenBank Protein Sequences or other public databases. Both BLASTP and BLASTX were run using default parameters of open gap penalty=11.0, gap extension penalty=1.0, and utilize the BLOSUM-62 matrix. See supra.

[0048] 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 and Altschul, Proc. Nat'l. Acad. Sci. USA, 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which indicates the probability that a match between two nucleotide sequences or two amino acid sequences will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability when comparing a test nucleic acid with the reference nucleic acid is, for example, less than about 0.1, less than about 0.01, or less than about 0.001.

[0049] The term "heterologous," as used herein, refers to a construct or state that is not native or does not exist in nature, e.g., a construct or state that can be achieved by replacing an existing native construct or state with a construct or state from another source. Similarly, expression of a protein in an organism other than the organism in which the protein is naturally expressed results in a heterologous expression system and a heterologous protein.

[0050] The term "antibody," as used herein, refers to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes, and having specificity for tumor antigens or molecules overexpressed in pathological conditions. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as subtypes of these genes and the myriad immunoglobulin variable region genes. Light chains (LC) are classified as either kappa or lambda light chains. Heavy chains (HC) are classified as gamma, mu, alpha, delta, or epsilon heavy chains, which define the immunoglobulin classes (IgG, IgM, IgA, IgD, and IgE, respectively). A typical immunoglobulin (e.g., antibody) structural unit is a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kD) and one "heavy" chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of approximately 100-110 or more amino acids that is primarily responsible for antigen recognition.

[0051] The term "Fc region," as used herein, defines the C-terminal region of an immunoglobulin heavy chain and can be generated by papain digestion of an intact antibody. The Fc region may be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin typically contains two constant domains, the CH2 domain and the CH3 domain, and optionally contains a CH4 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 antibody and antigen-antibody complexes (e.g., neonatal FcR (FcRn) binds to the Fc region of IgG at the acidic pH in the endosome, protecting the IgG from degradation and contributing to the long serum half-life of IgG). Substitutions of amino acid residues in the Fc portion to alter antibody effector functions are known in the art (see, e.g., Winter et al., U.S. Patent Nos. 5,648,260 and 5,624,821). Exemplary Fc amino acid substitutions that eliminate binding to FcγRs and C1q but retain FcRn binding include L234A / L235A / G237A and L234A / L235A / P329G mutations.

[0052] A "polynucleotide" refers to a polymer composed of nucleotide units. Polynucleotides encompass not only naturally occurring nucleic acids such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), but also nucleic acid analogs. Nucleic acid analogs include those containing unnatural bases, which are nucleotides joined with other nucleotides via bonds other than natural phosphodiester bonds, and those containing bases added through bonds other than phosphodiester bonds. That is, nucleic acid analogs include, but are not limited to, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized using automated DNA synthesizers, etc. The term "nucleic acid" typically refers to large polynucleotides. The term "oligonucleotide" typically refers to short polynucleotides, usually less than about 50 nucleotides. Where a nucleotide sequence is represented as a DNA sequence (i.e., A, T, G, C), it is understood that this also encompasses RNA sequences in which "U" is replaced with "T" (i.e., A, U, G, C).

[0053] 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 addition of nucleotides from the 5' to the 3' direction to a nascent RNA transcript is referred to as the transcription direction. The DNA strand having the same sequence as the mRNA is referred to as the "coding strand"; sequences on the DNA strand having the same sequence as the mRNA transcribed from the DNA and located 5' to the 5' end of the RNA transcript are referred to as "upstream sequences"; and sequences on the DNA strand having the same sequence as the RNA and located 3' to the 3' end of the coding RNA transcript are referred to as "downstream sequences."

[0054] "Complementary" refers to a topological compatibility, i.e., the identity of the interacting surfaces of two polynucleotides. That is, the two molecules can be described as complementary, and further, the contacting 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 is capable of hybridizing to the second polynucleotide under stringent hybridization conditions.

[0055] A "vector" is a polynucleotide that can be used to introduce another nucleic acid linked to it into a cell. One type of vector is a "plasmid," which is a linear or circular double-stranded DNA molecule into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication-deficient retroviruses, replication-deficient adenoviruses, and replication-deficient adeno-associated viruses), whose viral genome can contain additional DNA segments. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors containing a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell after introduction and are replicated along with the host genome. An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide.

[0056] A "control sequence" is a nucleic acid that affects the expression (e.g., amount, timing, or location) of a nucleic acid to which it is operably linked. A control sequence can, for example, exert its effect directly on the regulated nucleic acid or through the action of one or more other molecules (e.g., a polypeptide that binds to the control sequence and / or the nucleic acid). Examples of control sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Further examples of control sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, CA, and Baron et al., 1995, Nucleic Acids Res., Vol. 23, pp. 3605-06. A nucleotide sequence is "operably linked" to a control sequence if the control sequence affects the expression (e.g., amount, timing, or location) of the nucleotide sequence.

[0057] A "host cell" is a cell that can be used to express a polynucleotide of the present disclosure. A host cell can be prokaryotic, e.g., E. coli, or eukaryotic, e.g., a unicellular eukaryote (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. Typically, a host cell is a cultured cell that can be transformed or transfected with a polypeptide-encoding nucleic acid, allowing expression of the polypeptide-encoding nucleic acid in the host cell. The term "recombinant host cell" is sometimes used to refer to a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell can also be a cell that contains the nucleic acid but does not express it at the desired level, unless control sequences operably linked to the nucleic acid have been introduced into the host cell. The term host cell is understood to refer not only to the particular subject cell but also to the progeny and potential progeny of such a cell. Because certain modifications may occur in subsequent generations due to mutations, environmental influences, and the like, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term as used herein.

[0058] The term "isolated molecule" (where the molecule is, for example, a polypeptide or polynucleotide) refers to a molecule that, based on its origin or source of derivation, is (1) free from naturally occurring components that accompany it in its natural state; (2) substantially free from other molecules from the same species; (3) expressed by cells from a different species; or (4) not naturally occurring. That is, a molecule that is chemically synthesized or expressed in a cellular system different from the cell in which it naturally occurs would be "isolated" from its naturally occurring components. A molecule may be rendered substantially free of naturally occurring components by isolation using purification methods well known in the art. Molecular purity or homogeneity can be assessed by several means well known in the art. For example, the purity of a polypeptide sample can be assessed by visualization of the polypeptide by polyacrylamide gel electrophoresis and gel staining using methods well known in the art. For certain purposes, higher resolution may be achieved using HPLC or other purification means well known in the art.

[0059] A protein or polypeptide is "substantially pure," "substantially homogeneous," or "substantially purified" when at least about 60%-75% of a sample represents a single polypeptide species. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein typically comprises about 50%, 60%, 70%, 80%, or 90% (w / w) of a protein sample, more frequently about 95%, and preferably greater than 99% pure. Protein purity or homogeneity can be demonstrated by several means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample followed by visualization of a single polypeptide band by staining the gel using staining methods well known in the art. For certain purposes, higher resolution may be achieved by using HPLC or other purification means well known in the art.

[0060] A "linker" refers to a molecule that links two other molecules covalently or through ionic, van der Waals, or hydrogen bonds. For example, a nucleic acid molecule that hybridizes to the 5' end of one complementary sequence and to the 3' end of another complementary sequence, thereby linking two non-complementary sequences. A "cleavable linker" refers to a linker that can be degraded or otherwise cleaved to separate the two components connected by the cleavable linker. Cleavable linkers are generally cleaved by enzymes, typically peptidases, proteases, nucleases, lipases, etc. Cleavable linkers can also be cleaved by environmental factors, such as changes in temperature, pH, or salt concentration.

[0061] The term "label" or "labeling," as used herein, refers to the incorporation of another molecule into an antibody. In one embodiment, the label is a detectable marker, such as the incorporation of a radiolabeled amino acid or the addition of a biotinyl moiety to the polypeptide that is detectable by marked avidin (e.g., streptavidin containing a fluorescent marker or an enzymatic activity that is detectable by optical or calorimetric methods). In another embodiment, the label or marker can be a therapeutic label or marker, such as a drug conjugate or a toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, 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 fluorophores, rhodamine fluorophores, lanthanide fluorophores), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), magnetic agents such as gadolinium chelates, toxins such as pertussis toxin and taxol, Cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, 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, labels are attached by spacer arms of various lengths to avoid potential steric hindrance.

[0062] The term "immunotherapy" refers to cancer treatments, including, for example, treatment with depleting antibodies against specific tumor antigens; treatment with antibody-drug conjugates; treatment with agonist, antagonist, or blocking antibodies against costimulatory or co-inhibitory molecules (immune checkpoints), such as CD276, CD272, CTLA-4, PD-1, PD-L1, CD40, SIRPα, 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 (BiTE®), such as blinatumomab; treatment with IL-2, IL-12, IL-15, IL-21, GM-CSF, IFN-α, IFN-β, and IFN-β. treatment with dendritic cell vaccines or tumor antigen peptide vaccines; 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; treatment with immunostimulants such as the Toll-like receptor (TLR) agonist CpG and imiquimod, and treatment with vaccines such as BCG; the above combination therapies increase tumor cell killing by effector cells, i.e., there is synergy between the IL-15 construct and immunotherapy when administered simultaneously.

[0063] The term "effective amount" or "therapeutically effective amount," as used herein, refers to an amount of a compound or composition sufficient to treat a particular disorder, condition, or disease, such as improving, alleviating, mitigating, and / or delaying one or more of its symptoms. With respect to NHL and other cancers or other unwanted cell growth, an effective amount includes an amount sufficient to: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow to some extent, and preferably stop cancer cell invasion into peripheral organs; (iv) inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay tumor onset and / or recurrence; 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.

[0064] The terms "patient," "individual," and "subject" may be used interchangeably and refer to a mammal, preferably a human or non-human primate, but may also refer to domesticated mammals (e.g., dogs or cats), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and agricultural mammals (e.g., horses, cows, pigs, sheep). In various embodiments, a patient can be a human (e.g., an adult male, adult female, adolescent male, adolescent female, boy, girl) receiving treatment from a physician or other medical professional in a hospital, psychiatric facility, as an outpatient, or in other clinical settings. 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 disorders, AIDS patients, cancer patients and transplant patients taking certain immunosuppressants, and patients with genetic diseases affecting 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 with reported high mutation rates (Lawrence et al., Nature, 499(7457):214-218, 2013).

[0065] A "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in mammals. A pharmaceutical composition contains a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. A "pharmacologically effective amount" refers to an amount of agent effective to achieve the intended pharmacological result. A "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier, solvent, buffer, and excipient, such as phosphate-buffered saline, 5% aqueous dextrose, emulsions such as oil-in-water emulsions or water-in-oil emulsions, and various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 21st ed., 2005, Mack Publishing Co., Easton. A "pharmaceutically acceptable salt" refers to a salt that can be incorporated into a compound for pharmaceutical use, including, for example, metal salts (sodium salts, potassium salts, magnesium salts, calcium salts, etc.), ammonium salts, and organic amine salts.

[0066] The phrases "administering" or "cause to be administered" refer to the act of managing and / or authorizing the administration of the agent / compound in question to a patient by a medical professional (e.g., a physician) or a person managing the medical care of a patient. Administering 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. Where administering is described herein, "causing to be administered" is also contemplated.

[0067] "Resistant or refractory cancer" refers to tumor cells or cancers that do not respond to previous anti-cancer treatments, including chemotherapy, surgery, radiation therapy, stem cell transplantation, and immunotherapy. Tumor cells may be resistant or refractory from the beginning of treatment, or may become resistant or refractory over the course of treatment. Refractory tumor cells include tumors that do not respond to the initiation of treatment, or that show a short-term initial response but do not respond to treatment. Refractory tumor cells also include tumors that respond to treatment with an anti-cancer therapy but do not respond to subsequent treatments. For purposes of the present invention, refractory tumor cells also encompass tumors that appear to be inhibited by treatment with an anti-cancer therapy but recur within five years, and in some cases within ten years or longer, after treatment is discontinued. Anti-cancer treatments can include chemotherapy alone, radiation alone, targeted therapy alone, surgery alone, or a combination of these. For ease of explanation and not by way of limitation, it should be understood that the above-mentioned refractory tumor cells are interchangeable with resistant tumors.

[0068] The terms "treat," "treating," and "treatment" refer to a method of alleviating or eliminating at least one of a biological disorder and / or its attendant symptoms. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of occurrence of the symptoms of the disease, disorder, or condition. Furthermore, references to "treatment" herein encompass references to curative, symptomatic, and prophylactic treatment.

[0069] Aspects and embodiments of the present disclosure described herein are understood to encompass "consisting of" and / or "consisting essentially of" aspects and embodiments.

[0070] Any statement herein referring to "about" a value or parameter encompasses (and accounts for) the variation that surrounds the value or parameter itself. For example, a statement referring to "about X" encompasses the statement "X."

[0071] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include the plural unless the context clearly dictates otherwise. The embodiments and modifications of the disclosure described herein are understood to include "consisting of" and / or "consisting essentially of" embodiments and modifications.

[0072] IL-15 / IL-15RαSushi complex Interleukin-15 (IL-15) is a cytokine identified by two separate groups based on its ability to stimulate proliferation of the IL-2-dependent CTLL-2 T cell line in the presence of anti-IL-2 neutralizing antibodies (Steel et al., Trends in Pharmacological Sciences, Vol. 33(1): pp. 35-41, 2012). IL-15 and interleukin-2 (IL-2) interact via receptor (R) signaling components (IL-2 / 15Rβγ). cIL-15 and IL-2 share similar biological properties in vitro, as evidenced by their shared IL-15Rαβγ and IL-2Rαβγ receptors. However, the differential specificity of IL-15 relative to IL-2 is mediated by unique, individual α-chain receptors that complete the heterotrimeric high-affinity receptor complex IL-15Rαβγ and IL-2Rαβγ, allowing for differential responsiveness depending on the ligand and the expressed high-affinity receptor. Interestingly, both IL-15 and IL-15Rα transcripts have a much broader tissue distribution than IL-2 / IL-2Rα. Furthermore, multiple complex post-transcriptional regulatory mechanisms tightly control IL-15 expression. Thus, the complex regulation of IL-15 and IL-15Rα expression, as well as the differential expression patterns, suggest that the critical in vivo functions of this receptor / ligand pair may differ from those of IL-2 and IL-2Rα. To date, studies examining the biological properties of IL-15 have identified several important, nonredundant roles, including its importance in the development and function of natural killer (NK) cells, NK-T cells, and small intestinal intraepithelial lymphocytes. The role of IL-15 in autoimmune processes such as rheumatoid arthritis and malignancies such as adult T-cell leukemia suggests that dysregulation of IL-15 may have deleterious effects on the host (Fehniger et al., Blood 97:14-32, 2001).

[0073] 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 the immature or precursor form, and the mature form. Non-limiting examples of GenBank accession numbers for the amino acid sequences of various native mammalian interleukin-15 species include NP_000576 (human, immature), CAA62616 (human, immature), NP_001009207 (Felis catus, immature), AAB94536 (Rattus, immature), AAB41697 (Rattus, immature), NP_032383 (Mus musculus, immature), AAR19080 (dog), AAB60398 (Macaca mulatta, immature), AAI00964 (human, immature), AAH23698 (Mus musculus, immature), and AAH18149 (human). In various embodiments of the present invention, native IL-15 is an immature or precursor form of naturally occurring mammalian IL-15. In other embodiments, native IL-15 is a mature form of naturally occurring mammalian IL-15. In various embodiments, native IL-15 is a precursor form of naturally occurring human IL-15. In various embodiments, 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 below. MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 1)

[0074] The IL-15 receptor is a type I cytokine receptor consisting of beta (β) and gamma (γ) subunits (shared with the IL-2 receptor) and an alpha (α) subunit (which binds IL-15 with high affinity). Full-length human IL-15Rα is a type 1 transmembrane protein with multiple N-linked or O-linked glycosylation sites, including a 32-amino acid signal peptide, a 173-amino acid extracellular domain, a 21-amino acid transmembrane region, and a 37-amino acid cytoplasmic tail (Anderson et al., J. Biol. Chem. 270:29862-29869, 1995). Previously, a naturally occurring soluble form of the IL-15Rα chain, corresponding to the entire extracellular domain of IL-15Rα, was shown to act as a high-affinity IL-15 antagonist. In stark contrast to this finding, we found that a recombinant soluble sushi domain of IL-15Rα, which accounts for the majority of IL-15 binding affinity, acts as a potent IL-15 agonist by enhancing its binding and biological effects (proliferation and protection from apoptosis) via the IL-15Rβ / γ heterodimer, while leaving the IL-15 binding and function of the trimolecular IL-15Rα / β / γ membrane receptor unaffected. These results suggest that such a soluble sushi domain, when naturally produced, may be involved in the transpresentation mechanism of IL-15 (Mortier et al., J. Biol. Chem. 281(3):1612-1619, 2006).

[0075] 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, mature forms, and naturally occurring isoforms. Non-limiting examples of GenBank accession numbers for various native mammalian IL-15Rα amino acid sequences include NP_002180 (human), ABK41438 (rhesus monkey), NP_032384 (house mouse), Q60819 (house mouse), and Q13261 (human). In various embodiments, native IL-15Rα is the immature form of a naturally occurring mammalian IL-15Rα polypeptide. In various embodiments, native IL-15Rα is the mature form of a naturally occurring mammalian IL-15Rα polypeptide. In various embodiments, native IL-15Rα is a form of a naturally occurring mammalian IL-15Rα polypeptide. In various embodiments, native IL-15Rα is the full-length form of a naturally occurring mammalian IL-15Rα polypeptide. In various embodiments, native IL-15Rα is the immature form of a naturally occurring human IL-15Rα polypeptide. In various embodiments, native IL-15Rα is the mature form of a naturally occurring human IL-15Rα polypeptide. In various embodiments, native IL-15Rα is a form of a naturally occurring human IL-15Rα polypeptide. In various embodiments, native IL-15Rα is the full-length 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 domain is derived from the amino acid sequence of the human IL-15Rα sequence set forth in SEQ ID NO:3 below: MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 3)

[0076] In various embodiments, native IL-15Rα is the complete extracellular form of the 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 below. ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT (SEQ ID NO: 4)

[0077] 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 below: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 2) The IL-15Rα Sushi domain comprises the amino acid sequence of the mature human IL-15Rα polypeptide set forth in SEQ ID NO:5 below. ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP (SEQ ID NO: 5)

[0078] In various embodiments, the IL-15 domain of the IL-15 / IL-15RαSushi complex is an IL-15 variant (or mutant) comprising a sequence derived from the sequence of the mature human IL-15 polypeptide set forth in SEQ ID NO:2. IL-15 variants (or mutants) are designated using the original amino acid, the position of that original amino acid within the mature sequence, and the variant amino acid. For example, "huIL-15S58D" refers to human IL-15 containing 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 functions as an IL-15 agonist or antagonist. In various embodiments, an IL-15 variant with agonist activity also has superagonist activity. In various embodiments, an IL-15 variant can function as an IL-15 agonist or antagonist independently of binding to IL-15Rα. An IL-15 agonist is exemplified by equivalent or increased biological activity compared to wild-type IL-15. An IL-15 antagonist is exemplified by decreased biological activity compared to wild-type IL-15 or the ability to inhibit IL-15-mediated responses. In various embodiments, IL-15 variants exhibit increased or decreased activity and / or inhibit IL-15Rβγ. C In various embodiments, the sequence of the IL-15 variant has at least one amino acid change, e.g., a substitution or deletion, when compared to the native IL-15 sequence, which confers IL-15 agonist or IL-15 antagonist activity. In various embodiments, the amino acid substitution / deletion is at least one amino acid change, e.g., a substitution or deletion, at the IL-15Rβ and / or γ receptors. CThe amino acid substitutions / deletions are located within the domain of IL-15 that interacts with IL-15Rα. In various embodiments, the amino acid substitutions / deletions do not affect binding to the IL-15Rα polypeptide or the ability of the IL-15 variant to be produced. Suitable amino acid substitutions / deletions for generating IL-15 variants can be identified based on the known structure of IL-15, by comparing IL-15 with similar molecules, such as IL-2, with known structures, through rational or random mutagenesis and functional analysis, or other empirical methods, as provided herein. Furthermore, suitable amino acid substitutions can be conservative changes and insertions of additional amino acids, or non-conservative changes and insertions of additional amino acids. In various embodiments, the IL-15 variants of the present invention contain one or more amino acid substitutions at positions 30, 31, 32, 62, 63, 67, 68, or 108 of the mature human IL-15 sequence set forth in SEQ ID NO:2. In various embodiments, a D30T, V31Y, H32E, S58R, S58Q, T62D, V63A, V63R, V63K, I68H, I68F, I68D, I68K, I68Q, I68G, Q108A, Q108S, Q108E, Q108K, or Q108M ​​substitution or combination of substitutions results in an IL-15 variant with antagonist activity, and an S58D substitution results in an IL-15 variant with agonist activity.

[0079] In various embodiments, the IL-15 domain of the IL-15 / IL-15RαSushi complex is a human IL-15 variant polypeptide with a deletion at positions 111-114 (SEQ ID NO: 39). In various embodiments, the IL-15 domain of the IL-15 / IL-15RαSushi complex is a human IL-15 variant polypeptide with a deletion at positions 109-114 (SEQ ID NO: 40). In various embodiments, the IL-15 domain of the IL-15 / IL-15RαSushi complex is a human IL-15 variant polypeptide with a deletion at positions 108-114 (SEQ ID NO: 41). In various embodiments, the IL-15 domain of the IL-15 / IL-15RαSushi complex is a human IL-15 variant polypeptide with a deletion at positions 105-114 (SEQ ID NO: 42). In various embodiments, the IL-15 domain of the IL-15 / IL-15RαSushi complex is a human IL-15 variant polypeptide (SEQ ID NOs: 56-61) with one, two, three, four, five, or six amino acid deletions at the N-terminus. In various embodiments, the IL-15 domain of the IL-15 / IL-15RαSushi complex is a human IL-15 variant polypeptide with a combination of amino acid substitutions and deletions represented by SEQ ID NOs: 62 and 63. In various embodiments, the IL-15 domain of the IL-15 / IL-15RαSushi complex is a human IL-15 variant polypeptide (SEQ ID NO: 43) with a "GS" (SEQ ID NO: 12) insertion after position N95. In various embodiments, the IL-15 domain of the IL-15 / IL-15RαSushi complex is a human IL-15 variant polypeptide (SEQ ID NO: 44) with a "GGSGG" (SEQ ID NO: 153) insertion after position N95. In various embodiments, the IL-15 domain of the IL-15 / IL-15RαSushi complex is a human IL-15 variant polypeptide (SEQ ID NO: 45) with a "GSSGGSGGS" (SEQ ID NO: 154) insertion after position N95.

[0080] Fc domain IgG class immunoglobulins are the most abundant proteins in human blood. Their circulating half-life can reach as long as 21 days. Fusion proteins combining the Fc region of IgG with domains from other proteins, such as various cytokines or receptors, have been reported (see, e.g., 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 of a fusion protein is a homodimeric protein, in which the heavy chain variable region and CH1 domain are linked via cysteine ​​residues in the hinge region of the IgG Fc, resulting in a molecule similar to an IgG molecule lacking the light chain. The dimeric nature of fusion proteins containing the Fc domain can be advantageous for achieving higher-order interactions (i.e., bivalent or bispecific binding) with other molecules. Due to their structural homology, Fc fusion proteins exhibit in vivo pharmacokinetic profiles comparable to those of human IgG of the same isotype.

[0081] The term "Fc" refers to a molecule or sequence containing the sequence of a non-antigen-binding fragment of a full-length antibody, which may be in a monomeric or multimeric form. The original immunoglobulin source of a native Fc is preferably human and may be any immunoglobulin. Native Fc is composed of monomeric polypeptides that can be linked by covalent (i.e., disulfide) and non-covalent bonds into dimeric or multimeric forms. The number of intermolecular disulfide bonds between the monomeric subunits of a native Fc molecule ranges from one to four, depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is the disulfide-linked dimer resulting from papain digestion of IgG (Ellison et al., (1982), Nucleic Acids Res., 10:4071-9). The term "native Fc" as used herein refers collectively to the monomeric, dimeric, and multimeric forms of the Fc domain, which contains binding sites for Protein A, Protein G, various Fc receptors, and complement proteins.

[0082] In various embodiments, the term "Fc variant" refers to a molecule or sequence that has been modified from a native Fc but still contains a binding site for the salvage receptor, FcRn. Exemplary Fc variants and their interactions with the salvage receptor are described in International Publication Nos. WO 97 / 34631 (published September 25, 1997) and WO 96 / 32478, which are incorporated herein by reference. Additionally, the native Fc contains sites that may be removed because they confer structural features or biological activity not required for the fusion molecules of the invention. That is, in various embodiments, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal or C-terminal heterogeneity after expression 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).

[0083] The term "Fc domain" encompasses native Fc and Fc variant molecules and sequences, as defined above. Similar to Fc variants and native Fc, the term "Fc domain" encompasses molecules in monomeric or multimeric form, either digested from a full-length antibody or produced by recombinant gene expression or other means. In various embodiments, each Fc domain monomer in the Fc domain contains an amino acid substitution in the CH2 antibody constant domain to reduce interaction or binding between the Fc domain and an Fcγ receptor. In various embodiments, each subunit of the Fc domain contains three amino acid substitutions that reduce binding to an activating Fc receptor and / or effector function, wherein the amino acid substitutions are L234A, L235A, and G237A (SEQ ID NO: 6).

[0084] In various embodiments, two Fc domain monomers of the Fc domain each contain an amino acid substitution that promotes heterodimerization of the two monomers. In various other embodiments, heterodimerization of Fc domain monomers can be promoted by introducing different but compatible substitutions (e.g., a "knob-into-hole" pair of residues) into the two Fc domain monomers. This "knob-into-hole" technique is also disclosed in U.S. Pat. No. 8,216,805. In yet another embodiment, one Fc domain monomer contains the knob-type mutation T366W, and the other Fc domain monomer contains the hole-type mutations T366S, L358A, and Y407V. In various embodiments, two Cy residues that form stabilizing disulfide bridges (S354C on the "knob" side and Y349C on the "hole" side) were introduced (SEQ ID NOs: 7 and 8).

[0085] 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 linked to the IL-15 / IL-15RαSushi complex directly or via a peptide linker sequence to form an IL-15 fusion protein. As used herein, the term "fusion protein" refers to a protein having a heterologous polypeptide linked by recombinant DNA techniques. 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 to the fusion molecule. Such polypeptides include other polypeptides that bind to IgG Fc or neonatal Fcγ / receptors, human serum albumin, or polypeptides that bind to proteins with long serum half-lives, including IgG, immunoglobulins other than IgG, proteins, and non-protein drugs that have an extended in vivo half-life due to the presence of an IgG constant domain, or an FcRn-binding portion thereof, with one or more amino acid modifications that increase the affinity of the constant domain or fragment for FcRn. Such proteins and molecules with extended half-lives have the advantage that reduced amounts and / or less frequent administration are required for therapeutic, prophylactic, or diagnostic uses 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 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, or any combination thereof. In various embodiments, the Fc domain comprises amino acid changes that result in the Fc domain having altered complement fixation or Fc receptor binding properties.Amino acid changes that produce Fc domains with altered complement binding or Fc receptor binding properties are known in the art.

[0086] In various embodiments, the Fc domain may be mutated to further extend in vivo half-life. In various embodiments, each subunit of the Fc domain contains three amino acid substitutions (M252Y, S254T, and T256E) that enhance binding to human FcRn, as disclosed in U.S. Patent No. 7,658,921 (SEQ ID NO: 105). In various embodiments, each subunit of the Fc domain contains one amino acid substitution (N434A) that enhances binding to human FcRn, as disclosed in U.S. Patent No. 7,371,826 (SEQ ID NO: 106). In various embodiments, each subunit of the Fc domain contains one amino acid substitution (M428L and N434S) that enhances binding to human FcRn, as disclosed in U.S. Patent No. 8,546,543. In various embodiments, the half-life extending mutations can be combined with amino acid substitutions that reduce / eliminate binding to activating Fc receptors, thereby reducing / eliminating effector function.

[0087] In various embodiments, the Fc domain sequence used to generate the dimeric IL-15 / IL-15Rα complex-Fc fusion protein is an IgG1-Fc domain sequence with reduced / eliminated effector function and having the amino acid sequence set forth in SEQ ID NO:6.

[0088] In various embodiments, the Fc domain sequence used to generate the dimeric IL-15 / IL-15Rα complex-Fc fusion protein is an IgG1-Fc domain sequence having the amino acid sequence set forth in SEQ ID NO: 105, which has reduced / eliminated effector function and extended half-life.

[0089] In various embodiments, the Fc domain sequence used to generate the dimeric IL-15 / IL-15Rα complex-Fc fusion protein is an IgG1-Fc domain sequence having the amino acid sequence set forth in SEQ ID NO: 106, which has reduced / eliminated effector function and extended half-life.

[0090] In various embodiments, the heterodimeric Fc domain sequence used to generate the monovalent IL-15 / IL-15Rα complex-Fc fusion protein is a Knob-Fc domain sequence with reduced / eliminated effector function and having the sequence set forth in SEQ ID NO:7.

[0091] In various embodiments, the heterodimeric Fc domain sequence used to generate the monovalent IL-15 / IL-15Rα complex-Fc fusion protein is a Hole-Fc domain sequence with reduced / eliminated effector function and having the sequence set forth in SEQ ID NO:8.

[0092] In various embodiments, the heterodimeric Fc domain sequence used to generate the monovalent IL-15 / IL-15Rα complex-Fc fusion protein is a Knob-Fc domain having the amino acid sequence set forth in SEQ ID NO: 107, which has reduced / eliminated effector function and extended half-life.

[0093] In various embodiments, the heterodimeric Fc domain sequence used to generate the monovalent IL-15 / IL-15Rα complex-Fc fusion protein is a Hole-Fc domain having the amino acid sequence set forth in SEQ ID NO: 108, which has reduced / eliminated effector function and extended half-life.

[0094] Tumor-associated antigens, antibodies, and protein / peptide conjugates In various embodiments, the heterologous protein can comprise an antibody, antibody fragment, or protein or peptide capable of binding to a tumor-associated antigen (TAA) on diseased cells or tissue. A TAA can be any molecule, macromolecule, combination of molecules, etc., to which an immune response is desired. A TAA can be a protein comprising one or more polypeptide subunits. For example, the protein can be a dimer, trimer, or higher multimer. In various embodiments, two or more subunits of the protein can be linked by a covalent bond, such as a disulfide bond. In various embodiments, the subunits of the protein can be held together by non-covalent interactions. Thus, a TAA can be any peptide, polypeptide, protein, nucleic acid, lipid, carbohydrate, or small organic molecule, or any combination thereof, to which one of skill in the art desires to induce an immune response. In various embodiments, the TAA is a peptide comprising about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 amino acids. In various embodiments, the peptide, polypeptide, or protein is a molecule that is typically administered to a subject by injection. In various embodiments, after administration, the tumor-specific antibody or binding protein functions as a targeting moiety to direct the fusion molecule to an affected area, such as a cancer site, where the active domain can interact with its cognate receptor on affected cells or tissue.

[0095] Any of the aforementioned markers can be used as disease-associated targets or TAA targets of the constructs of the present invention. In various embodiments, one or more disease-associated targets or variants thereof, or TAA, TAA variants, or TAA mutants contemplated for use in the constructs and methods of the present disclosure are selected from or derived from the list shown in Table 2. TIFF0007807076000002.tif194170TIFF0007807076000003.tif232170TIFF0007807076000004.tif157170

[0096] Further examples of tumor-associated antigens include TRP-1, TRP-2, MAG-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-BSO (LAGE), SCP-1, Hom / Mel-40, H-Ras, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA19-9, CA72-4, CAM17.1, Numa, K-ras, β-ka These include tenin, CDK4, Muni-1, p16, TAGE, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, β-HCG, BCA225, BTAA, CA15-3 (CA27.29 / BCAA), CA195, CA242, CA-50, CAM43, CD68 / KF1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein / cyclophilin C-associated protein), TAAL6, TAG72, TLP, and TPS.

[0097] Immune checkpoint regulators Numerous immune checkpoint protein antigens have been reported to be expressed on various immune cells, including, for example, CD152 (expressed on activated CD8+ T cells, CD4+ T cells, and regulatory T cells), CD279 (expressed on tumor-infiltrating lymphocytes, activated T cells (both CD4 and CD8), regulatory T cells, activated B cells, activated NK cells, allergic T cells, monocytes, and dendritic cells), CD274 (expressed on T cells, B cells, dendritic cells, macrophages, vascular endothelial cells, and pancreatic islet cells), and CD223 (expressed on activated T cells, regulatory T cells, angergic T cells, NK cells, NKT cells, and plasmacytoid dendritic cells) (see, e.g., Pardoll, D., Nature Reviews Cancer, 12:252-264, 2012). Antibodies that bind to antigens identified as immune checkpoint proteins are known to those skilled in the art. For example, various anti-CD276 antibodies have been described in the art (see, e.g., US Patent Application Publication No. 20120294796 (Johnson et al) and the references cited therein). Various anti-CD272 antibodies have been described in the art (see, e.g., US Patent Application Publication No. 20140017255 (Mataraza et al) and the references cited therein). Various anti-CD152 / CTLA-4 antibodies have been described in the art (see, e.g., US Patent Application Publication No. 20130136749 (Korman et al) and the references cited therein). Various anti-LAG-3 / CD223 antibodies have been described in the art (see, e.g., US Patent Application Publication No. 20110150892 (Thudium et al) and the references cited therein). Various anti-CD279 / PD-1 antibodies have been described in the art (see, e.g., U.S. Patent No. 7,488,802 (Collins et al.) and references cited therein). Various anti-CD274 / PD-L1 antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20130122014 (Korman et al.) and references cited therein).Various anti-TIM-3 antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20140044728 (Takayanagi et al.) and the references cited therein). Various anti-B7-H4 antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20110085970 (Terrett et al.) and the references cited therein). Each of these documents is incorporated by reference in its entirety herein with respect to the specific antibodies and sequences taught therein.

[0098] In various embodiments, the heterologous protein may comprise an antibody, antibody fragment, or protein or peptide that exhibits binding to an immune checkpoint protein antigen present on the surface of an immune cell, hi various embodiments, the immune checkpoint protein antigen is selected from the group consisting of, but not limited to, CD276, CD272, CD152, CD223, CD279 (PD-1), CD274 (PD-L1), CD40, SIRPα, CD47, OX-40, GITR, ICOS, CD27, 4-1BB, TIM-3, B7-H4, Siglec-7, Siglec-8, Siglec-9, Siglec-15, and VISTA.

[0099] In various embodiments, the antibody is a humanized anti-FAP antibody comprising the heavy and light chain sequences set forth in SEQ ID NOs: 109-110. In various embodiments, the antibody is an antagonistic Programmed Death-1 (PD-1) antibody or antibody fragment. In various embodiments, the antibody is an antagonistic humanized PD-1 antibody comprising the heavy and light chain amino acid sequences set forth in SEQ ID NOs: 111-112. In various embodiments, the antibody is an antagonistic human PD-1 antibody comprising the heavy and light chain amino acid sequences set forth in SEQ ID NOs: 113-114. In various embodiments, the antibody is an antagonistic Programmed Death Ligand-1 (PD-L1) antibody or antibody fragment.

[0100] Linker In various embodiments, the heterologous protein (e.g., an Fc domain or antibody / antibody fragment) is covalently linked to the IL-15 polypeptide of the IL-15 / IL-15RαSushi (or functional fragment thereof) complex by 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 (or any number therebetween) that is relatively free of secondary structure. In various embodiments, the linker is enriched in G / S content (e.g., at least about 60%, at least about 70%, at least about 80%, or at least about 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 set forth in SEQ ID NOs: 9-12, 47, and 153-154. Each peptide linker sequence can be independently selected.

[0101] Examples of novel IL-15 / IL-15RαSushi complex fusion proteins In various embodiments, exemplary IL-15 / IL-15 RaSushi heterodimeric Fc fusion proteins of the invention are listed in Table 3A along with the fusion protein ID number and sequence numbers of fusion protein chain 1 and chain 2. TIFF0007807076000005.tif58170

[0102] In various embodiments, exemplary monovalent IL-15 / IL-15RαSushi (non-covalent) Fc fusion proteins of the invention are listed in Table 3B along with the fusion protein ID number and the sequence numbers of fusion protein chain 1, chain 2, and chain 3. TIFF0007807076000006.tif83170

[0103] In various embodiments, exemplary bivalent IL-15 / IL-15RαSushi (non-covalent) Fc fusion proteins of the invention are listed in Table 3C along with the fusion protein ID number and the sequence numbers of fusion protein chain 1, chain 2, and chain 3. TIFF0007807076000007.tif58170

[0104] In various embodiments, exemplary bivalent IL-15 / IL-15RαECD (non-covalent) Fc fusion proteins of the invention are listed in Table 3D along with the fusion protein ID number and sequence numbers of fusion protein chain 1 and chain 2. TIFF0007807076000008.tif51170

[0105] In various embodiments, exemplary monovalent IL-15 (non-covalent) / IL-15RαSushiFc fusion proteins of the invention are listed in Table 3E along with the fusion protein ID number and the sequence numbers of fusion protein chain 1, chain 2, and chain 3. TIFF0007807076000009.tif52170

[0106] 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 and chain 2 (Fc-linker-IL-15Rα-Sushi+) having the amino acid sequence set forth in SEQ ID NO: 23.

[0107] 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 the sequences set forth in 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, SEQ ID NO:45, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, and SEQ ID NO:81.

[0108] In various embodiments, various types of exemplary IL-15 / IL-15RαSushiFc fusion proteins of the present invention, which contain the S58D mutation in IL-15 and are illustrated in FIG. 1, are listed in Table 3F along with the fusion protein ID number and the sequence numbers of fusion protein chain 1, chain 2, and chain 3. TIFF0007807076000010.tif83170

[0109] In various embodiments, additional IL-15 variant / IL-15Rα complex-Fc fusion proteins comprising two or more heterodimeric chains are listed in Table 3G. TIFF0007807076000011.tif203170

[0110] In various embodiments, the use of an attenuated potency IL-15 variant in an IL-15 / IL15Rα-antibody fusion protein can facilitate the establishment of a stoichiometric balance between IL-15 and the targeting antibody to achieve an optimal dose where the antibody achieves sufficient target occupancy while the IL-15 moiety does not cause excessive pathway activation, minimizing peripheral activation, reducing antigen sinks, and facilitating tumor targeting via the antibody arm.

[0111] In various embodiments, additional IL-15 variant / IL-15Rα complex-PD-1 antagonist antibody fusion proteins comprising two or more heterodimeric chains are described in Table 3H. TIFF0007807076000012.tif105170

[0112] Polynucleotides In another aspect, the present disclosure provides an isolated nucleic acid molecule comprising a polynucleotide encoding the 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, an antibody, antibody fragment, IL-15 antibody fusion protein, or IL-15 / IL-15RαSushi-antibody fusion protein targeting a TAA or immune checkpoint regulator. The subject nucleic acids may be single-stranded or double-stranded. Such nucleic acids may be DNA or RNA molecules. Examples of DNA include cDNA, genomic DNA, synthetic DNA, PCR-amplified DNA, and combinations thereof. Genomic DNA encoding the IL-15 / IL-15RαSushi complex may be obtained from genomic libraries covering multiple species. Synthetic DNA can be obtained by chemically synthesizing overlapping oligonucleotide fragments and then assembling the fragments to reconstitute part or all of the coding region and flanking sequences. RNA can be obtained from a prokaryotic expression vector that directs high-level mRNA synthesis, such as a vector using a T7 promoter, and an RNA polymerase. cDNA can be obtained from a library prepared from mRNA isolated from various tissues that express IL-15. The DNA molecules of the present disclosure encompass not only full-length genes but also polynucleotides and fragments thereof. Full-length genes may also include sequences encoding an N-terminal signal sequence. Such nucleic acids can be used in methods, such as those for generating novel IL-15 / IL-15Rα Sushi-fusion proteins.

[0113] 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, hi various embodiments, the nucleic acid molecule further comprises a polynucleotide encoding a linker or hinge linker described herein.

[0114] In various embodiments, the recombinant nucleic acid of the present disclosure may be operably linked to one or more regulatory nucleotide sequences within an expression construct. Regulatory sequences are art-recognized and selected to direct expression of the IL-15 / IL-15RαSushi-fusion protein. Thus, the term regulatory sequence encompasses promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990). Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, a promoter sequence, a leader or signal sequence, a ribosomal binding site, transcription start and stop sequences, translation start and stop sequences, and an enhancer or activator sequence. Constitutive or inducible promoters known in the art are contemplated by the present disclosure. The promoter may be a naturally occurring promoter or a hybrid promoter that combines elements of two or more promoters. The expression construct may be present intracellularly or episomally, such as a plasmid, or the expression construct may be inserted into a chromosome. In various embodiments, the expression vector contains a selectable marker gene to allow for the selection of transformed host cells. Selectable marker genes are well known in the art and vary depending on the host cell used.

[0115] In another embodiment of the present disclosure, the subject nucleic acids are provided in an expression vector comprising a nucleotide sequence encoding an IL-15 / IL-15RαSushi complex operably linked to at least one regulatory sequence. The term "expression vector" refers to a plasmid, phage, virus, or vector for expressing a polypeptide from a polynucleotide sequence. Suitable vectors for expression in host cells are readily available, and insertion of nucleic acid molecules into the vector is accomplished using standard recombinant DNA techniques. Such vectors can contain a variety of expression control sequences that, when operably linked, control expression of the DNA sequence and can be used in the vector to express an IL-15 / IL-15RαSushi-Fc or IL-15 / IL-15RαSushi-antibody fusion protein. Useful expression control sequences include, for example, the SV40 early and late promoters, the tet promoter, adenovirus or cytomegalovirus immediate-early promoters, the RSV promoter, the lac, trp, TAC, or TRC systems, the T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter region of lambda phage, the regulatory region for the fd coat protein, promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoter for acid phosphatase (e.g., PhoS), the promoter for yeast alpha mating factor, the polyhedrin promoter of baculovirus systems, and other sequences known to regulate the expression of genes in prokaryotic or eukaryotic cells or their viruses, as well as various combinations thereof. It will be understood that the design of the expression vector may vary depending on factors such as the choice of the host cell to be transformed and / or the type of protein desired to be expressed. Additionally, consideration should be given to the vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers.

[0116] The cloned gene, or a portion thereof, can be ligated into a vector suitable for expression in either prokaryotic or eukaryotic cells (yeast, avian, insect, or mammalian cells), or both, to produce the recombinant nucleic acid of the present disclosure. Expression vehicles for producing the recombinant IL-15 / IL-15RαSushi complex include plasmids and other vectors. For example, suitable vectors include the following various plasmids for expression in prokaryotic cells, such as E. coli: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids.

[0117] Some mammalian expression vectors contain both prokaryotic sequences that facilitate propagation of the vector in bacteria and one or more eukaryotic transcription units that are expressed in eukaryotic cells. pcDNAI / amp-derived vectors, pcDNAI / neo-derived vectors, pRc / CMV-derived vectors, pSV2gpt-derived vectors, pSV2neo-derived vectors, pSV2-dhfr-derived vectors, pTk2-derived vectors, pRSVneo-derived vectors, pMSG-derived vectors, pSVT7-derived vectors, pko-neo-derived vectors, and pHyg-derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors have been modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, for transient expression of proteins in eukaryotic cells, derivatives of viruses such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived, and p205) can be used. Examples of other viral (including retroviral) expression systems can be found in the discussion of gene therapy delivery systems below. Various methods used in preparing plasmids and transforming host organisms are well known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells, as well as general recombinant methods, see Chapters 16 and 17 of Molecular Cloning: A Laboratory Manual, 2nd ed., Sambrook, Fritsch, and Maniatis (eds.) (Cold Spring Harbor Laboratory Press, 1989). 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 the B-gal-containing pBlueBacIII).

[0118] In various embodiments, vectors will be designed for producing a subject IL-15 / IL-15RαSushi-Fc fusion protein or IL-15 / IL-15RαSushi-antibody fusion protein in CHO cells, such as, for example, the Pcmv-Script vector (Stratagene, La Jolla, CA), the pcDNA4 vector (Invitrogen, Carlsbad, CA), and the pCI-neo vector (Promega, Madison, WI). As described below, subject genetic constructs can be used to direct expression of a subject IL-15 / IL-15RαSushi-Fc fusion protein or IL-15 / IL-15RαSushi-antibody fusion protein in cells grown in culture, e.g., to produce and purify the protein (including the fusion protein or variant protein).

[0119] The present disclosure also relates to host cells transfected with a recombinant gene comprising a nucleotide sequence encoding the amino acid sequence of one or more of the subject IL-15 / IL-15RαSushi-Fc fusion proteins or IL-15 / IL-15RαSushi-antibody fusion proteins. The host cell can be any prokaryotic or eukaryotic cell. For example, the IL-15 / IL-15RαSushi complex of the present disclosure can be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those of skill in the art.

[0120] Accordingly, the present disclosure further relates to methods for producing the subject IL-15 / IL-15RαSushi-Fc fusion protein or IL-15 / IL-15RαSushi-antibody fusion protein. For example, the IL-15 / IL-15RαSushi complex can be expressed by culturing host cells transfected with an expression vector encoding the IL-15 / IL-15RαSushi complex under appropriate conditions. After secretion, the IL-15 / IL-15RαSushi complex can be isolated from a mixture of cells and medium containing the IL-15 / IL-15RαSushi-Fc fusion protein or IL-15 / IL-15RαSushi-antibody fusion protein. Alternatively, the IL-15 / IL-15RαSushi complex may be retained in the cytoplasm or membrane fraction, and the cells may be harvested, lysed, and the protein isolated. The cell culture medium includes host cells, medium, and other by-products. Suitable media for cell culture are well known in the art.

[0121] The polypeptides and proteins of the present disclosure can be purified according to protein purification methods well known to those of skill in the art. These methods include, at some level, crude fractionation into proteinaceous and non-proteinaceous fractions. After separation of the peptide polypeptide from other proteins, the peptide or polypeptide of interest can be further purified by chromatographic and electrophoretic methods to achieve partial or complete purification (i.e., purification to homogeneity). The terms "isolated polypeptide" or "purified polypeptide," as used herein, are intended to refer to a composition isolatable from other components, where the polypeptide has been purified to any degree relative to its naturally available state. A purified polypeptide, therefore, also refers to a polypeptide that has been separated from the environment in which it may naturally occur. Generally, "purified" refers to a polypeptide composition that has undergone fractionation to remove various other components and that substantially retains its expressed biological activity. When the term "substantially purified" is used, this designation refers to a peptide or polypeptide composition in which the polypeptide or peptide forms the majority of the composition, e.g., comprises about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 85% or more, or about 90% or more of the protein in the composition.

[0122] Various methods suitable for purification are well known to those of skill in the art. These methods include, for example, precipitation using ammonium sulfate, PEG, antibodies (immunoprecipitation), or heat denaturation, followed by centrifugation; chromatography, such as affinity chromatography (protein A column), ion exchange chromatography, gel filtration chromatography, reverse-phase chromatography, hydroxyapatite chromatography, and hydrophobic interaction chromatography; isoelectric focusing; gel electrophoresis; and combinations of these methods. As is well known in the art, it is contemplated that the order in which the various purification steps are performed, or certain steps are omitted, may still be suitable for preparing a substantially purified polypeptide.

[0123] Pharmaceutical Composition In another aspect, the present disclosure provides a pharmaceutical composition comprising an IL-15 / IL-15RαSushi-Fc fusion protein or an IL-15 / IL-15RαSushi-antibody fusion protein in admixture with a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are well known and understood by those skilled in the art and have been extensively described (see, e.g., Remington's Pharmaceutical Sciences, 18th ed., A.R. Gennaro (ed.), Mack Publishing Co., 1990). Pharmaceutically acceptable carriers may be included for the purpose of altering, maintaining, or preserving the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution rate, release rate, adsorption, or permeability of the composition. Such pharmaceutical compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the polypeptide.Suitable pharmaceutically acceptable carriers include 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 borate, bicarbonate, Tris-HCl, citrate, phosphate, other organic acids, etc.); bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, 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; flavoring agents, and diluting agents. agent; emulsifier; hydrophilic polymer (such as polyvinylpyrrolidone); low molecular weight polypeptide; salt-forming counterion (such as sodium); preservative (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvent (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohol (such as mannitol or sorbitol); suspending agent; surfactant or wetting agent (such as pluronic, PEG, sorbitan ester, polysorbate, e.g., polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stabilizer (sucrose or sorbitol); tonicity enhancing agent (such as alkali metal halide (preferably sodium chloride or potassium chloride), mannitol, sorbitol); delivery vehicle vehicle); diluent; excipient, and / or pharmaceutical adjuvant.

[0124] The primary solvent or carrier in a pharmaceutical composition may be aqueous or non-aqueous in nature. For example, a suitable solvent or carrier may be distilled water for injection, physiological saline, or artificial cerebrospinal fluid, to which other substances typically contained in compositions for parenteral administration may be added. Further exemplary solvents include neutral buffered saline or normal saline mixed with serum albumin. Other exemplary pharmaceutical compositions include Tris buffer at about pH 7.0-8.5 or acetate buffer at about pH 4.0-5.5, which may further contain sorbitol or a suitable substitute. In one embodiment of the present disclosure, a composition may be prepared for storage in the form of a lyophilized cake or aqueous solution by mixing a selected composition having the desired purity with any formulation agent (Remington's Pharmaceutical Sciences, supra). Additionally, therapeutic compositions may be formulated as lyophilized products using appropriate pharmaceutical excipients, such as sucrose. The optimal pharmaceutical composition can be determined by one of skill in the art based on the intended route of administration, delivery mode, and desired dose.

[0125] If parenteral administration is intended, the therapeutic pharmaceutical composition may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired IL-15 / IL-15RαSushi complex in a pharmaceutically acceptable solvent. A particularly suitable solvent for parenteral injection is sterile distilled water, in which the polypeptide is formulated as a sterile, isotonic solution, properly preserved. In various embodiments, pharmaceutical preparations suitable for injection may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's 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. Additionally, suspensions of the active compound may be prepared as appropriate oily injection suspensions. Optionally, the suspension may contain suitable stabilizers, i.e., agents that increase the solubility of the compound, thereby allowing for the preparation of highly concentrated solutions.

[0126] In various embodiments, therapeutic pharmaceutical compositions can be formulated for targeted delivery using colloidal dispersion systems.Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including 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 egg yolk phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.Liposome targeting can also be based on, for example, organ specificity, cell specificity, and organelle specificity, and is known in the art.

[0127] In various embodiments, oral administration of the pharmaceutical composition is contemplated. Pharmaceutical compositions administered in this manner can be formulated with or without carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. In solid dosage forms for oral administration (such as capsules, tablets, pills, dragees, powders, granules, and the like), one or more therapeutic compounds of the present disclosure may be mixed with one or more pharmaceutically acceptable carriers, such as, for example, sodium citrate or dicalcium 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 acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; and (5) solution retarding agents, such as paraffin. (6) absorption enhancers, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8) adsorbents, such as kaolin clay and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. For capsules, tablets, and pills, the pharmaceutical compositions may also contain buffering agents. Similar types of solid compositions may also be used as fillers in soft and hard gelatin capsules, employing excipients such as lactose or milk sugar and high molecular weight polyethylene glycols. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.In addition to the active ingredient, the liquid dosage form may contain inert excipients commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (specifically cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol sorbitan fatty acid esters, and mixtures thereof. In addition to inert excipients, oral compositions may contain auxiliary agents such as wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, coloring agents, fragrances, and preservatives.

[0128] In various embodiments, the pharmaceutical composition is intended for topical administration to the skin or mucosa. Topical formulations may further include one or more of a variety of agents known to be effective as skin or stratum corneum penetration enhancers. Examples include 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methyl alcohol, isopropyl alcohol, dimethyl sulfoxide, and azone. Additional agents may be included to make the formulation cosmetically acceptable. Examples include fats, waxes, oils, dyes, fragrances, preservatives, stabilizers, and surfactants. Keratolytic agents, such as those known in the art, may also be included. Examples include salicylic acid and sulfur. Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and, if required, any preservatives, buffers, or propellants. Ointments, pastes, creams, and gels may contain, in addition to a compound of the presently disclosed subject matter (e.g., an IL-15 / IL-15RαSushi-Fc fusion protein), pharmaceutical excipients such as animal fats, vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0129] Additional pharmaceutical compositions contemplated for use herein include formulations comprising polypeptides in sustained- or controlled-release formulations. In various embodiments, the pharmaceutical compositions may be formulated in nanoparticles as sustained-release hydrogels or incorporated into oncolytic viruses. Methods for such nanoparticles include, for example, encapsulation in nanoparticles composed of polymers with hydrophobic backbones and hydrophilic arms as drug carriers, encapsulation in microparticles, incorporation into liposomes in emulsions, and conjugation to other molecules. Examples of nanoparticles include mucoadhesive nanoparticles coated with chitosan or carbopol (Takeuchi et al., Adv. Drug Deliv. Rev. 47(1):39-54, 2001) and nanoparticles comprising the charged combination polyester, poly(2-sulfobutyl-vinyl alcohol) and poly(D,L-lactic-co-glycolic acid) (Jung et al., Eur. J. Pharm. Biopharm. 50(1):147-160, 2000). Albumin-based nanoparticle compositions have been developed as drug delivery systems for delivering hydrophobic drugs such as taxanes. See, for example, U.S. Patent Nos. 5,916,596, 6,506,405, 6,749,868, 6,537,579, 7,820,788, and 7,923,536. Abraxane®, an albumin-stabilized nanoparticle formulation of paclitaxel, was approved in the United States in 2005 for the treatment of metastatic breast cancer, and has since been approved in various other countries.

[0130] Methods for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot preparations, are also known to those skilled in the art.

[0131] The effective amount of a pharmaceutical composition used therapeutically will vary depending on the type and purpose of the treatment. As will be understood by those skilled in the art, appropriate therapeutic dosage levels will vary, in part, depending on the molecule being delivered, the indication for which the polypeptide is intended to be used, the route of administration, and the patient's size (body weight, body surface, or organ size) and condition (age and overall health). Clinicians can therefore adjust the dosage and route of administration to achieve optimal therapeutic efficacy. Typical dosages can range from about 0.0001 mg / kg to about 100 mg / kg or more, depending on the factors discussed above. Preferably, the polypeptide composition may be injected or administered subcutaneously or intravenously. Long-release pharmaceutical compositions may be administered every 3 to 4 days, weekly, biweekly, or monthly, depending on the half-life and clearance rate of the particular formulation. The frequency of administration will depend on the pharmacokinetic parameters of the polypeptide in the formulation used. Typically, the composition is administered until a dose that achieves the desired effect is reached. The composition may therefore be administered as a single dose, or as multiple doses over time (at the same or different concentrations per dose), or as a continuous infusion. Further refinement of the appropriate dosage is performed periodically. Appropriate doses may be confirmed using appropriate dose-response data.

[0132] The pharmaceutical composition may be administered by any known route, such as oral, intravenous, intraperitoneal, intratumoral, intracerebral (intracerebroparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional injection, intramedullary, intrathecal, intravesical, intraventricular, transdermal, subcutaneous, or intraperitoneal administration; as well as intranasal, enteral, topical, sublingual, transurethral, ​​vaginal, or rectal administration, sustained-release systems, or implanted devices. If desired, the composition may be administered by bolus injection, continuous infusion, or implanted. Alternatively or additionally, the composition may be administered locally by implantation of a membrane, sponge, or other suitable material to which the molecule of interest is adsorbed or encapsulated. Where an implantation device is used, the device may be implanted in any suitable tissue or organ, and delivery of the molecule of interest may be via diffusion, timed-release bolus, or continuous administration.

[0133] therapeutic use 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 regimen or in a combination therapy regimen) of an IL-15 / IL-15RαSushi-Fc fusion protein or IL-15 / IL-15RαSushi-antibody fusion protein of the present disclosure in a pharmaceutically acceptable carrier, whereby the growth and / or proliferation of cancer cells is inhibited. In particular, the IL-15 / IL-15RαSushi-Fc fusion protein of the present disclosure is useful in treating disorders characterized as cancer. Such disorders include, but are not limited to, solid tumors, such as breast cancer, respiratory tract cancer, brain cancer, reproductive cancer, gastrointestinal cancer, urinary tract cancer, eye cancer, liver cancer, skin cancer, head and neck cancer, thyroid cancer, parathyroid cancer, and distant metastases thereof, lymphoma, sarcoma, multiple myeloma, and leukemia. Examples of breast cancer include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, intraductal carcinoma in situ, and lobular carcinoma in situ. Examples of cancer of the respiratory tract include, but are not limited to, small cell lung carcinoma, non-small cell lung carcinoma, bronchial adenoma, and pleuropulmonary blastoma. Examples of brain cancer include, but are not limited to, brainstem glioma, hypothalamic glioma, cerebellar astrocytoma, cerebral astrocytoma, neuroblastoma, medulloblastoma, ependymoma, neuroectodermal tumor, and pineal tumor. Tumors of the male reproductive organs include, but are not limited to, prostate cancer and testicular cancer. Tumors of the female reproductive organs include, but are not limited to, endometrial cancer, cervical cancer, ovarian cancer, vaginal cancer, and vulvar cancer, as well as uterine sarcoma. Digestive tract tumors 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. Urinary tract tumors include, but are not limited to, bladder cancer, penile cancer, kidney cancer, renal pelvis cancer, ureter 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 (with or without fibrolamellar atypia), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular carcinoma and cholangiocarcinoma. Skin cancer includes, but is not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer. Head and neck cancer includes, but is not limited to, nasopharyngeal carcinoma and cancer of the lip and oral cavity. Lymphoma includes, but is not limited to, AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and lymphoma of the central nervous system. Sarcomas include, but are not limited to, sarcoma of the soft tissue, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma. Leukemia includes, but is not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.In some embodiments, cancer is a cancer 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.

[0134] The present disclosure provides methods for treating refractory or resistant liquid or solid tumors by enhancing the therapeutic effect of existing cancer treatments as an adjunct therapy.

[0135] The present disclosure also provides methods for treating viral infections 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 or IL-15 / IL-15RαSushi-antibody fusion protein of the present disclosure in a pharmaceutically acceptable carrier, wherein such administration inhibits viral growth and / or replication.

[0136] "Therapeutically effective amount" or "therapeutically effective dose" refers to that amount of the therapeutic agent being administered which will relieve to some extent one or more of the symptoms of the disorder being treated.

[0137] First, IC 50 Therapeutic effective doses can then be assessed from cell culture assays using the IC measured in cell culture in animal models. 50 The dose can be formulated to achieve a circulating plasma concentration range including: 100 mg / kg / day, ...

[0138] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, or several divided doses (multiple or repeated or maintenance doses) may be administered over time, with the dose being proportionally increased or decreased as required by the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to physically discrete units suited as a uniform dose for the mammalian subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present disclosure are determined primarily by the unique characteristics of the antibody and the particular therapeutic or prophylactic effect to be achieved.

[0139] That is, as will be understood by those skilled in the art, based on the disclosure provided herein, dosages and administration regimens can be adjusted according to methods well known in the therapeutic field. That is, the maximum tolerated dose can be easily established, and the effective amount for providing a detectable therapeutic effect to a subject can be determined, as can the time requirements for administering each agent so as to provide a detectable therapeutic effect to a subject. Thus, although certain dosages and administration regimens are exemplified herein, these examples are not intended to limit the dosages and administration regimens that can be administered to a subject in the practice of the present disclosure.

[0140] Dosage values ​​vary depending on the type and severity of the condition to be improved and may include single or multiple doses. It is further understood that for any particular subject, specific dosing regimens will be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. Furthermore, dosing regimens using the compositions of the present disclosure may be based on a variety of factors, including the type of disease, the subject's age, weight, sex, medical condition, severity of the condition, route of administration, and the particular antibody used. Thus, dosing regimens may vary but are routinely determined using standard methods. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or laboratory values. Thus, the present disclosure encompasses intra-subject dose escalation as required by those skilled in the art. It is understood that determining appropriate dosages and dosing regimens is well known in the relevant art and would be accomplished by one of ordinary skill in the art given the teachings disclosed herein.

[0141] Exemplary, non-limiting daily dose ranges for a therapeutically or prophylactically effective amount of an IL-15 / IL-15RαSushi-fusion protein of the present disclosure include 0.0001-100 mg / kg body weight, 0.0001-90 mg / kg body weight, 0.0001-80 mg / kg body weight, 0.0001-70 mg / kg body weight, 0.0001-60 mg / kg body weight, 0.0001-50 mg / kg body weight, 0.0001-40 mg / kg body weight, 0.0001-30 mg / kg body weight, 0.0001-20 mg / kg body weight, 0.0001-30 mg / kg body weight, 0.0001-40 mg / kg body weight, 0.0001-50 mg / kg body weight, 0.0001-60 mg / kg body weight, 0.0001-70 mg / kg body weight, 0.0001-80 mg / kg body weight, 0.0001-90 mg / kg body weight, 0.0001-100 mg / kg body weight, 0.0001-120 mg / kg body weight, 0.0001-140 mg / kg body weight, 0.0001-160 mg / kg body weight, 0.0001-180 mg / kg body weight, 0.0001-20 mg / kg body weight, 0.0001-20 mg / kg body weight, 0.0001-30 mg / kg body weight, 0.0001-30 mg / kg body weight, 0.0001-40 mg / kg body weight, 0.0001-20 mg / kg body weight, 0.0001-30 mg / kg body weight, 0.0001-4 001~10mg / kg body weight, 0.0001~5mg / kg body weight, 0.0001~4mg / kg body weight, 0.0001~3mg / kg body weight, 0.0001~2mg / kg body weight, 0.0001~1mg / kg body weight, 0.0010~50mg / kg body weight, 0.0010~40mg / kg body weight, 0.0010~30mg / kg body weight, 0.0010~20mg / kg body weight, 0.0010~10mg / kg body weight, 0.0010~5mg / kg body weight, 0.0010~4mg / kg body weight, 0.0010~3mg / kg body weight, 0.0010-2mg / kg body weight, 0.0010-1mg / kg body weight, 0.01-50mg / kg body weight, 0.01-40mg / kg body weight, 0.01-30mg / kg body weight, 0.01-20mg / kg body weight, 0.01-10mg / kg body weight, 0.01-5mg / kg body weight, 0.01-4mg / kg body weight, 0.01-3mg / kg body weight, 0.01-2mg / kg body weight, 0.01-1mg / kg body weight, 0.1-50mg / kg body weight, 0.1-40mg / kg body weight, 0.1-30mg / kg body weight The dosage can be 0.1-20 mg / kg body weight, 0.1-10 mg / kg body weight, 0.1-5 mg / kg body weight, 0.1-4 mg / kg body weight, 0.1-3 mg / kg body weight, 0.1-2 mg / kg body weight, or 0.1 mg / kg body weight, 1-50 mg / kg body weight, 1-40 mg / kg body weight, 1-30 mg / kg body weight, 1-20 mg / kg body weight, 1-10 mg / kg body weight, 1-5 mg / kg body weight, 1-4 mg / kg body weight, 1-3 mg / kg body weight, 1-2 mg / kg body weight, or 1-1 mg / kg body weight. Note that the dosage value may vary depending on the type and severity of the condition to be improved.It is further understood that for any particular subject, specific dosage regimens will be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

[0142] The toxicity and therapeutic index of the pharmaceutical compositions of the present disclosure are 50 (a dose lethal to 50% of the population) and ED 50 The LD (the dose that is therapeutically effective in 50% of a population) can be determined by standard pharmaceutical techniques in cell culture or experimental animals. The dose ratio between the toxic dose and the therapeutically effective dose is the therapeutic index, and the LD 50 / ED 50 Compositions that exhibit large therapeutic indices are generally preferred.

[0143] The frequency of administration of the IL-15 / IL-15RαSushi-fusion protein pharmaceutical composition will depend on the nature of the therapy and the particular disease being treated. Subjects can be treated at regular intervals, such as weekly or monthly, until the desired therapeutic result is achieved. Exemplary administration frequencies include, but are not limited to, once or twice weekly; once or twice every other week; once every two weeks; once every three weeks; once weekly for two weeks, then monthly; once weekly for three weeks, then monthly; once monthly; once every two months; once every three months; once every four months; once every five months; or once every six months, or once yearly.

[0144] Combination therapy As used herein, the terms "co-administration," "co-administered," and "in combination with," in reference to an IL-15 / IL-15RαSushi-fusion protein of the present disclosure and one or more other therapeutic agents, are intended to mean, and refer to, and include: the simultaneous administration of such a combination of an IL-15 / IL-15RαSushi-fusion protein of the present disclosure and a therapeutic agent to a subject in need of treatment, where such components are combined into a single dosage form and each of said components is released to said subject at substantially the same time; and the substantially simultaneous administration of such a combination of an IL-15 / IL-15RαSushi-fusion protein of the present disclosure and a therapeutic agent to a subject in need of treatment, where such components are formulated separately from one another into separate dosage forms and, when ingested by said subject at substantially the same time, each of said components is released at substantially the same time. sequential administration of such a combination of an IL-15 / IL-15RaSushi-fusion protein of the present disclosure and a therapeutic agent to a subject in need of treatment, where such components are formulated separately from one another into separate dosage forms that, when ingested by the subject at successive times with a significant time interval between each administration, result in the components being released to the subject at substantially different times; and sequential administration of such a combination of an IL-15 / IL-15RaSushi-fusion protein of the present disclosure and a therapeutic agent to a subject in need of treatment, where such components are combined into a single dosage form that, when released in a sustained manner, result in the components being released to the subject simultaneously, sequentially, and / or overlappingly at the same and / or different times, and where each portion may be administered by the same or different routes.

[0145] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention in combination with a second treatment, including, but not limited to, immunotherapy, cytotoxic chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, and stem cell transplantation. For example, such a method can be used prophylactically to prevent cancer, prevent cancer recurrence and metastasis after surgery, and as an adjunct therapy to other conventional cancer treatments. The present disclosure recognizes that the effectiveness of conventional cancer treatments (e.g., chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery) can be enhanced through the use of the combination methods described herein.

[0146] A wide range of conventional compounds have been shown to have anti-cancer activity. These compounds are used as pharmaceuticals in chemotherapy to cause solid tumor regression, inhibit metastasis and further growth, or reduce the number of malignant T cells in leukemic or bone marrow malignancies. Although chemotherapy has been effective in treating various types of malignancies, many anti-cancer compounds induce undesirable side effects. It has been shown that when two or more different treatments are combined, the treatments may act synergistically, allowing for a reduction in the dose of each treatment, thereby reducing the adverse side effects that each compound exhibits at higher doses. In other examples, treatment-resistant malignancies may respond to combination therapy with two or more different treatments.

[0147] In various embodiments, a second anti-cancer agent, such as a chemotherapeutic agent, will be administered to the patient. An exemplary list of chemotherapeutic agents includes daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, 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, oxaliplatin, and the like. In various embodiments, the chemotherapeutic agents include, but are not limited to, fluprexin, pentostatin, cladribine, cytarabine, gemcitabine, pralatrexate, mitoxantrone, diethylstilbestrol (DES), fludarabine, ifosfamide, hydroxyurea, taxanes (such as paclitaxel and doxetaxel), and / or anthracycline antibiotics, as well as 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 , about 20mg / m 2 , about 30mg / m 2 , about 40mg / m 2 , about 50mg / m 2 , about 60mg / m 2 , about 75mg / m 2 , about 80mg / m 2 , about 90mg / m 2 , about 100mg / m 2 , about 120mg / m 2 , about 150mg / m 2 , about 175mg / m 2 , about 200mg / m 2 , about 210mg / m 2 , about 220mg / m 2 , about 230mg / m 2 , about 240mg / m 2 , about 250mg / m 2 , about 260mg / m 2, and approximately 300 mg / m 2 Any of the following.

[0148] In various embodiments, the combination therapy of the present disclosure may further comprise administering to the subject a therapeutically effective amount of an immunotherapy, for example, treatment with a depleting antibody against a specific tumor antigen; treatment with an antibody-drug conjugate; treatment with an agonist, antagonist, or blocking antibody against a costimulatory or co-inhibitory molecule (immune checkpoint) such as CD276, CD272, CTLA-4, PD-1, PD-L1, CD40, SIRPα, CD47, OX-40, CD137, GITR, LAG3, ICOS, CD27, 4-1BB, TIM-3, B7-H4, Siglec7, Siglec8, Siglec9, Siglec15, and VISTA; Treatment with bispecific T cell-engaging antibodies (BiTE®) such as tumomab; treatment involving the administration of biological response modifiers such as IL-2, IL-7, IL-12, IL-21, GM-CSF, IFN-α, IFN-β, and IFN-γ; treatment with therapeutic vaccines such as sipuleucel-T; treatment with dendritic cell vaccines or tumor antigen peptide vaccines; treatment with NK cells; treatment with TCR-T cells; treatment with chimeric antigen receptor (CAR)-T cells; treatment with CAR-NK cells; treatment with iPS-induced NK cells, iPS-induced TCR-T cells, iPS-induced CAR-T cells, or iPS-induced CAR-NK cells; dendritic cells These include, but are not limited to, treatment with tumor-infiltrating lymphocytes (TILs); treatment with adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR transgenic); treatment with vaccines such as Bacillus Calmette-Guerin (BCG); treatment with TALL-104 cells; and treatment with immunostimulants such as the Toll-like receptor (TLR) agonist CpG and imiquimod. The above combination therapies enhance tumor cell killing by effector cells, i.e., synergistic effects exist between the IL-15 / IL-15RαSushi-Fc fusion protein and immunotherapy when administered simultaneously.

[0149] In various embodiments, the combination therapy comprises administering the IL-15 / IL-15RαSushi-Fc fusion protein or IL-15 / IL-15RαSushi-antibody fusion protein and the second pharmaceutical composition simultaneously, either in the same pharmaceutical composition or in separate pharmaceutical compositions. In various embodiments, the IL-15 / IL-15RαSushi-fusion protein composition and the second pharmaceutical composition are administered sequentially, i.e., the IL-15 / IL-15RαSushi-fusion protein composition is administered before or after the second pharmaceutical composition. In various embodiments, the administration of the IL-15 / IL-15RαSushi-fusion protein composition and the second pharmaceutical composition is concurrent, i.e., the administration of the IL-15 / IL-15RαSushi-fusion protein composition overlaps with the administration of the second pharmaceutical composition. In various embodiments, the administration of the IL-15 / IL-15RαSushi-fusion protein composition and the second pharmaceutical composition is not concurrent. For example, in various embodiments, administration of the IL-15 / IL-15RαSushi-fusion protein composition is terminated before the second pharmaceutical composition is administered. In various embodiments, administration of the second pharmaceutical composition is terminated before the IL-15 / IL-15RαSushi-fusion protein composition is administered. [Example]

[0150] The following examples are provided in order to more fully illustrate the present disclosure and are not to be construed as limiting the scope of the disclosure.

[0151] Example 1 Construction, expression, and purification of IL-15 / IL-15RαSushi-Fc fusion protein 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 poly(A) signal sequence was present at the 3' end of the CDS. A leader sequence was added to the N-terminus of the construct to ensure proper signaling and processing for secretion.

[0152] The fusion proteins were produced by co-transfecting HEK293-F cells growing in suspension with the mammalian expression vectors using polyethyleneimine (PEI, 25,000 molecular weight, linear, Polysciences). When two or more expression vectors were present, the vectors were transfected at a 1:1 ratio. For transfection, HEK293 cells were cultured in serum-free FreeStyle™ 293 Expression Medium (ThermoFisher). For production in 1000 ml shake flasks (maximum working volume 330 ml), 0.8 × 10 HEK293 cells were transfected 24 hours prior to transfection. 6 Cells were seeded at a density of 1000 cells / ml. A total of 330 μg of expression vector DNA was mixed with 16.7 ml of Opti-MeM medium (Thermo Fisher Scientific). 0.33 mg of PEI diluted in 16.7 ml of Opti-MeM medium was added, and the mixture was vortexed for 15 seconds and then left at room temperature for 10 minutes. The DNA / PEI solution was then added to the cells and incubated at 37°C in an incubator with an 8% CO2 atmosphere. Sodium butyrate (MilliporeSigma) was added to the cell culture at a final concentration of 2 mg / L on day 4 to help maintain protein expression. After 6 days of culture, the supernatant was collected and purified by centrifugation at 2200 rpm for 20 minutes. The solution was sterile filtered (0.22 μm filter, Corning).

[0153] Alternatively, constructs were produced in ExpiCHO cells (Thermo Fisher) according to the manufacturer's instructions.

[0154] Secreted proteins were purified from cell culture supernatants using Protein A affinity chromatography. The cell culture supernatant was loaded onto a HiTrap MabSelect SuRe 5 ml column (GE Healthcare) equilibrated with 5 column volumes (CV) of phosphate-buffered saline (pH 7.2) (Thermo Fisher Scientific). 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 and buffer exchanged into PBS (pH 7.2) using an Amicon® Ultra-15 Ultracel 10K (Merck Millipore).

[0155] The purity and molecular weight of the purified molecules were analyzed by SDS-PAGE in the presence and absence of reducing agents and Coomassie (Imperial™ Protein Stain, Thermo Fisher Scientific) staining. The NuPAGE® Pre-Cast Gel System (4-12% Bis-Tris, Thermo Fisher Scientific) was used according to the manufacturer's instructions. The aggregation level of the molecules was analyzed using an Agilent 1200 high-performance liquid chromatography (HPLC) system. Samples were injected onto an AdvanceBio size-exclusion column (300 Å, 4.6 × 150 mm, 2.7 μm, LC column, Agilent) at 25°C using 150 mM sodium phosphate (pH 7.0) as the mobile phase.

[0156] The protein concentration of the purified protein samples was determined by measuring the absorbance at 280 nm using a Nanodrop spectrophotometer (Thermo Fisher Scientific) and dividing 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 Biosciences) according to the manufacturer's instructions.

[0157] As an example showing the protein profiles of isolated IL-15 / IL-15Rα-Fc 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 architecture as P-0234 but differs only by an S58D substitution in the IL-15 domain.

[0158] In both monovalent and bivalent Fc fusions, the IL-15Rα sushi+ domain, with a theoretical molecular weight of 8.6 kDa, was noncovalently associated with IL-15 or IL-15 variants fused to the Fc domain, which dissociated under denaturing conditions and migrated to the expected position as a sharp band (Fig. 2A). The presence of the IL-15Rα-sushi+ band on the gel confirmed the noncovalent association of IL-15 with IL-15Rα during cell culture growth; this association was maintained during Protein A purification under low pH elution conditions.

[0159] The size-exclusion chromatograms in Figure 2B show that the two fusion forms have a low tendency to aggregate, with only 1–2% aggregation observed for all three fusion proteins after the initial Protein A capture step without further polishing. The sharpness of the main peaks further suggests that the association between IL-15 and IL-15Rα is tight, albeit noncovalent, under native buffer conditions.

[0160] Furthermore, the expression levels of the fusion proteins were comparable (within 2-fold) to those of the Fc-only proteins using the same vector and culture conditions. This high yield and low aggregation demonstrated a favorable development suitability profile for both monovalent and bivalent forms of the IL-15 / IL-15Rα (non-covalent) Fc fusion protein. Furthermore, as exemplified by P-0313, amino acid substitutions in IL-15 did not affect the expression profile of the fusion proteins; P-0313 exhibited similar purity and aggregation tendency to its wild-type counterpart, P-0234 (Figure 2).

[0161] Example 2 Purity-focused development suitability assessment of different fusion protein forms underscores the role of a properly complexed IL-15Rα domain in enhancing the development suitability profile of fusion proteins SEC analysis of Protein A-purified samples was used to assess the impact of different fusion types on protein aggregation propensity and purity. We found that although protein expression levels can vary between different batches due to variations in cell growth, protein aggregation propensity and purity are intrinsic properties associated with a particular protein, and therefore lot-to-lot variability appeared to be small, as illustrated in Figures 3F and 3H for P-0234.

[0162] First, we evaluated the effect of IL-15Rα complex formation on the protein purity of IL-15-Fc fusion proteins based on five types of molecules. P-0162 is a C-terminal monomeric IL-15-only Fc fusion protein containing the Hole-Fc-Linker1-IL-15 chain (SEQ ID NO: 13) and the empty Knob-Fc chain (SEQ ID NO: 7). P-0197 is a C-terminal monovalent IL-15 / IL-15Rα (non-covalent)-Fc fusion, the schematic of which is shown in Figure 1B. P-0197 differs from P-0162 only by the presence of the free IL-15Rα Sushi+ domain non-covalently complexed with IL-15. P-0153 is an N-terminal monomeric IL-15 / IL-15Rα fusion in the form of a heterodimeric Fc fusion, the cartoon diagram of which is shown in Figure 1A. P-0167 (SEQ ID NOs: 13 and 55) and P-0198 are the dimeric counterparts of P-0162 and P-0197, respectively. Schematics of the size exclusion separation of the five molecules are shown in Figures 3A-3E.

[0163] As can be seen in Figure 3A, the IL-15-Fc monomeric fusion without IL-15Rα had a monomer content of 84.5%, with the majority of impurities being smaller in molecular weight. In contrast, P-0197 contained a monomer content of 98.6% (Figure 3B), clearly demonstrating the contribution of the free IL-15Rα Sushi domain to such a significant improvement in purity. The effect of the free IL-15Rα Sushi domain on the protein quality of the IL-15-Fc fusion protein was further clarified in the dimeric form, as shown by the SEC chromatograms of P-0167 and P-0198 in Figures 3D and 3E, respectively. P-0167 contained a broad, irregular peak with shoulders on both sides of the main peak. More notably, P-0167 showed significantly reduced in vitro activity in activating NK and T cells from fresh human PBMCs, which was likely due to protein misfolding. However, P-0198, which contains the noncovalently bound IL-15Rα Sushi domain in a dimeric form, showed a sharp major peak with 90.5% monomer content (Figure 3E). Interestingly, when the IL-15Rα Sushi domain was not free but covalently fused to a matching heterodimeric Fc, as in P-0153, its complex formation with IL-15-Fc did not result in any improvement in protein purity; rather, the protein sample contained more than 25% dimers and higher molecular weight soluble aggregates (Figure 3C). The fusion of both IL-15 and IL-15Rα to the Fc domain appears to create a spatial constraint that restricts their physiological interaction. 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 bind IL-15 in a favorable, non-restrictive manner.

[0164] Second, the impact of IL-15Rα ECD (SEQ ID NO: 4) versus IL-15Rα Sushi+ (SEQ ID NO: 5) on the suitability of IL-15 / IL-15Rα-Fc fusion proteins for development was assessed by comparing P-0234 and P-0220. Both constructs are C-terminal Fc fusions sharing the same structure as shown in Figure 1C, with P-0234 containing the IL-15Rα Sushi+ domain and P-0220 containing the IL-15Rα ECD. Their SEC chromatograms are shown in Figures 3F and 3G, respectively. P-0220, a protein containing the IL-15Rα ECD domain, was not only less pure than its IL-15Rα Sushi+-containing counterpart, P-0234 (88.5% vs. 100%), but also expressed at a 2.5-fold lower level in the same batch of cells. In summary, it is clear that the IL-15RαSushi+ domain is a preferred partner over the IL-15RαECD in constructing more development-compatible IL-15 / IL-15Rα fusion proteins.

[0165] We further evaluated the influence of the fusion terminus on the development suitability of IL-15 / IL-15Rα-Fc fusion proteins. P-0234 and P-0223 are dimeric IL-15 / IL-15Rα (non-covalent)-Fc fusion proteins in which the IL-15 / IL-15Rα Sushi complex is attached to the C-terminus and N-terminus of the Fc, respectively. Their SEC chromatograms (Figures 3H and 3I) show only a subtle difference in purity (98.5% vs. 93.9%). However, purified P-0223 consistently contained a broad peak of higher molecular weight species and a small, distinct peak containing smaller molecular weight impurities, which were virtually absent in P-0234. Although the difference was small, P-0234, with its C-terminal fusion, certainly had a better SEC purity profile and is the preferred form in terms of development suitability, given that both molecules are expressed at comparable levels.

[0166] In conclusion, complexing the IL-15Rα subunit with an IL-15-Fc fusion significantly improved expression and purity and reduced aggregation; such improvements required that IL-15Rα properly associate with IL-15 while minimizing spatial constraints. Furthermore, a truncated form of the IL-15Rα ECD, IL-15RαSushi+, appeared preferable to the full-length ECD based on both productivity and purity assessments. Furthermore, positioning the IL-15 / IL-15Rα complex at the C-terminus of the Fc is advantageous over N-terminal fusions in achieving high purity. Therefore, the dimeric IL-15 / IL-15Rα (noncovalent) C-terminal Fc fusion, exemplified by P-0234, incorporates all of these favorable components and is the preferred form.

[0167] Example 3 Noncovalent binding of IL-15Rα enhances receptor binding and functional activity of IL-15 variant / IL-15Rα-Fc fusion proteins IL-15 binds with high affinity to its specific receptor, IL-15Rα, both of which are expressed on antigen-presenting cells. IL-15Rα binds IL-15, which then binds IL-15 to IL-15Rβ and γ on responding lymphocytes, including NK cells, T cells, and B cells. c IL-15 is trans-presented to a heterodimeric receptor complex composed of IL-15 and IL-15Rαβγ. Formation of the ligand-trimeric receptor complex (IL-15-IL-15Rαβγ) initiates an intracellular signaling cascade, resulting in downstream biological effects. The complex biological properties of the IL-15 receptor have prevented the design of optimal IL-15 fusion proteins that achieve conformationally efficient ligand-trimeric receptor signaling complexes.

[0168] We hypothesized that covalently linking IL-15 to the N- or C-terminus of the Fc portion of human IgG would be more advantageous in extending its in vivo half-life than noncovalently linking IL-15 to an IL-15Rα-Fc fusion protein. Furthermore, we 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 a high-affinity ligand-trimeric receptor signaling complex. Furthermore, noncovalent association of the IL-15Rα domain with the IL-15-Fc fusion protein would preserve the native IL-15-IL-15Rα binding and the optimal conformation for trans-presentation of IL-15. Furthermore, the extremely high binding affinity between IL-15 and the IL-15Rα domain makes the generation of such a fusion protein complex feasible.

[0169] Various types of IL-15-Fc fusion proteins containing the IL-15 / IL-15Rα domain complex in various configurations were constructed. The biological activity of the fusion proteins in stimulating lymphocyte activation was analyzed by measuring the binding activity of the fusion proteins to the IL-15Rβ subunit and by measuring CD69 expression on human CD8 cells and human NK cells. Exemplary structural diagrams of the fusion proteins are shown in Figure 1.

[0170] Binding activity was tested by ELISA assay. Briefly, Nunc Maxisorp (Thermo Fisher Scientific) plates were coated overnight at 4°C with 1 μg / well (100 μl / well) of huIL-15Rβ-6His in bicarbonate buffer (pH 9.4) (Thermo Fisher Scientific). After washing three times with PBS / 0.05% Tween 20, SuperBlock (300 μl / well) was added and the plates were incubated at room temperature for 2 hours to block nonspecific binding. After washing, 100 μl / well of each IL-15 compound was added in a 3-fold serial dilution in blocking buffer and incubated at room temperature for 1 hour. After washing, Fc fusions were detected by incubation with a horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc secondary antibody at a 1:5000 dilution in blocking buffer (Thermo Fisher Scientific) (100 μl / well) for 1 hour at room temperature. After washing, TMB substrate (Thermo Fisher Scientific) was added (100 μl / well). The plate was sealed and incubated in the dark at room temperature for 5–20 minutes. The reaction was stopped by adding 2N sulfuric acid (Ricca Chemical) (50 μl / well), and the absorbance was read at 450–590 nm.

[0171] Biological activity was measured by measuring the induction of CD69 expression on human NK cells and human CD8+ T cells. CD69 is a cell surface glycoprotein that is induced early during lymphocyte activation. To analyze the number / percentage of NK cells or CD8+ T cells expressing CD69 after IL-15 treatment, an ex vivo human peripheral blood mononuclear cell (PBMC) assay was established. Specifically, human PBMCs were isolated by Ficoll-Hypaque centrifugation from buffy coats purchased from the Oklahoma Blood Institute. 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 300 × g and resuspended in FACS buffer. After blocking Fc receptors with Human TruStain FcX (1:50 dilution), cells were stained with anti-human CD56-FITC, anti-human CD69-PE, and anti-human CD8-APC antibodies (1:50 dilution). After 30 minutes of incubation with these antibodies at room temperature, cells were collected, washed, resuspended in FACS buffer, and prepared for flow cytometry analysis. CD69 expression on CD56+ NK cells and CD8+ T cells was measured. Data are expressed as the percentage of CD69-positive cells in the gated population.

[0172] P-0157 is an N-terminal bivalent IL-15 (non-covalent) / IL-15Rα Sushi-Fc fusion protein; P-0153 is a C-terminal IL-15 / IL-15Rα Sushi heterodimeric Fc fusion protein; and P-0162 is a C-terminal monovalent Fc-IL-15 fusion protein with uncomplexed IL-15Rα Sushi. ELISA binding assays demonstrated that incorporation of the IL-15Rα Sushi domain significantly increased the binding strength of the IL-15-Fc fusion proteins (P-0157 and P0153) to IL-15Rβ compared with the fusion protein with uncomplexed IL-15Rα (P-0162) (Figure 4), suggesting a critical role for IL-15Rα in promoting the interaction of the fusion proteins with the receptor. Furthermore, receptor binding activity was reduced when both IL-15 and IL-15Ra were covalently conjugated to Fc in a heterodimeric form (P-0153), in contrast to when IL-15 was noncovalently bound to an IL-15Rα-Fc fusion protein (P-0157) (Fig. 4), suggesting that conformationally restricted fusion forms may negatively affect receptor binding activity.

[0173] Consistent with the binding potency, the incorporation of IL-15Rα also increased the biological activity of the IL-15 fusion proteins compared to fusion proteins lacking IL-15Rα. P-0197 is a C-terminal monovalent IL-15 / IL-15Rα (noncovalent)-Fc fusion protein, and P-0162 shares the same structure as P-0197 without IL-15Rα. P-0197 showed a 10-fold and 6-fold increase in potency compared to P-0162 in inducing CD69+ NK cells (Figure 5A) and CD8+ T cells (Figure 5B), respectively, due to the inclusion of IL-15Rα.

[0174] Finally, we compared the biological activities of three C-terminal monovalent IL-15 / IL-15Rα-Fc fusion proteins: P-0165 is an Fc fusion protein in which IL-15 is noncovalently bound to IL-15Rα fused to the Fc domain; P-0197 is the reversed fusion protein in which IL-15 is noncovalently bound to IL-15Rα fused to the Fc domain; and P-0153 is a heterodimeric structure in which both IL-15 and IL-15Rα are fused to the Fc domain. As shown in Figure 6, fusion proteins formed by noncovalent complexation with IL-15 or IL-15Rα showed better potency in inducing CD69+ NK cells (Figure 6A) and CD8+ T cells (Figure 6B) than both fusion proteins in which IL-15 / IL-15Rα were covalently fused to Fc and fusion proteins in which IL-15 / IL-15Rα were heterodimerized to Fc. The data suggest that optimal conformational binding between IL-15 and IL-15Rα is crucial for IL-15 fusion proteins to bind to its receptor and exert its biological activity. Increased conformational restriction, such as in heterodimeric Fc fusions, negatively impacts biological activity.

[0175] Example 4 Effect of linker on activity of IL-15 / IL-15Rα-Fc fusion protein The selection of an appropriate linker for linking protein domains is crucial in fusion protein design. The peptide linker not only provides spatial distance between the fusion protein domains, allowing them to fold independently, but also directly influences the structural stability and functional properties of the fusion protein. Here, we investigate the effects of linker flexibility and length on the biological activity of IL-15 / IL-15Rα-Fc fusion proteins.

[0176] Biological activity was confirmed by measuring the induction of CD69 expression on human NK cells and CD8+ T cells in an ex vivo human PBMC FACS-based assay as previously described. P-0165 and P-0166 are monomeric IL-15 (non-covalent) / IL-15Rα-Fc fusions with 10-amino acid rigid and flexible linkers, respectively. P-0197, P-0207, and P-0217 are all bivalent IL-15 / IL-15Rα (non-covalent)-Fc fusion proteins. P-0197 contains a rigid linker, P-0207 contains a 10-amino acid GS-rich flexible linker, and P-0217 contains a 15-amino acid flexible linker. The results (Figures 7A and 7B) showed that the rigidity or length of the peptide linkers linking Fc to IL-15 (P-0165 and P-0166) or Fc to IL-15Ra (P-0197, P-0207, and P-0217) did not affect the biological activity of the fusion proteins tested.

[0177] Example 5 Effect of valency on the activity of IL-15 / IL-15Rα fusion proteins Most therapeutic Fc fusion proteins are homodimeric because the Fc of IgG1 naturally homodimerizes via disulfide bonds formed in the hinge region. Dimeric proteins offer advantages in avidity, stability, quantity, size, and function. However, manipulation of the Fc can lead to the generation of monomeric Fc fusion proteins. This change may affect bioactivity, pharmacokinetics, and side effects, or reduce the size of the dimeric protein, potentially increasing tissue permeability. To assess the effect of valency on the biological activity of IL-15 / IL-15Rα-Fc fusion proteins, we constructed both monomeric and homodimeric forms with various IL-15 / IL-15Rα-Fc fusion configurations and tested their biological activity.

[0178] Biological activity was confirmed by measuring the induction of CD69 expression on human NK cells and CD8+ T cells in an ex vivo human PBMC FACS-based assay as previously described. Results showed that across all fusion types tested (Table 4), the homodimeric forms of the IL-15 / IL-15RaFc fusion proteins exhibited an approximately two-fold enhancement in biological activity compared to their respective monomeric counterparts, suggesting that the valency of the dimeric form may confer a functional advantage. TIFF0007807076000013.tif128170

[0179] Example 6 Effect of N- or C-terminal Fc fusion on the biological activity of IL-15 Fc fusion proteins can be constructed by placing the IL-15 / IL-15Rα complex, linked by a spacer linker, at either the N- or C-terminus of Fc. The optimal scaffold was determined based on whether the fusion protein folded and expressed correctly and whether biological activity was maintained. IL-15 / IL-15Rα-Fc fusion proteins were generated by attaching the IL-15 / IL-15Rα complex to either the N- or C-terminus of Fc, spaced apart by a linker. IL-15 / IL-15Rα complexes were also constructed differently, with respect to C- and N-terminal fusions. P-0218 is a C-terminal bivalent IL-15 (noncovalent) / IL-15Rα-Fc fusion protein, while the standard is the N-terminal counterpart of P-0218, which contains an additional 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.

[0180] 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 IL-15 compounds. 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 quiescent phase (G0), making it a marker of cell proliferation.

[0181] An in vitro human PBMC assay was established. Briefly, purified human PBMCs were treated with serial dilutions of IL-15 test compounds and incubated at 37°C for 5 days. On day 5, cells were washed once with FACS buffer (1% FBS / PBS) and initially stained with Fc blockers 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 thoroughly resuspended in 200 μl / well of 1x Foxp3 Fixation and Permeabilization Working Solution and incubated for 30 minutes in the dark at room temperature. After centrifugation, 200 μl of 1x Permeabilization Buffer was added to each well for further washing. The cell pellet was resuspended in permeabilization buffer containing anti-human Ki67-PE (1:10 dilution). After a 30-minute incubation at room temperature, cells were collected, washed, resuspended in FACS buffer, and prepared for flow cytometry analysis. Data are expressed as the percentage of Ki67-positive cells in the gated population.

[0182] As shown in Figure 8, the C-terminal Fc fusions (P-0218 and P-0234) consistently demonstrated stronger induction of Ki67-positive CD8+ T cells, i.e., CD8+ T cell proliferation, than their N-terminal Fc fusion counterparts (standard and P-0223) (Figures 8A and 8B). The data suggest that the C-terminus of Fc is the preferred binding site for the IL-15 / IL-15Rα complex, allowing for the preservation of biological activity.

[0183] Example 7 Effect of receptor-α domain selection on the activity of IL-15 / IL-15RαFc fusion proteins IL-15 binds to the extracellular domain (ECD) of IL-15Rα, primarily through a conserved protein-binding motif called the sushi domain. In constructing fusion proteins, a truncated version of this IL-15Rα may be preferable to reduce size and structural complexity. To confirm binding specificity and affinity, all or part of the ECD domain (the sushi domain with an additional 12 amino acids) that confers IL-15 binding was constructed into fusion proteins, and functional activity was measured.

[0184] P-0234 and P-0220 are C-terminal dimeric IL-15 / IL-15Rα (non-covalent) fusion proteins, where IL-15Rα is sushi and the complete ECD, respectively. P-0223 and P-0224 are N-terminal dimeric IL-15 / IL-15Rα (non-covalent)-Fc fusion proteins, where IL-15Rα is sushi and the complete ECD, respectively. P-0221 and P-0222 are N-terminal monovalent IL-15 / IL-15Rα (non-covalent)-Fc fusion proteins, where IL-15Rα is sushi and the complete ECD, respectively. The results showed that fusion proteins noncovalently complexed with the IL-15Rα sushi domain, regardless of whether the fusion was N- or C-terminal, dimeric, or monomeric, were more potent at inducing CD69+ NK cells than those complexed with the complete IL-15Rα ECD (Figure 9). The data suggest that the IL-15Rα sushi domain is preferable to the complete ECD in constructing IL-15 / IL-15Rα-Fc fusion proteins and conferring an optimal conformation for IL-15 interaction with its signaling receptor.

[0185] Example 8 Binding activity of IL-15 mutants and variant fusion proteins to IL-15Rβ In searching for IL-15 agonists, superagonists, or antagonists, deletions, insertions, or point mutations were introduced into the human IL-15 peptide sequence at the contact interface between IL-15 and receptor β or receptor γ. These variants were introduced into various forms of IL-15 / IL-15Rα-Fc fusion proteins, and their binding activity to IL-15Rβ was quantified by enzyme-linked immunosorbent assay (ELISA) as previously described.

[0186] Table 5 shows the IL-15Rβ binding activity of C-terminal IL-15 variant / IL-15Rα heterodimeric Fc fusion proteins. IL-15 variants were derived from human IL-15 peptides with amino acid deletions, insertions, or point mutations. Truncating three amino acids from the C-terminus of IL-15 retained the binding activity of the fusion protein to IL-15Rβ compared to the full-length wild-type IL-15 fusion protein. However, further truncating six or nine amino acids from the C-terminus of IL-15 resulted in a gradual decrease in fusion protein binding activity. GS insertions of various lengths after N95 resulted in a decrease in the IL-15Rβ binding activity of the fusion protein.

[0187] Table 6 shows the IL-15Rβ-binding activity of monomeric IL-15 variant (non-covalent) / IL-15Rα-Fc fusion proteins containing a single amino acid substitution at positions 58, 62, 63, 67, or 68 in the human IL-15 domain. P-0185, an Fc fusion protein containing the IL-15(I67V) variant, exhibited binding activity to IL-15Rβ similar to that of the wild-type fusion protein. P-0182, a fusion protein containing an IL-15 variant with a serine-to-aspartic acid substitution at position 58 (S58D), exhibited a four-fold increase in binding activity 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 proteins.

[0188] Table 7 shows the IL-15Rβ binding activity of dimeric IL-15 variant / IL-15Rα (non-covalent)-Fc fusion proteins in which human IL-15 contains a single amino acid substitution at position 58 or 68 or an amino acid insertion after N95. Similarly, as seen in P-0182 (Table 4), P-0313, which contains the same IL-15 variant with S58D, showed a two-fold increase in binding activity to IL-15Rβ when compared to its respective wild-type fusion protein, P-0234. This data reinforces the idea that the S58D substitution in the IL-15 peptide may modulate IL-15 into a superagonist by enhancing receptor binding activity.

[0189] In summary, we generated IL-15 variant-Fc fusion proteins and confirmed their differential IL-15Rβ binding activity. Some IL-15 variants showed reduced binding activity to IL-15Rβ compared with their wild-type counterparts (Tables 5–7). Some variants, such as P-0173 and P-0185, retained binding activity to IL-15Rβ similar to that of the wild-type variant (Table 5). A single point mutation, in which serine at position 58 in the human IL-15 domain was replaced with aspartic acid, enhanced the binding activity of the fusion proteins (P-0182 and P-0313) to IL-15Rβ (Tables 6 and 7). TIFF0007807076000014.tif72170TIFF0007807076000015.tif65170TIFF0007807076000016.tif43170

[0190] Example 9 T cell activation activity of IL-15S58D / IL-15RαFc fusion protein Fc fusion proteins of IL-15 variants / IL-15Rα complexes were evaluated for their functional activity in stimulating lymphocyte activation. As previously described, an ex vivo human PBMC assay was established to analyze the number / percentage (%) of CD8+ T cells expressing the lymphocyte activation marker CD69.

[0191] P-0234 is a C-terminal dimeric IL-15 / IL-15Rαsushi (non-covalent)-Fc fusion protein optimized by combining favorable configurations, including covalent linkage of IL-15 to Fc, C-terminal fusion, dimeric valency, and non-covalent IL-15Rαsushi complex formation. P-0313 is the S58D counterpart of P-0234. P-0313 shares the same fusion configuration as P-0234 but differs only by the S58D substitution in the IL-15 polypeptide. P-0313 showed increased IL-15Rβ binding activity compared to its wild-type counterpart, P-0234, as described above (Table 7). Consistent with its enhanced binding activity to IL-15Rβ, variant P-0313 carrying the S58D mutation also showed increased CD69-positive T cell induction activity (7 pM vs. 12 pM for P-0313 and P-0234, respectively), confirming that P-0313 exhibits superagonist activity.

[0192] Example 10 Signaling activity of IL-15S58D / IL-15Rα-Fc fusion protein The IL-15S58D variant showed increased binding activity to IL-15Rβ and enhanced stimulatory activity of CD69+ lymphocytes. In this study, we investigated the signaling activity of the IL-15S58D / IL-15Rα fusion protein in stimulating intracellular phosphorylation of signal transducer and activator of transcription 5 (STAT5) in NK cells and T cells.

[0193] STAT5 phosphorylation was measured by intracellular FACS analysis in an ex vivo human PBMC assay after treatment with IL-15 compounds. 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 treatment, cells were washed once with FACS buffer (1% FBS / PBS) and incubated in 150 μl / well of prewarmed Cytofix fixation buffer at 37°C for 15 minutes. Fixed cells were washed again and resuspended in 150 μl / well of pre-chilled Perm buffer II at 4°C for 30 minutes. After blocking Fc receptors with Human TruStain FcX (1:50 dilution), 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 45 min of incubation with the antibodies at room temperature, cells were collected, washed, resuspended in FACS buffer, and prepared for flow cytometry analysis. Data are expressed as the percentage of pSTAT5-positive cells in the gated population.

[0194] The S58D mutation was introduced into two types of Fc fusion proteins: a bivalent IL-15 (non-covalent) / IL-15Rα-Fc fusion and a bivalent IL-15 / IL-15Rα (non-covalent)-Fc fusion protein. P-0218 and P-0314 are C-terminal dimeric IL-15 (non-covalent) / IL-15Rα-Fc fusion proteins containing wild-type and 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 and S58D variant IL-15, respectively. Regardless of the fusion composition, the IL-15(S58D) variant fusion proteins (P-0314 and P-0313) showed an approximately two-fold increase in stimulatory activity of STAT5 phosphorylation in CD8 T cells (Fig. 10A), CD4 T cells (Fig. 10B), and NK cells (Fig. 10C) compared with their respective wild-type fusion proteins (P-0218 and P-0234). The data confirmed that the S58D substitution in the IL-15 peptide confers superagonistic activity to various IL-15 / IL-15Rα fusion proteins.

[0195] Example 11 Cell proliferation activity of IL-15S58D / IL-15-RαFc fusion protein After confirming their increased ability to bind IL-15Rβ, stimulate STAT5 phosphorylation, and induce CD69 expression, IL-15S58D variant-Fc fusion proteins were tested for their ability to stimulate cell proliferation in comparison with the wild-type fusion protein by measuring Ki67 expression in NK cells and CD8+ T cells. Human PBMCs were treated with increasing amounts of IL-15 fusion molecules, and Ki67 expression was measured by intracellular FACS analysis gated on the CD56+ NK cell and CD8+ T cell populations, as previously described.

[0196] Similar to the observations regarding STAT5 phosphorylation, the IL-15S58D variant fusion protein also showed a two-fold increase in stimulatory activity for Ki67 expression in CD8+ T cells (Fig. 11A), CD4+ T cells (Fig. 11B), and CD56+ NK cells (Fig. 11C) compared with the wild-type fusion protein (Fig. 11B). These data confirm that the introduction of the S58D mutation in the IL-15 domain enhances a range of biological activities, including receptor binding, intracellular signaling, activation of cell surface markers, and activation of cell proliferation.

[0197] Example 12 A 4-day repeated-dose study of P-0234 compared to rhIL-15 and a standard in mice IL-15 / IL-15Rα-Fc fusion proteins demonstrated potent binding to IL-15Rβ, inducing intracellular signaling cascades, and stimulating the proliferation of NK lymphocytes and CD8 T lymphocytes in vitro and ex vivo. Here, we investigated the effects of serum exposure and various IL-15 compounds on the proliferation and proliferation of NK cells in mice. Proteins tested included recombinant human native IL-15 (rhIL-15), P-0234 (a C-terminal bivalent IL-15 / IL-15Rα (noncovalent)-Fc fusion protein), and a standard compound (an N-terminal bivalent IL-15 (noncovalent) / IL-15Rα-Fc fusion protein containing the N72D mutation in IL-15).

[0198] Seven-week-old female Balb / c mice were obtained from Charles River Laboratories and acclimated in-house for at least 7 days prior to testing. Mice received daily intraperitoneal injections containing equimolar amounts of IL-15 compounds for 4 days. Treatments included vehicle, 0.03 mg / kg rhIL-15 (40 pmol / kg), 0.1 mg / kg and 0.5 mg / kg standard (40 pmol / kg and 200 pmol / kg), and 0.1 mg / kg and 0.5 mg / kg P-0234 (40 pmol / kg and 200 pmol / kg). Each group contained five mice. Body weights were recorded daily before and during treatment. Mice were sacrificed 1 hour after the last injection, and terminal blood was collected by cardiac puncture.

[0199] Heparinized whole blood and spleens were collected for NK cell phenotyping and Ki67 intracellular staining. After erythrocyte lysis and Fc receptor blocking with purified anti-mouse CD16 / CD32 (1:50 dilution), mononuclear blood and spleen cells in single-cell suspension were stained with NK cell surface markers, including anti-mouse CD3-FITC and anti-mouse CD49b-APC (1:50 dilution), for 30 minutes at room temperature in the dark. For intracellular Ki67 staining, cell pellets were thoroughly resuspended in 200 μl / well of 1x Foxp3 fixation / permeabilization working solution and incubated for 30 minutes at room temperature in the dark. Cells were washed with 200 μl of 1x permeabilization buffer, and Fc receptors were blocked with purified anti-mouse CD16 / CD32 (1:50 dilution). Cells were then stained with Ki67-PE, and anti-mouse CD3-FITC and anti-mouse CD49b-APC (1:50 dilution) for the NK cell population. After a 30-minute incubation, cells were harvested, washed, resuspended in FACS buffer, and prepared for flow cytometry analysis. Statistical analysis was performed using one-way ANOVA with Tukey's post hoc multiple comparisons in GraphPad Prism software.

[0200] Figure 12 shows the serum concentrations of IL-15 1 hour after the last injection of IL-15 compound. IL-15 was measured using a commercially available ELISA kit (R&D Systems, Inc.; catalog number DY247) that detects IL-15, according to the manufacturer's instructions. After four daily doses of compound, cumulative serum concentrations of IL-15 were highest in mice treated with P-0234, intermediate in mice treated with standard, and lowest in mice treated with rhIL-15, when administered at equimolar doses (Figure 12). Comparing P-0234 to standard administered at 0.1 mg / kg and 0.5 mg / kg, P-0234 consistently demonstrated 6-fold and 4-fold higher serum concentrations than standard. 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 standard compound (0.1 mg / kg dose and 0.5 mg / kg dose, respectively), and 72±14 and 443±57 (ng / ml) for P-0234 (0.1 mg / kg dose and 0.5 mg / kg dose, respectively). The superior serum exposure suggests that P-0234 may exhibit a longer in vivo half-life and serum retention compared to the standard and rhIL-15.

[0201] Although there was no difference in total body weight between treatment groups, high-dose standard-treated mice lost nearly 6% 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 to the vehicle group, suggesting that potential dose-limiting toxicity was identified for the standard compound but not for P-0234.

[0202] All tested IL-15 compounds increased the percentage of Ki67-positive NK cells in peripheral blood (Figure 14A), suggesting enhanced NK cell proliferation. However, a significant increase in the percentage of NK cells among CD3-negative peripheral blood lymphocytes was only observed in P-0234-treated mice at both dose levels tested (Figure 14B). An increase in the percentage of NK cells was also observed in rhIL-15-treated mice and low-dose standard-treated mice, but the effect did not reach statistical significance (Figure 14B). Interestingly, a decrease in the number of NK cells in peripheral blood was observed in high-dose standard-treated mice (Figure 14B). This reversal of the pharmacodynamic dose-response suggests toxicity and is consistent with the weight loss observed in this group.

[0203] The effects of IL-15 compounds on lymphocyte proliferation and proliferation were also examined in the spleen, a lymphoid organ. Similar to the results observed in peripheral blood, all IL-15 compounds increased splenic Ki67-positive NK cells compared with vehicle (Figure 15A). Only the low-dose standard and P-0234 significantly increased the total number of NK cells in the spleen (Figure 15B). Similarly, a dose-response reversal was observed for NK cell proliferation in the spleen with the standard (Figure 15B), which was associated with high and sustained CD69 expression on splenic NK cells measured 4 days after termination (Figure 15C). These data suggest that the standard compounds may overstimulate NK cells and exhaust them. Because IL-15 is noncovalently bound to the IL-15Rα-Fc fusion protein, IL-15 may dissociate from the fusion complex with the standard compounds, resulting in lymphocyte overstimulation, cell exhaustion, toxicity, and weight loss.

[0204] Example 13 Pharmacokinetic and Pharmacodynamic Effects of IL-15 / IL-15Rα-Fc Fusion Protein in Mice After a Single Injection A dose-response study using P-0313, a C-terminal bivalent IL-15S58D / IL-15Rα (non-covalent)-Fc fusion protein, was conducted in Balb / C mice after a single injection. The effects on peripheral blood lymphocyte proliferation and proliferation were monitored over time. Additionally, the pharmacokinetics and pharmacodynamics (PK / PD) of P-0313 were compared with those of a standard N-terminal bivalent IL-15 (non-covalent) / IL-15Rα-Fc fusion protein containing the N72D mutation in IL-15 after a single injection.

[0205] Seven-week-old female Balb / c mice were obtained from Charles River Laboratories and acclimated in-house for at least 7 days prior to testing. Mice were intraperitoneally (ip) injected with vehicle, standard (0.3 mg / kg), or P-0313 (0.01 mg / kg, 0.03 mg / kg, 0.1 mg / kg, and 0.3 mg / kg) at time 0. Blood samples were collected at -24 hours (pre-dose) and 1, 4, 24, 72, 120, and 192 hours post-injection. Body weights were recorded daily before and during treatment. Each group contained five mice.

[0206] Immunophenotyping was performed using heparinized whole blood, and the volume was recorded. After erythrocyte lysis using BD pharm lysis buffer, total viable mononuclear blood cells were counted by excluding dead cells with trypan blue and then subjected to intracellular staining for Ki67. The cell pellet was thoroughly resuspended in 200 μl / well of 1x Foxp3 fixation / permeabilization working solution and incubated at room temperature in the dark for 30 minutes. After centrifugation, 200 μl of 1x permeabilization buffer was added to each well for further washing. After Fc receptor blocking 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-Percpcy5.5 (1:50 dilution). After a 30-minute incubation, cells were collected, washed, resuspended in FACS buffer, and analyzed by flow cytometry. Statistical analysis was performed with one-way analysis of variance with Tukey's multiple comparison test in GraphPad prism software.

[0207] The serum concentrations of the above compounds were measured using two different ELISA assays. An in-house ELISA assay was developed to measure the complex of IL-15 and Fc. The commercial ELISA assay measures IL-15 alone and contains both capture and detection antibodies reactive with human IL-15. For the in-house ELISA assay, MaxiSorp plates were coated with anti-IL-15 antibody (R&D Systems MAB647) overnight at 4°C. The plates were blocked with SuperBlock. Various dilutions of standards and samples were added to the plates and incubated for 1 hour at room temperature. Active compounds were detected with anti-human IgG Fc-HRP, and the signal was developed using Ultra TMB Substrate Solution. Values ​​were calculated using interpolation from nonlinear regression curve fitting in GraphPad Prism.

[0208] During the first 24 hours, both compounds were detectable in serum at comparable serum concentrations. Peak concentrations were observed 4 hours after intraperitoneal administration. By 72 hours, only P-0313 remained measurable, while the standard was undetectable in all three mice (Figures 16A-B). Similar results were obtained using two different ELISA assays, confirming that P-0313 had a superior pharmacokinetic profile compared with the standard. This result confirmed the previous findings shown in Example 12, where the IL-15 / IL-15Rα (non-covalent)-Fc fusion protein P-0234 exhibited higher serum exposure than the standard. These data strongly support the superiority of the IL-15 / IL-15Rα (non-covalent)-Fc fusion construct over the IL-15 (non-covalent) / IL-15Rα-Fc fusion protein construct in extending the in vivo half-life of IL-15.

[0209] No significant changes in body weight were observed in any of the treatment groups (Figure 17).

[0210] A dose-dependent increase in Ki67 expression was observed in NK cells and CD8+ T cells from P-0313-treated mice (Figures 18A and 18B). The effect peaked at 72 hours and persisted for up to 120 hours with the standard treatment, and even up to 192 hours with P-0313 (Figure 18A), demonstrating that P-0313 has a longer duration of action than the standard treatment. In addition, P-0313 showed similar Ki67 induction at doses 3- to 10-fold lower than the standard treatment, demonstrating that P-0313 is more effective than the standard treatment (Figures 18A and 18B). A significant response was observed in NK cells at a dose 10-fold lower than that for CD8+ T cells, indicating that NK cells are more sensitive to P-0313 treatment than CD8+ T cells.

[0211] Consistent with the increase in the proliferation marker Ki67, a dose-dependent proliferation of NK cells and CD8+ T cells in the blood was observed in the P-0313-treated group (Figures 19A and 19B). Cell proliferation was observed at 72 hours and peaked at 120 hours. P-0313 increased NK cells by 4-fold, 15-fold, 50-fold, and 163-fold from baseline at doses of 0.01 mg / kg, 0.03 mg / kg, 0.1 mg / kg, and 0.3 mg / kg, respectively (Figure 19A). It also increased CD8+ T cells by 10-fold and 50-fold from baseline at doses of 0.1 mg / kg and 0.3 mg / kg (Figure 19B). In contrast, standard increased peripheral NK cells by 28-fold and CD8+ T cells by 12-fold only at the 0.3 mg / kg dose (Figures 19A and 19B).

[0212] In summary, P-0313 exhibited superior pharmacokinetic and pharmacodynamic properties to the standard compound on the proliferation and expansion of NK cells and CD8 T cells.

[0213] Example 14 Effect of IL-15 / IL-15Rα-Fc fusion protein on inhibiting lung metastasis of mouse colon cancer To investigate the anti-metastatic effect and immune response of IL-15 / IL-15Rα-Fc fusion protein in tumor models, 1 × 10 5CT26-WT (ATCC CRL-2638) mouse colon carcinoma cells were intravenously injected into female Balb / C mice (10–12 weeks old). Starting the following day, 0.03 mg / kg or 0.1 mg / kg of P-0313 or 0.3 mg / kg of the standard compound was administered intravenously for 5 days (days 1, 6, and 11 after cell implantation). A vehicle (PBS) was included as a negative control, and each group contained eight mice. On day 15, blood samples were collected for lymphocyte phenotyping and liver enzyme measurements. On day 16, all mice were sacrificed and tissues were removed. Lungs were inflated with 15% India ink and destained with Fekete's solution (10% formaldehyde, 5% glacial acetic acid, and 60% ethanol). Lung tumor nodules were counted for all lungs under a light microscope, and the difference in the number of tumor nodules between the treatment group and the vehicle control group represented the anti-metastatic effect.

[0214] To study immune responses, peripheral blood was collected from mice on day 15 into heparinized tubes, and the volume of blood used in the assay was recorded for each mouse. After erythrocyte lysis with BD pharm lysis buffer, total viable mononuclear blood cells were counted by excluding dead cells with trypan blue. These cells were then used for immune cell phenotyping and Ki67 proliferation analysis, as previously described. After fixation, permeabilization, and antibody staining, cells were collected, washed, resuspended in FACS buffer, and analyzed by flow cytometry.

[0215] Figure 20A shows representative photographs of lungs from each group to illustrate lung nodules. Lung metastatic lesions were counted and quantified using a microscope (Figure 20B). As shown in Figure 20, the standard molecule administered at 0.3 mg / kg inhibited lung metastasis and reduced the number of lung nodules by 84%, confirming the effectiveness of IL-15 pathway activation in suppressing the formation and growth of lung metastases. Notably, administration of the IL-15 / IL-15Rα-Fc complex P-0313 at a three-fold lower dose (0.1 mg / kg) completely inhibited the development of lung metastases, with zero nodules observed in all eight treated mice (Figures 20A and 20B). The superiority of P-0313 over the standard in suppressing the formation and growth of lung metastases is consistent with the enhanced pharmacokinetic and pharmacodynamic effects revealed above (Examples 12 and 13). Because the IL-15 moiety of P-0313 is covalently linked to the Fc domain, P-0313 exhibited a significant improvement in IL-15 serum half-life compared with a control containing IL-15 noncovalently linked to the Fc chain via the IL-15RaSushi domain (Example 13). A 0.03 mg / kg dose of P-0313 also suppressed lung metastasis with an inhibitory effect of approximately 35% (Figures 20A and 20B). This finding further emphasizes that P-0313 is effective at doses much lower than the control, but it also clearly demonstrates the importance of the biodistribution and bioavailability of the IL-15 / IL-15Rα-Fc complex for its anticancer activity in vivo.

[0216] After three repeated doses every 5 days, the proliferation 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 correlated with a significant increase in spleen weight in this treatment group (Figure 22). In contrast, only minimal CD8+ T cell proliferation was observed in the 0.3 mg / kg standard treatment group; no increase in circulating NK cell numbers was observed compared to the control group (Figures 21A and 21B). However, the standard treatment group showed a significant increase in spleen weight (Figure 22). The data suggest that proliferated lymphocytes may be migrating to lymphoid tissues for storage after repeated doses, or that cell exhaustion may have occurred. Since all three treatment groups exhibited tumor-suppressive effects, the data suggest that either CD8+ T cells or NK cells may be the effector subset responsible for the tumor-suppressive effect. However, the complete eradication of lung metastases seen in the 0.1 mg / kg P-0313 treatment group suggested that effects involving both NK cells and CD8+ T cells induced the strongest tumor growth inhibition.

[0217] Treatment-associated hepatotoxicity was assessed by measuring liver weight and serum concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). As shown in Figures 23A-C, no increase in liver weight, ALT, or AST levels was observed in any of the treatment groups compared with the vehicle group. The data suggest that the potent antitumor effect of the IL-15 / IL-15Rα-Fc fusion protein is not accompanied by hepatotoxicity.

[0218] Example 15 Effect of IL-15 / IL-15Rα-Fc fusion protein on established CT26 solid tumor growth in mice To further investigate the antitumor activity and immune response of the IL-15 / IL-15Rα-Fc fusion protein in established tumor models, 1 × 10 IL-15Rα-Fc fusion protein was injected into the right flank of female Balb / C mice (10–12 weeks old). 5 CT26 cells were subcutaneously injected. On day 11, the average tumor volume was approximately 70 mm 3At this time, 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. On day 16, an additional intraperitoneal injection of each test drug was administered (for a total of two doses). Tumors were measured three times weekly using calipers, and tumor volume was calculated as follows: volume = 0.5 × (width). 2 × (length). On day 19, non-terminal peripheral blood was collected into heparinized tubes for immune response studies. On day 21, all mice were sacrificed and tissues were harvested.

[0219] As shown in Figure 24A, PBS-treated mice rapidly developed large subcutaneous tumors. Treatment of mice with either 0.1 mg / kg or 0.05 mg / kg P-0313 was approximately equipotent in delaying tumor growth (Figure 24A). Tumor growth curves were plotted for individual mice in all three treatment groups (Figures 25A-C). It is clear that in the two dose groups tested, mice responded well to P-0313 treatment, exhibiting tumor growth delay and synchronous inhibition, especially in the early stages (Figures 25A-C). At 21 days post-tumor inoculation, the average tumor volume in PBS-treated mice was 820 mm. 3 In contrast, P-0313-treated mice had 410 mm 3 (Figure 25A, **P<0.01; one-way ANOVA with Tukey's post-test). It is noteworthy that the high dose (0.1 mg / kg) of P-0313 initially showed a greater reduction in tumor burden than the low dose group, but the difference gradually diminished as treatment progressed.

[0220] P-0313-treated mice showed similar weight gain as PBS-treated mice throughout the 21-day study (Figure 24B), suggesting that P-0313 was well tolerated and without apparent toxicity at the two doses tested.

[0221] Next, we investigated the effects of P-0313 on CD8+ T cell and NK cell populations in peripheral blood and the spleen. Administration of P-0313 to tumor-bearing mice potently induced the proliferation of NK cells and CD8+ T cells (Figures 26A and 26B), and induced the proliferation of NK cells and CD8+ T cells in a dose-dependent manner (Figures 26C and 26D), resulting in immune cell responses comparable to those observed in non-tumor-bearing mice (Example 13). A higher fold change was observed in NK cells between these two lymphocyte populations (approximately 100-fold in the 0.1 mg / kg dose group and approximately 38-fold in the 0.05 mg / kg dose group). CD8+ T cells expanded approximately 5.4-fold at the 0.1 mg / kg dose and approximately 2.7-fold in the 0.05 mg / kg dose group.

[0222] P-0313 also enhanced the proliferation of both NK cells and CD8+ T cells in the spleen, similar to that in peripheral blood (Figures 27A and 27B), although the magnitude / fold change in the spleen was not as great. A larger fold change was observed in NK cells (approximately 10-fold in the 0.1 mg / kg dose group and approximately 8-fold in the 0.05 mg / kg dose group). CD8+ T cells expanded approximately 2.7-fold at the 0.1 mg / kg dose, with negligible expansion in the 0.05 mg / kg dose group.

[0223] Collectively, these data demonstrate that P-0313 treatment significantly slows and inhibits solid tumor growth, and that this tumor-suppressive effect correlates 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 possesses an Fc region lacking effector function, its in vivo antitumor activity is not due to direct tumor cell killing, but rather to a potent immune response against tumor cells via robust activation of cytotoxic CD8+ T cells and NK cells.

[0224] Example 16 Effect of IL-15 / IL-15Rα-Fc fusion protein on unestablished CT26 solid tumor growth in Balb / C mice To confirm the antitumor activity of P-0313, a similar study was performed in a non-established CT26 tumor model. 5 Three days after subcutaneous implantation of CT26 cells, mice were intraperitoneally injected with vehicle (PBS) or P-0313 (0.1 mg / kg) every 5 days for a total of 5 injections. Mice were sacrificed on day 25, and tumor measurements were performed 2-3 times a week.

[0225] Similar to what was seen in the established CT26 tumor model (Example 15), P-0313 demonstrated marked inhibition of tumor growth (Figure 28A) and a significant reduction in solid tumor burden (Figure 28B). After five repeated doses, P-0313-treated mice showed a modest increase in spleen weight (Figure 29A), but no significant reduction in body weight gain (Figure 29B), indicating that P-0313 was well tolerated.

[0226] Overall, these data further demonstrate that P-0313 is an effective immunotherapeutic agent against solid and liquid tumors, as well as tumor cell metastasis, with a well-tolerated safety profile.

[0227] Example 17 Construction of IL-15 antagonist variant / IL-15RαFc fusion protein As disclosed in Example 8 and related Tables 5-7, a significant number of IL-15 variants, including amino acid substitutions, insertions, and deletions, resulted in reduced binding to IL-15Rβ in ELISA assays. In this example, additional IL-15Rβγ-disrupting substitutions were introduced into the IL-15 moiety with the goal of achieving optimally attenuated potency in the form of an IL-15 / IL-15RαFc fusion protein. The inventors reasoned that lower potency might prevent pathway overactivation and reduce undesirable target sinks, and thus, IL-15 variant / IL-15RαFc fusion proteins with attenuated activity might result in reduced toxicity and improved pharmacokinetics.

[0228] Three IL-15 residues at the interface with IL-15Rβ, S58, V63, and I68, and one γc-interacting residue, Q108, were targeted for potency-attenuating mutagenesis. IL-15 variants with various lengths of N-terminal deletions were also constructed.

[0229] The amino acid substitutions and deletions and the corresponding IL-15 variant / IL-15RαFc fusion proteins are listed in Table 8. TIFF0007807076000017.tif203170

[0230] Example 18 Construction of IL-15 antagonist variant / IL-15Rα antibody fusion protein Various IL-15 antagonist variant / IL-15RαSushi antibody fusion proteins will be prepared and evaluated. It is understood in the art that the use of recombinant antibody-cytokine fusion proteins (immunocytokines) can enhance the therapeutic index of cytokines by targeting them to the site of disease. In addition to tumor-targeting antibodies, immune checkpoint-blocking antibodies that circumvent immunosuppressive effects in the tumor microenvironment or immune stimulatory antibodies to enhance existing responses can also be used to construct antibody-cytokine fusion proteins. Immune checkpoint-blocking antibody-cytokine fusion proteins are expected to further enhance immune system activity against tumors. The inventors of the present invention propose that the use of IL-15 variants with attenuated activity may facilitate the establishment of a stoichiometric balance between the cytokine and antibody arms. Furthermore, attenuated cytokine activity is expected to minimize peripheral activation, alleviate antigen sinks, and facilitate tumor targeting via the antibody arm.

[0231] Following this concept, various IL-15 / IL-15RαSushi-antibody fusion proteins were constructed. Exemplary antibodies used to prepare the fusion proteins include various PD-1 antagonist antibodies having the sequences set forth in SEQ ID NOS: 111-120, PD-L1 blocking antibodies (SEQ ID NOS: 121-122), anti-CTLA4 antibodies (SEQ ID NOS: 123-124), CD40 agonist antibodies (SEQ ID NOS: 125-126), the L19 antibody against the fibronectin extra-domain (SEQ ID NOS: 127-128), rituximab against CD20 (SEQ ID NOS: 129-130), the antibody trastuzumab against Her-2 (SEQ ID NOS: 131-132), cetuximab against EGFR (SEQ ID NOS: 133-134), and an anti-FAP antibody for tumor targeting and retention (SEQ ID NOS: 109-110).

[0232] To prepare IL-15 / IL-15RαSushi-antibody fusion proteins, the CH1-CH2-CH3 (antibody residues 118-447 based on EU numbering) domains of the heavy chain of the antibodies listed above were replaced with the IgG1 sequence set forth in SEQ ID NO: 135, which contains L234A, L235A, and G237A mutations that abolish binding to FcγR and C1q but retain FcRn binding or PK. IL-15 variant peptides were fused to the C-terminus of the Fc domain via peptide linkers with the sequences listed in Table 6. Alternatively, to express monovalent IL-15 variants, the CH1-CH2-CH3 domains of the heavy chain of the antibodies listed above were replaced with heterodimeric chains set forth in SEQ ID NOs: 136-137. The IL-15 variant peptide is fused via a peptide linker to the C-terminus of a knob-containing heterodimer heavy chain engineered using knob-into-holes technology. Half-life-extending mutations, such as N434A, can also be incorporated into the Fc chain of homodimers or heterodimers. Yet another approach to generating bivalent IL-15 / IL-15RαSushi-antibody fusion proteins is to fuse the IL-15 variant peptide to the C-terminus of the light chain via a peptide linker. In all cases, IL-15Rα is coexpressed with the IL-15 moiety to form a noncovalent complex. Exemplary IL-15 variant / IL-15RαSushi-PD-1 antagonist antibody fusion proteins are listed in Table 9, and their illustrations are shown in Figure 30. TIFF0007807076000018.tif112170

[0233] Gene synthesis, expression vector construction, and protein production, purification, and characterization were performed according to the same procedures detailed in Example 1. All IL-15 variant / IL-15Rα-PD-1 antagonist antibody fusion proteins were expressed with decent productivity comparable to the parental antibody with low aggregation tendency (data not shown).

[0234] Example 19 IL-15 variant / IL-15Rα fusion proteins exhibit varying levels of potency modulation in in vitro functional activity. Exemplary IL-15 variant / IL-15Rα Fc fusion proteins (monomer or dimer) were screened for their ability to stimulate cell proliferation by measuring Ki67 expression in CD8+ T cells. Human PBMCs were treated with increasing doses of IL-15 fusion molecules, and Ki67 expression was determined by intracellular FACS analysis gated on the CD8+ T cell population, as previously described. P-0313, a bivalent IL-15 / IL-15Rα sushi (non-covalent) Fc fusion containing the S58D substitution, which has enhanced binding and functional activity, has been extensively characterized and was used as a positive control.

[0235] As shown in Figure 31A, substitution of IL-15 residue S58 with amino acids of varying size, solubility, and ionization properties only modestly modulated the potency to stimulate CD8 T cell proliferation. The EC of exemplary variants in stimulating CD8 T cell proliferation 50 The values ​​are summarized in Table 10A. Compared to P-0313, all variants showed slightly reduced potency, with reductions ranging from 1.7-fold to 7-fold. TIFF0007807076000019.tif71170

[0236] Similarly, an exemplary IL-15 variant / IL-15RαFc fusion containing a substitution at position V63 resulted in only a moderate attenuation of CD8 T cells, as shown in Figure 31B. EC 50 The values ​​and fold reduction compared to P-0313 are summarized in Table 10B. TIFF0007807076000020.tif56170

[0237] In contrast to residues S58 and V63, amino acid substitutions at position I68, which form van der Waals interactions with multiple IL-15Rβ residues, yield variants with a broad spectrum of potency to expand CD8 T cells and inhibit EC50 ranged from 0.4 nM to approximately 300 nM, a 700-fold difference. Compared to P-0313, the level of attenuation of activity ranged from 10-fold to ~7000-fold. The data are shown in Figure 31C and summarized in Table 10C. TIFF0007807076000021.tif70170

[0238] Yet another residue targeted to attenuate IL-15 functional activity is Q108, a critical hotspot residue that interacts with multiple residues in γc. Both exemplary IL-15Q108 mutant / IL-15RαFc fusion proteins, P-0763 and P-0793, carrying the Q108S and Q108A mutations, respectively, showed a significant decrease (>1000-fold) in CD8 T cell proliferation (Figure 31D and Table 10D). TIFF0007807076000022.tif52170

[0239] In addition to substituting IL-15 residues at the interface with the IL-15Rβ and / or γc receptor subunits to achieve the desired attenuation of activity, N-terminal deletions provide another means to achieve the same goal. The N-terminus of IL-15 is part of an α-helix that contains key residues that interact with IL-15Rβ and γc, such as Asp8 and Lys10. Bivalent IL-15 variant / IL-15Rα (non-covalent) Fc fusion proteins P-0866, P-0867, P-0868, and P-822, which contain one, two, three, and four amino acid deletions, respectively, at the N-terminus of IL-15, were analyzed in a human PBMC assay. As shown in Figure 32, the single amino acid deletion did not affect the potency of P-0866 to stimulate CD8+ T cell proliferation compared to its wild-type IL-15 counterpart, P-0234. In contrast, the two amino acid deletions in P-0867 resulted in an approximately 100-fold decrease in potency (EC 50(24.8 nM vs. 289 pM for P-0234). Deleting one additional amino acid from P-0868 did not further reduce function. However, deleting one extra amino acid or a total of four amino acids essentially eliminated the biological activity of the fusion protein P-0822.

[0240] Furthermore, combinations of IL-15 amino acid substitutions that disrupt IL-15Rβ and / or γc interactions appeared to result in varying levels of potency attenuation. Exemplary combination mutants incorporating V63A and one of the preferred mutations at I68 listed in Table 8, including V68H, I68Q, and I68G, were constructed and evaluated for CD8+ T cell proliferation in human PBMCs. Combinations of substitutions that disrupt IL-15Rβ resulted in progressively reduced CD8+ T cell proliferation (Table 11). Compared to P-0313, there was a 4.2-fold reduction in potency due to the V63A substitution, and V63A consistently resulted in 2.1-fold, 4.3-fold, and 3.8-fold attenuation of CD8+ T cell proliferation in the presence of the I68H, I68Q, and I68G mutations, respectively. The data are also shown in Figure 33A. Similarly, adding a V63A amino acid substitution in the presence of an N-terminal amino acid deletion also resulted in a three-fold attenuation of CD8 T cell proliferation, as exemplified by P-0886 and P-0888, which are illustrated in Figure 33B. Both compounds are IL-15 variant / IL-15RαPD-1 antibody fusion proteins containing two amino acid deletions at the N-terminus of IL-15. P-0888 contains an additional V63A mutation. TIFF0007807076000023.tif89170

[0241] As will be appreciated by one of skill in the art, additional variants with varying levels of attenuation of potency can be obtained by combining the preferred mutations listed in Table 8, e.g., V63K / Q108M, I68H / Q108K, and any additional combination variants are within the spirit and scope of the present invention.

[0242] CD8T cell Ki67EC 50Representative IL-15 variant / IL-15RαSushiFc fusion proteins, P-0736, P-0772, P-0737, P-768, P-0793, and P-0764, with potencies ranging from 3.6 nM to 69 nM, were further tested for their ability to stimulate NK cell proliferation by measuring Ki67 expression as previously described. Data are presented in Figures 34A and 34B, and EC 50 The values ​​are summarized in Table 12. TIFF0007807076000024.tif100170

[0243] Similarly, as observed in CD8 T cells, representative IL-15 variant / IL-15RαSushiFc fusion proteins also exhibited EC values ​​ranging from 0.42 nM to 9.7 nM. 50 The compounds showed a wide range of potency in stimulating NK cell proliferation. Compared to P-0313, the potency of each molecule was significantly attenuated, ranging from 100- to 2000-fold for CD8 T cells and from 30- to 750-fold for NK cells. Although each molecule followed a similar potency ranking in CD8 T cell proliferation, all compounds were more reactive with NK cells.

[0244] Finally, Figure 35 clearly shows that the IL-15 / IL-15Rα complexes in either Fc- or antibody-fused forms exhibit identical biological activity. Figure 35A shows P-0773 and P-0870, both containing the V63A / I68H amino acid substitution, as IL-15 / IL-15Rα Fc and antibody fusions, respectively. Figure 35B shows P-0867 and P-0886, a similar pair consisting of two amino acid deletions at the N-terminus of IL-15.

[0245] In summary, and importantly, introducing amino acid substitutions or deletions that disrupt IL-15Rβγc, individually or in combination, into the IL-15 moiety resulted in IL-15 variants with a broad spectrum of potency to stimulate cytotoxic lymphocytes, including CD8+ T cells and NK cells. The level of attenuation of activity with different mutations or combinations of mutations was similar for Fc and antibody fusion proteins.

[0246] All of the articles and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the articles and methods of the present disclosure have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes may be made therein without departing from the spirit and scope of the present disclosure. All such modifications and equivalents, whether now existing or later developed, apparent to those skilled in the art are deemed to be within the spirit and scope of the present disclosure as defined by the appended claims. All patents, patent applications, and publications cited in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All patents, patent applications, and publications are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The disclosure illustratively described herein may be suitably practiced in the absence of any element not specifically disclosed herein. That is, while the present disclosure has been specifically disclosed by preferred embodiments and optional features, it is to be understood that modifications and variations of the concepts disclosed herein may be made by those skilled in the art, and that such modifications and variations are deemed to be within the scope of the present disclosure as defined by the appended claims. [Sequence table] The amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for amino acids as prescribed by 37 CFR §1.822. SEQ ID NO: 1 is the amino acid sequence of human IL-15 precursor. SEQ ID NO: 2 is the amino acid sequence of the mature form of human IL-15. SEQ ID NO: 3 is the human IL-15Rα amino acid sequence. SEQ ID NO: 4 is the amino acid sequence of the extracellular domain of human IL-15Rα. SEQ ID NO: 5 is the amino acid sequence of the sushi domain+ of human IL-15Rα. SEQ ID NO: 6 is the human IgG1-Fc amino acid sequence. SEQ ID NO: 7 is the amino acid sequence of Knob-Fc. SEQ ID NO: 8 is the amino acid sequence of Hole-Fc. SEQ ID NOs: 9 to 12 are the amino acid sequences of various peptide linkers. SEQ ID NO: 13 is the amino acid sequence of the Hole-Fc-Linker1-IL-15 chain. SEQ ID NO: 14 is the amino acid sequence of Knob-Fc-linker1-IL-15Rα-Sushi+ chain. SEQ ID NO: 15 is the amino acid sequence of IL-15-Linker 4-Hole-Fc chain. SEQ ID NO: 16 is the amino acid sequence of IL-15Rα-Sushi+-Linker 4-Knob-Fc chain. SEQ ID NO: 17 is the amino acid sequence of the Knob-Fc-linker2-IL-15Rα-Sushi+ chain. SEQ ID NO: 18 is the amino acid sequence of the Hole-Fc-linker2-IL-15 chain. SEQ ID NO: 19 is the amino acid sequence of IL-15-Linker3-Hole-Fc chain. SEQ ID NO: 20 is the amino acid sequence of the Fc-linker3-IL-15 chain. SEQ ID NO: 21 is the amino acid sequence of the IL-15-linker3-Fc chain. SEQ ID NO: 22 is the amino acid sequence of the Knob-Fc-linker2-IL-15Rα-Sushi+ chain. SEQ ID NO: 23 is the amino acid sequence of the Fc-linker2-IL-15Rα-Sushi+ chain. SEQ ID NOs: 24 to 45 are the amino acid sequences of various IL-15 variant polypeptides. SEQ ID NO: 46 is the amino acid sequence of the Fc-linker3-IL-15S58D chain. SEQ ID NO: 47 is the amino acid sequence of the peptide linker. SEQ ID NO: 48 is the amino acid sequence of the Hole-Fc-linker3-IL-15-S58D chain. SEQ ID NO: 49 is the amino acid sequence of IL-15-S58D-linker 3-Hole-Fc chain. SEQ ID NO: 50 is the amino acid sequence of IL-15-S58D-linker3-Fc chain. SEQ ID NO: 51 is the amino acid sequence of IL-15Rα-Sushi-Linker2-Knob-Fc chain. SEQ ID NO: 52 is the amino acid sequence of IL-15Rα-Sushi-linker2-Fc chain. SEQ ID NO: 53 is the amino acid sequence of the Hole-Fc-Linker1-IL-15-S58D chain. SEQ ID NO: 54 is the amino acid sequence of the Hole-Fc-linker3-IL-15 chain. SEQ ID NO: 55 is the amino acid sequence of the Knob-Fc-linker1-IL-15 chain. SEQ ID NOs: 56-63 are the amino acid sequences of various IL-15 variant polypeptides containing N-terminal deletions. SEQ ID NOs: 64 and 65 are the amino acid sequences of the two authentic polypeptide chains. SEQ ID NOs: 66 to 81 are the amino acid sequences of various IL-15 variant polypeptides. SEQ ID NOs: 82-104 are the amino acid sequences of various IL-15 variant Fc fusion constructs. SEQ ID NOs: 105 to 106 are amino acid sequences of human IgG1-Fc containing sequences that reduce / eliminate effector functions and extend half-lives. SEQ ID NO: 107 is the amino acid sequence of Knob-Fc with extended half-life. SEQ ID NO: 108 is the amino acid sequence of the extended half-life Hole-Fc. SEQ ID NOs: 109 to 110 are the amino acid sequences of the heavy and light chains of the humanized anti-FAP antibody. SEQ ID NOs: 111-112 are the amino acid sequences of the heavy and light chains of the humanized PD-1 antagonist antibody. SEQ ID NOs: 113-114 are the amino acid sequences of the heavy and light chains of human PD-1 antagonist antibodies. SEQ ID NOs: 115-116 are the amino acid sequences of the heavy and light chains of the PD-1 antagonist antibody. SEQ ID NOs: 117-118 are the amino acid sequences of the heavy and light chains of the PD-1 antagonist antibody. SEQ ID NOs: 119-120 are the amino acid sequences of the heavy and light chains of the PD-1 antagonist antibody. SEQ ID NOs: 121-122 are the amino acid sequences of the heavy and light chains of PD-L1 antagonist antibodies. SEQ ID NOs: 123-124 are the amino acid sequences of the heavy and light chains of a CTLA-4 antagonist antibody. SEQ ID NOs: 125 to 126 are the amino acid sequences of the heavy and light chains of a CD40 agonist antibody. SEQ ID NOs: 127 and 128 are the amino acid sequences of the heavy and light chains of an anti-fibronectin antibody. SEQ ID NOs: 129 to 130 are the amino acid sequences of the heavy and light chains of the CD20 antagonist antibody. SEQ ID NOs: 131-132 are the amino acid sequences of the heavy and light chains of a Her-2 / neu antagonist antibody. SEQ ID NOs: 133-134 are the amino acid sequences of the heavy and light chains of the EGFR antagonist antibody. SEQ ID NO: 135 is the amino acid sequence of a human IgG1 CH1CH2CH3 domain sequence with reduced / eliminated Fc effector function. SEQ ID NO: 136 is the amino acid sequence of a human IgG1 CH1CH2CH3 domain knob chain sequence with reduced / eliminated Fc effector function. SEQ ID NO: 137 is the amino acid sequence of a human IgG1 CH1CH2CH3 domain hole chain sequence with reduced / eliminated Fc effector function. SEQ ID NOs: 138-148 are the amino acid sequences of various exemplary IL-5 / IL-15RαSushi-antibody fusion proteins. SEQ ID NOs: 149-152 are the amino acid sequences of various IL-15 loss variant Fc fusion constructs. SEQ ID NOs: 153 to 154 are the amino acid sequences of various peptide linkers. [Sequence table] Human IL-15 precursor sequence MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 1) Human IL-15 mature sequence NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 2) Human IL-15Rα sequence MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 3) Human IL-15Rα extracellular domain ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT (SEQ ID NO: 4) Human IL-15Rα sushi domain+ ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP (SEQ ID NO: 5) Human IgG1-Fc with reduced effector function DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 6) Knob-Fc DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 7) Hole-Fc DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 8) Peptide linker sequence EPKSSDKTHTSPPSP (SEQ ID NO: 9) Peptide linker sequence GGGGSGGGGS (SEQ ID NO: 10) Peptide linker sequence GGGGSGGGGSGGGGS (SEQ ID NO: 11) Peptide linker sequence GSGS (SEQ ID NO: 12) Hole-Fc-linker1-IL-15 chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGEPKSSDKTHTSPPSPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 13) Knob-Fc-IL-linker1-IL-15Rα-Sushi chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGEPKSSDKTHTSPPSPITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP (SEQ ID NO: 14) IL-15-Linker 4-Hole-Fc Chain NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 15) IL-1�Rα-Sushi+-Linker4-Knob-Fc chain ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 16) Knob-Fc-Linker2-IL-15Rα-+ chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP (SEQ ID NO: 17) Hole-Fc-Linker 2-IL-15 chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO: 18) IL-15-Linker 3-Hole-Fc chain NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:19) Fc-Linker 3-IL-15 chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:20) IL-15-Linker 3-Fc chain NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 21) Knob-Fc-Linker 2-IL-15Rα-Sushi+ chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP (SEQ ID NO: 22) Fc-Linker 2-IL-15Rα-Sushi+ chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP (SEQ ID NO: 23) Human IL-15S58D variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDADIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 24) Human IL-15T62D variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDDVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 25) Human IL-15V63F variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTFENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 26) Human IL-15I67V variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLVILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 27) Human IL-15I68F variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIFLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 28) Human IL-15I68K variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIKLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 29) Human IL-15I68D variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIDLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 30) Human IL-15I68H variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIHLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 31) Human IL-15Q108A variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVAMFINTS (SEQ ID NO: 32) Human IL-15Q108M ​​variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVMMFINTS (SEQ ID NO: 33) Human IL-15Q108S variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVSMFINTS (SEQ ID NO: 34) Human IL-15Q108S / D30T variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESTVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVSMFINTS (SEQ ID NO: 35) Human IL-15Q108S / V31Y variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDYHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVSMFINTS (SEQ ID NO: 36) Human IL-15Q108S / H32E variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVEPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVSMFINTS (SEQ ID NO: 37) Human IL-15Q108S / D30T / V31Y / H32E variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESTYEPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVSMFINTS (SEQ ID NO: 38) Human IL-15 deletion 111-114 variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF (SEQ ID NO: 39) Human IL-15 deletion 109-114 variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ (SEQ ID NO: 40) Human IL-15 deletion 108-114 variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIV (SEQ ID NO: 41) Human IL-15 deletion 105-114 variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV (SEQ ID NO: 42) Human IL-15 N95 post-"GS" insertion variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNGSIKEFLQSFVHIVQMFINTS (SEQ ID NO: 43) Human IL-15 N95 post-'GGSGG' insertion variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNGGSGGIKEFLQSFVHIVQMFINTS (SEQ ID NO: 44) Human IL-15 post-N95 "GSSGGSGGS" insertion variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNGSSGSSGGSIKEFLQSFVHIVQMFINTS (SEQ ID NO: 45) Fc-linker3-IL-15S58D chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDADIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 46) Peptide linker sequence GGGGSGGGG (SEQ ID NO: 47) Hole-Fc-linker3-IL-15S58D chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDADIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 48) IL-15S58D-Linker 3-Hole-Fc chain NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDADIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 49) IL-15S58D-Linker 3-Fc chain NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDADIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 50) IL-15Rα-Sushi-Linker 2-Knob-Fc chain ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSGGGGSCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 51) IL-15Rα-Sushi-Linker 2-Fc chain ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSGGGGSCPPCPAPEAAGAPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 52) Hole-Fc-Linker1-IL-15-S58D chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGEPKSSDKTHTSPPSPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDADIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 53) Hole-Fc-linker3-IL-15 chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 54) Knob-Fc-linker1-IL-15 chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGEPKSSDKTHTSPPSPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 55) Human IL-15 variant with one amino acid deletion at the N-terminus WVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 56) Human IL-15 variant with two amino acids deleted at the N-terminus VNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 57) Human IL-15 variant with three amino acids deleted at the N-terminus NVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 58) Human IL-15 variant with four amino acids deleted at the N-terminus VISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 59) Human IL-15 variant with 5 amino acids deleted at the N-terminus ISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 60) Human IL-15 variant with six amino acids deleted at the N-terminus SDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 61) Human IL-15V63A variant with two amino acids deleted at the N-terminus VNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTAENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 62) Human IL-15I68H variant with two amino acids deleted at the N-terminus VNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIHLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 63) standard chain 1 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIREPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 64) standard chain 2 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 65) Human IL-15S58G variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDAGIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 66) Human IL-15S58H variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDAHIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 67) Human IL-15S58R variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDARIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 68) Human IL-15S58Q variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDAQIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 69) Human IL-15S58I variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDAIIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 70) Human IL-15S58P variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDAPIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 71) Human IL-15V63A variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTAENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 72) Human IL-15V63K variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTKENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 73) Human IL-15V63R variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTRENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 74) Human IL-15I68Q variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIQLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 75) Human IL-15I68G variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIGLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 76) Human IL-15Q108E variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVEMFINTS (SEQ ID NO: 77) Human IL-15Q108K variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVKMFINTS (SEQ ID NO: 78) Human IL-15V63A / I68H variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTAENLIHLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 79) Human IL-15V63A / I68Q variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTAENLIQLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 80) Human IL-15V63A / I68G variant polypeptide NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTAENLIGLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 81) Fc-linker3-IL-15S58G chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDAGIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 82) Fc-linker3-IL-15S58 heavy chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDAHIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 83) Fc-linker3-IL-15S58R chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDARIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 84) Fc-linker3-IL-15S58Q chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDAQIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 85) Fc-linker3-IL-15S58I chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDAIIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 86) Fc-linker3-IL-15S58P chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDAPIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 87) Fc-Linker 3-IL-15 V63A chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTAENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 88) Fc-Linker 3-IL-15 V63K chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTKENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 89) Fc-linker3-IL-15V63R chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTRENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 90) Fc-linker3-IL-15I68F chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIFLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 91) Fc-Linker 3-IL-15 I68D chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIDLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 92) Fc-Linker 3-IL-15 I68H chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIHLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 93) Fc-Linker 3-IL-15 I68K chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIKLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 94) Fc-Linker 3-IL-15 I68Q chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIQLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 95) Fc-Linker 3-IL-15 I68G chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIGLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 96) Hole Fc-Linker 1-IL-15 Q108S chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGEPKSSDKTHTSPPSPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVSMFINTS (SEQ ID NO: 97) Hole Fc-Linker 1-IL-15 Q108A chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGEPKSSDKTHTSPPSPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVAMFINTS (SEQ ID NO: 98) Knob Fc-Linker 3-IL-15 Q108M chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVMMFINTS (SEQ ID NO: 99) Knob Fc-Linker 3-IL-15 Q108E chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVEMFINTS (SEQ ID NO: 100) Knob Fc-Linker 3-IL-15 Q108K chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVKMFINTS (SEQ ID NO: 101) Fc-Linker 3-IL-15 V63A / I68H chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTAENLIHLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 102) Fc-Linker 3-IL-15 V63A / I68Q chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTAENLIQLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 103) Fc-linker 3-IL-15V63A / I68G chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTAENLIGLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 104) Human IgG1-Fc with reduced / eliminated effector functions and extended half-life DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 105) Human IgG1-Fc with reduced / eliminated effector functions and extended in vivo half-life DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPG (SEQ ID NO: 106) Knob-Fc with extended in vivo half-life DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPG (SEQ ID NO: 107) Hole-Fc with extended in vivo half-life DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPG (SEQ ID NO: 108) Humanized anti-FAP antibody heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTENIIHWVRQAPGQGLEWMGWFHPGSGSIKYAQKFQGRVTMTADKSTSTVYMELSSLRSEDTAVYYCARHGGTGRGAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 109) Humanized anti-FAP antibody kappa light chain DIQMTQSPSSLSASVGDRVTITCRASRSISTSAYSYMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSRELPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 110) Human PD-1 antagonist antibody heavy chain EVQLVQSGAEVKKPGASVKVSCKASGYRFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTNTAYMELRSLRSDDTAVYYCARDADYSSGSGYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 111) Human PD-1 antagonist antibody Lλ SYELTQPPSVSVSPGQTARITCSGDALPKQYAYWYQQKPGQAPVMVIYKDTERPSGIPERFSGSSSGTKVTLTISGVQAEDEADYYCQSADNSITYRVFGGGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 112) Humanized PD-1 antagonist antibody-HC EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATISGGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPDSSSGVAYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 113) Humanized PD-1 antagonist antibody - LκDIVMTQSPLSLPVTPGEPASITCKASQDVETVVAWYLQKPGQSPRLLIYWASTRHTGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYSRYPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 114) Humanized PD-1 antagonist antibody-HC QGQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGVIESETGGTAYNQKFKGRAKITADKSTSTAYMELSSLRSEDTAVYYCTREGITTVATTYYWYFDVW GQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 115) Humanized PD-1 antagonist antibody-Lκ DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 116) Humanized PD-1 antagonist antibody-HC QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGT TVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 117) Humanized PD-1 antagonist antibody-Lκ EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 118) Human PD-1 antagonist antibody-HC QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVS SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 119) Human PD-1 antagonist antibody-Lκ EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 120) Humanized PD-L1 antagonist antibody-HC EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 121) Humanized PD-L1 antagonist antibody-Lκ DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 122) Human CTLA-4 antagonist antibody-HC QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 123) Human CTLA-4 antagonist antibody-Lκ EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 124) Human CD40 agonist antibody-HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCV ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 125) Human CD40 agonist antibody-Lκ DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 126) Humanized anti-fibronectin antibody-HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 127) Humanized anti-fibronectin antibody-Lκ EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 128) Chimeric anti-CD20 antibody-HC QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGT TVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 129) Chimeric anti-CD20 antibody-Lκ QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 130) Humanized anti-Her2 antibody-HC EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 131) Humanized anti-Her2 antibody-Lκ DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 132) Chimeric anti-EGFR antibody-HC QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTL VTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 133) Chimeric anti-EGFR antibody-Lκ DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 134) Human IgG1 CH1-CH2-CH3 domains with reduced / abolished effector function ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 135) Human IgG1 CH1-CH2-CH3 domains with knob chains that have reduced / eliminated effector functions ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 136) Human IgG1 CH1-CH2-CH3 domains with Hole chains that have reduced / eliminated effector functions ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 137) PD-1 antagonist antibody - HC - IL-15 Q108S chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATISGGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPDSSGVAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVSMFINTS (SEQ ID NO: 138) PD-1 antagonist antibody-HC-IL-15Q108S knob chain (SEQ ID NO: 139) PD-1 antagonist antibody-HC-IL-15Q108A knob chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATISGGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPDSSGVAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVAMFINTS (SEQ ID NO: 140) PD-1 antagonist antibody HC-IL-15V63A / I68H chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATISGGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPDSSGVAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTAENLIHLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:141) PD-1 antagonist antibody-HC-IL-1�I68G knob lock EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATISGGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPDSSGVAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIGLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 142) PD-1 antagonist antibody - HC - IL-15 V63A / I68G knob lock EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATISGGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPDSSGVAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTAENLIGLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 143) PD-1 antagonist antibody - HC hole chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATISGGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPDSSSGVAYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 144) PD-1 antagonist antibody-HC-IL-15V63A / I68G chain (SEQ ID NO: 145) PD-1 antagonist antibody-HC-IL-15 N-terminal 2 amino acid deletion chain (SEQ ID NO: 146) PD-1 antagonist antibody-HC-IL-15 N-terminal 4 amino acid deletion chain (SEQ ID NO: 147) PD-1 antagonist antibody - HC-IL-15V63A and N-terminal two amino acid deletion chain (SEQ ID NO: 148) Fc-linker 3-IL-15 N-terminal 1 amino acid deletion chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 149) Fc-linker 3-IL-15 N-terminal 2-amino acid deletion chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 150) Fc-linker 3-IL-15 N-terminal 3-amino acid deletion chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 151) Fc-linker 3-IL-15 N-terminal 4 amino acid deletion chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 152) Peptide linker sequence GGSGG (SEQ ID NO: 153) Peptide linker sequence GSSGGSGGS (SEQ ID NO: 154)

Claims

1. 1. An isolated interleukin-15 (IL-15) fusion protein complex, the complex comprising: (1) an IL-15 variant polypeptide covalently linked by a peptide linker to an Fc domain, and (2) an IL-15 receptor alpha ("IL-15Rα") domain non-covalently linked to said IL-15 variant polypeptide, thereby forming an Fc-IL-15 / IL-15Rα fusion protein; the IL-15 variant polypeptide consists of the amino acid sequence set forth in SEQ ID NO:2 with amino acid residue positions V63 and I68 substituted with other amino acids, the amino acid substitutions being selected from the group of V63A, V63K, V63R, I68Q, I68G, and I68H substitutions, and the IL-15Rα domain is an IL-15 receptor αSushi ("IL-15RαSushi") domain consisting of the amino acid sequence set forth in SEQ ID NO:5; An isolated interleukin-15 (IL-15) fusion protein complex.

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

3. 3. The isolated interleukin-15 (IL-15) fusion protein complex of claim 1, wherein the Fc domain is an Fc domain having an amino acid sequence 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, and each IL-15 polypeptide is covalently linked to the Fc domain by a peptide linker.

4. 4. The isolated interleukin-15 (IL-15) fusion protein complex of any one of claims 1 to 3, wherein the IL-15 variant polypeptide is selected from the group consisting of polypeptides having the amino acid sequences set forth in SEQ ID NOs: 79 to 81.

5. 5. The isolated interleukin-15 (IL-15) fusion protein complex of any one of claims 1 to 4, wherein the complex is in a dimeric or monomeric form.

6. 6. The isolated interleukin-15 (IL-15) fusion protein complex of any one of claims 1 to 5, wherein the IL-15 variant polypeptide consists of the amino acid sequence set forth in SEQ ID NO:

79.

7. 1. An isolated interleukin-15 (IL-15) fusion protein complex, the complex comprising: (1) an IL-15 variant polypeptide covalently linked by a peptide linker to an antibody (Ab), and (2) an IL-15 receptor alpha ("IL-15Rα") domain non-covalently linked to the IL-15 variant polypeptide, forming an Ab-IL-15 / IL-15Rα fusion protein; the IL-15 variant polypeptide is covalently attached to the C-terminus of the antibody; or the IL-15 variant polypeptide is covalently attached to the N-terminus of the antibody; the IL-15 variant polypeptide consists of the amino acid sequence set forth in SEQ ID NO:2 with amino acid residue positions V63 and I68 substituted with other amino acids, wherein the amino acid substitutions are selected from the group of V63A, V63K, V63R, I68Q, I68G, and I68H substitutions; the antibody is an antagonist Programmed Death-1 (PD-1) antibody or antibody fragment; the IL-15Rα domain is an IL-15 receptor α Sushi ("IL-15Rα Sushi") domain consisting of the amino acid sequence set forth in SEQ ID NO:5; An isolated interleukin-15 (IL-15) fusion protein complex.

8. 8. The isolated interleukin-15 (IL-15) fusion protein complex of claim 7, wherein the antibody is an antagonist PD-1 antibody selected from antibodies comprising heavy and light chain amino acid sequences set forth in SEQ ID NOs:111 and 112, heavy and light chain amino acid sequences set forth in SEQ ID NOs:113 and 114, heavy and light chain amino acid sequences set forth in SEQ ID NOs:115 and 116, heavy and light chain amino acid sequences set forth in SEQ ID NOs:117 and 118, and heavy and light chain amino acid sequences set forth in SEQ ID NOs:119 and 120.

9. 9. The isolated interleukin-15 (IL-15) fusion protein complex of any one of claims 7 to 8, wherein the IL-15 variant polypeptide is selected from the group consisting of polypeptides having the amino acid sequences set forth in SEQ ID NOs: 79 to 81.

10. 10. The isolated interleukin-15 (IL-15) fusion protein complex of any one of claims 7 to 9, wherein the complex is in a dimeric or monomeric form.

11. 11. The isolated interleukin-15 (IL-15) fusion protein complex of any one of claims 7 to 10, wherein the fusion protein complex comprises the amino acid sequence set forth in SEQ ID NO:79, SEQ ID NO:114, SEQ ID NO:141, and SEQ ID NO:

5.

12. A pharmaceutical composition comprising the isolated interleukin-15 (IL-15) fusion protein complex of any one of claims 1 to 11 in admixture with a pharmaceutically acceptable carrier.

13. 13. Use of the pharmaceutical composition of claim 12 in the preparation of a medicament for use in treating cancer or cancer metastasis in a subject, wherein the cancer is colorectal cancer.

14. 14. The use of claim 13, wherein the medicament further provides a second therapy capable of treating cancer or cancer metastasis, and which enhances tumor cell killing by effector cells.

Images