Long-acting interleukin-15 receptor agonist in combination with another pharmacologically active agent

Abstract

To provide a combination treatment, a composition and a kit comprising (a) a long-acting IL-15 receptor agonist and (b) one or more antibodies (mAb) targeting a tumor antigen, and related methods of preparation and use, e.g., in treatment of conditions responsive to therapy effective to provide sustained immune activation and anti-tumor activity.SOLUTION: In some embodiments, antibodies bind specifically to a tumor antigen selected from a phosphoprotein, a transmembrane protein, a glycoprotein, a glycolipid and a growth factor. In further embodiments of the methods, a subject has a solid cancer.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 USC 119(e) of U.S. Provisional Patent Application No. 62 / 758,344, filed November 9, 2018; U.S. Provisional Patent Application No. 62 / 789,924, filed January 8, 2019; U.S. Provisional Patent Application No. 62 / 818,003, filed March 13, 2019; U.S. Provisional Patent Application No. 62 / 825,437, filed March 28, 2019; U.S. Provisional Patent Application No. 62 / 843,036, filed May 3, 2019; U.S. Provisional Patent Application No. 62 / 848,372, filed May 15, 2019; and U.S. Provisional Patent Application No. 62 / 924,015, filed October 21, 2019, the disclosures of which are incorporated herein by reference.

[0002] The present disclosure is directed (among other things) to therapeutic combinations and compositions comprising a long-acting interleukin-15 ("IL-15") receptor agonist and another pharmacologically active agent, i.e., an antibody, such as a monoclonal antibody, and related methods of use, e.g., in the treatment of conditions responsive to effective therapies, e.g., to provide sustained immune activation and anti-tumor activity. [Background technology]

[0003] Interleukin-15 ("IL-15") is a pleiotropic cytokine first described by Grabstein et al. (Grabstein et al. (1994) Science 264:965-968). Human IL-15, secreted as a 162 amino acid precursor, contains a 29 amino acid leader sequence and a 19 amino acid prosequence; the mature protein is therefore 114 amino acids long. Member of the four α-helical bundle cytokine family, IL-15 binds to a heterotrimeric receptor in which a unique α subunit (IL-15Rα) confers receptor specificity to IL-15, and the β and γ subunits of this receptor share characteristics with one or more other cytokine receptors. Giri et al. (1995) EMBO J. 14:3654-3663.

[0004] As a cytokine, IL-15 exerts effects on both the innate and adaptive immune systems (DiSabitino et al. (2011) Cytokine Growth Factor Rev. 22:19-33). With respect to the innate immune system, which generally defends the host against foreign invaders, IL-15, among other properties, induces the development of and maintains the survival of natural killer cells ("NK cells") and natural killer T cells ("NK-T cells"). Consistent with their role in the innate immune system, NK cells do not specifically attack invading pathogens; rather, NK cells destroy compromised host cells (such as tumor cells or virus-infected cells). NK-T cells produce immunomodulatory cytokines, in particular interferon-γ, which leads to a general activation of the immune response.

[0005] With respect to the adaptive immune system, which defends the host against specific foreign invaders of interest after an initial encounter with a particular pathogen, IL-15 is required for the maintenance of immunoregulatory cytokine-producing helper T cells. Importantly, IL-15 also supports the long-term maintenance of "antigen-experienced" memory T cells, which have the capacity to rapidly replicate and thus generate a more rapid and potent immune response upon re-exposure to a particular foreign pathogen that invades the host.

[0006] Finally, despite its specific role within both the innate and adaptive immune systems, IL-15 exerts important and broad effects across both categories of the immune system. In particular, IL-15 inhibits or reduces apoptosis (or cell death) of several cell types associated with both categories of the immune system, including dendritic cells, neutrophils, eosinophils, mast cells, CD4+ T cells, and B cells. IL-15-mediated responses have also been shown to play a role in the development, function, and survival of CD8+ T cells and intestinal intraepithelial lymphocytes.

[0007] Because IL-15 stimulates the growth and maintenance of many cells in the immune system that can fight cells that appear foreign (or "non-self") to the host, the use of IL-15 in the treatment of cancer patients has been proposed (Steel et al. (2012) Trends Pharmacol. Sci. 33(1):35-41). For example, IL-15-based agonists have been proposed for the treatment of myeloma (Wong et al. (2013) OncoImmunology 2(11), e26442:1-3). In addition, IL-15 drug therapy has been proposed for the treatment of viral infections, such as HIV infection.

[0008] Long-acting IL-15 receptor agonists comprising at least one water soluble polymer (e.g., polyethylene glycol) moiety stably covalently attached to an IL-15 amino group have been described as offering improved properties and in vivo profiles, such as, for example, potent immunostimulatory effects, reduced systemic toxicity, improved stability and / or pharmacokinetics, improved therapeutic efficacy, among other improvements, compared to IL-15 and other IL-15 receptor agonists (PCT Application No. 2018 / 032817, incorporated herein by reference in its entirety).

[0009] The production and use of monoclonal antibodies (mAbs) to treat certain cancers, including both hematopoietic and solid tumors, is well established (Scott et al., 2012, Cancer Immunity, vol. 12, p. 14). The mechanism of action of tumor-associated mAbs includes one or more of the following: direct action on tumor cells, immune-mediated action, and vascular and stromal ablation. Tumor-associated antigens targeted by mAbs include cluster of differentiation (CD) antigens (e.g., CD20, CD30, CD33, CD52), glycoproteins (e.g., EpCAM, CEA, gpA33, mucins, etc.), glycolipids (e.g., gangliosides such as GD2, GD3, and GM2), vascular targets (e.g., VEGF, VEGFR), growth factors (e.g., ErbB1 / EGFR, ErbB2 / HER2, ErbB3, c-MET, IGF1R), and stromal extracellular matrix antigens (e.g., FAP, tenascin). Although several mAbs (e.g., rituximab, ofatumumab, ZEVALIN®, BEXXAR®, gemtuzumab ozogamicin, brentuximab vedotin, cetuximab, and panitumumab) have been approved by the USFDA for use in oncology research, certain types of cancer cells are more vulnerable to monoclonal antibody-based therapies than others. However, despite the aforementioned approaches, there remains a need for improved anti-cancer immunotherapies. The present disclosure addresses these and other needs by providing, inter alia, combination therapies comprising a long-acting IL-15 receptor agonist and at least one mAb directed against a tumor antigen, which are believed to be novel and not fully proposed by the art, a combination having several advantageous characteristics described in more detail below, as well as compositions and kits comprising such combinations, and related methods of preparation and use. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Grabstein et al. (1994) Science 264:965-968 [Non-Patent Document 2] Giri et al. (1995)EMBO J.14:3654-3663 [Non-Patent Document 3] DiSabitino et al. (2011) Cytokine Growth Factor Rev.22:19-33 [Non-Patent Document 4] Steel et al.(2012)Trends Pharmacol.Sci.33(1):35-41 [Non-Patent Document 5] Wong et al. (2013) OncoImmunology 2(11),e26442:1-3 [Non-Patent Document 6] Scott et al.,2012,Cancer Immunity,vol.12,p.1 Summary of the Invention [Means for solving the problem]

[0011] In a first aspect, provided herein is a method of treating a subject with cancer. Specifically, the method comprises administering to the subject a long-acting IL-15 receptor agonist and (b) a monoclonal antibody, e.g., a monoclonal antibody that targets, i.e., binds, to tumor cells, wherein steps (a) and (b) are performed simultaneously or sequentially, in any order. In some embodiments, the long-acting IL-15 receptor agonist has the following structure: [ka] Here, the structure is [CH3O(CH2CH2O) n (CH2) m C(O)NH] n’ - IL-15, wherein IL-15 is an interleukin-15 moiety, (n) is an integer between about 150 and about 3,000, (m) is an integer selected from 2, 3, 4, and 5, (n') is 1, and ~NH~ represents an amino group of the IL-15 moiety; or a pharma- ceutically acceptable salt form thereof. In some particular embodiments, (m) in formula (I) is 2 or 3. In preferred embodiments, (m) in formula (I) is 3. In some particular embodiments, (n) in formula (I) has an average value of about 227, or an average value of about 340, or an average value of about 454, or an average value of about 681, or an average value of about 909. In one or more embodiments, (n) has a value of about 909.

[0012] In some embodiments, the antibody is an antibody that specifically binds to a tumor antigen selected from a phosphorylated protein, a transmembrane protein, a glycoprotein, a glycolipid, and a growth factor.

[0013] In further embodiments of this method, the subject has a solid cancer, hi some embodiments, the solid cancer is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, colorectal cancer, gastric cancer, malignant melanoma, multiple myeloma, liver cancer, lymphoma, small cell lung cancer, non-small cell lung cancer, thyroid cancer, kidney cancer, bile duct cancer, brain cancer, cervical cancer, maxillary sinus cancer, bladder cancer, esophageal cancer, Hodgkin's disease, and adrenocortical carcinoma (including metastatic forms of any of the foregoing).

[0014] In some other embodiments, the subject has lymphoma or leukemia.

[0015] In some further embodiments, the subject has multiple myeloma.

[0016] In some embodiments of this method, step (a) is performed before step (b). In other embodiments, step (b) is performed before step (a). In still further embodiments, steps (a) and (b) are performed simultaneously or substantially simultaneously. The method further includes one or more additional dosing cycles of one or both of the long-acting IL-15 receptor agonist and the monoclonal antibody.

[0017] In some embodiments, the administering is effective to stimulate NK activation and proliferation to a greater extent than observed when the long-acting IL-15 receptor agonist is administered as a single agent, as measured in a suitable animal model. In some additional embodiments, the administering is effective to support CD8 T cell survival and memory formation to a greater extent than observed when the long-acting IL-15 receptor agonist is administered as a single agent, as measured in a suitable animal model. In some further embodiments, the administering is effective to cause a reduction in the number of tumor cells, as measured in a suitable animal model, that is greater than observed when the long-acting receptor agonist is administered as a single agent (i.e., as monotherapy) and that is greater than observed when the monoclonal antibody is administered as a single agent (examples of which are provided herein). In some related embodiments, the administering causes a 3-fold or greater reduction, more preferably a 5-fold or greater reduction, more preferably a 7-fold or greater reduction in the number of tumor cells in the subject, as compared to administration of an equivalent dose of the long-acting receptor agonist as a single agent. In some further embodiments, the administration is effective to induce proliferation of NK cells (i.e., increase the number of NK cells) and activate their tumor cell killing capabilities, for example, in bone marrow tissue.

[0018] In some embodiments, the long-acting IL-15 receptor agonist is administered subcutaneously. In additional embodiments, the monoclonal antibody is administered intravenously.

[0019] In one or more embodiments, the antibody is a targeted monoclonal antibody that utilizes an antibody-dependent cellular cytotoxicity (ADCC, also known as antibody-dependent cell-mediated cytotoxicity) mechanism of action.

[0020] In some embodiments, the monoclonal antibody is selected from an anti-CD19 antibody, an anti-CD20 antibody, and an anti-CD38 antibody. In further embodiments, the monoclonal antibody that specifically binds to a glycoprotein is selected from an anti-SLAMF7 antibody, an anti-EpCAM antibody, an anti-gpA3 antibody3, and an anti-FBP antibody. In additional embodiments, the monoclonal antibody that specifically binds to a growth factor is selected from an anti-VEGF antibody, an anti-VEGFR antibody, and an anti-EGFR antibody. In yet some additional embodiments, the antibody is an anti-BCMA antibody. In some additional embodiments, the antibody is an antibody that targets multiple myeloma.

[0021] In a second aspect, provided herein is a therapeutic combination for use in treating a condition such as cancer, comprising a long-acting IL-15 receptor agonist and a monoclonal antibody, such as a monoclonal antibody that targets or binds to tumor cells (including but not limited to the monoclonal antibodies described herein).

[0022] In some related and more specific embodiments, the long-acting IL-15 receptor agonist has the structure: [ka] wherein IL-15 is an interleukin-15 moiety, (n) is an integer between about 150 and about 3,000, (m) is an integer selected from 2, 3, 4, and 5, (n') is 1, and ~NH~ represents an amino group of the IL-15 moiety; or a pharma- ceutically acceptable salt form thereof. In yet some additional embodiments, the monoclonal antibody is an antibody that specifically binds to a tumor antigen selected from a phosphorylated protein, a transmembrane protein, a glycoprotein, a glycolipid, and a growth factor. In some embodiments, (m) in formula (I) is 2 or 3. In some particular embodiments, (m) in formula (I) is 3. In some further embodiments, (n) in formula (I) has a value of about 227, or about 340, or about 454, or about 681, or about 909. In one or more embodiments, (n) has a value of about 909.

[0023] In a third aspect, a kit is provided herein. In an embodiment, the kit comprises a therapeutic combination of a long-acting IL-15 receptor agonist and a monoclonal antibody described herein, with instructions for use, wherein the long-acting IL-15 receptor agonist and the monoclonal antibody are each contained in one or more individual unit dosage forms. The kit and therapeutic combination are useful, for example, for treating a subject with cancer.

[0024] Additional aspects and embodiments are set forth in the following description and claims. The embodiments described herein are intended to apply equally to each of the aspects described herein and, where applicable, should be considered both alone and in combination, unless otherwise indicated. In certain embodiments, for example, the following are provided: (Item 1) 1. A method of treating a subject having cancer, comprising: (a) a long-acting IL-15 receptor agonist having the following structure: [ka] wherein IL-15 is an interleukin-15 moiety, n is an integer from about 150 to about 3,000, m is an integer selected from 2, 3, 4, and 5, n' is 1, and -NH- represents an amino group of the IL-15 moiety; and (b) a monoclonal antibody that specifically binds to a tumor antigen selected from a phosphorylated protein, a transmembrane protein, a glycoprotein, a glycolipid, and a growth factor, the monoclonal antibody having antibody-dependent cellular cytotoxicity (ADCC) as a mechanism of action; (wherein steps (a) and (b) are performed simultaneously or sequentially, in any order) to said subject. (Item 2) 2. The method of claim 1, wherein the long-acting IL-15 receptor agonist is a pharma- ceutically acceptable salt. (Item 3) The method according to item 1 or 2, wherein (m) in formula (I) is 2 or 3. (Item 4) The method according to any one of items 1 to 3, wherein (m) in formula (I) is 3. (Item 5) 5. The method according to any one of items 1 to 4, wherein (n) in formula (I) has a value of about 909. (Item 6) 6. The method according to any one of items 1 to 5, wherein the cancer is a solid cancer. (Item 7) 7. The method of claim 6, wherein the solid cancer is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, colorectal cancer, gastric cancer, malignant melanoma, liver cancer, small cell lung cancer, non-small cell lung cancer, thyroid cancer, kidney cancer, cancer of the bile duct, brain cancer, cervical cancer, maxillary sinus cancer, bladder cancer, esophageal cancer, Hodgkin's disease, and adrenocortical carcinoma, including metastatic forms of any of the foregoing. (Item 8) 6. The method according to any one of items 1 to 5, wherein the cancer is a hematopoietic tumor. (Item 9) 9. The method of claim 8, wherein the hematopoietic tumor is selected from the group consisting of multiple myeloma, non-Hodgkin's lymphoma, leukemia, and lymphoma. (Item 10) 10. The method according to any one of items 1 to 9, wherein step (a) is carried out before step (b). (Item 11) 10. The method according to any one of items 1 to 9, wherein step (b) is carried out before step (a). (Item 12) 10. The method according to any one of items 1 to 9, wherein step (a) and step (b) are carried out simultaneously or substantially simultaneously. (Item 13) 13. The method of any one of items 1 to 12, wherein the administering is effective to stimulate NK activation, as measured in a suitable animal model, to a greater extent than is observed when the long-acting IL-15 receptor agonist is administered as a single agent. (Item 14) 14. The method of any one of items 1 to 13, wherein the administering is effective to stimulate NK proliferation, as measured in a suitable animal model, to a greater extent than is observed when the long-acting IL-15 receptor agonist is administered as a single agent. (Item 15) 15. The method of any one of items 1 to 14, wherein the administering is effective to support CD8+ T cell survival and memory formation to a greater extent than is observed when the long-acting IL-15 receptor agonist is administered as a single agent, as measured in a suitable animal model. (Item 16) 16. The method according to any one of items 1 to 15, wherein the long-acting IL-15 receptor agonist is administered subcutaneously. (Item 17) 17. The method according to any one of items 1 to 16, wherein the monoclonal antibody is administered intravenously. (Item 18) 18. The method according to any one of items 1 to 17, wherein the monoclonal antibody is selected from an anti-CD19 antibody, an anti-CD20 antibody, and an anti-CD38 antibody. (Item 19) 18. The method according to any one of items 1 to 17, wherein the monoclonal antibody that specifically binds to the glycoprotein is selected from an anti-SLAMF7 antibody, an anti-EpCAM antibody, an anti-gpA3 antibody 3, and an anti-FBP antibody. (Item 20) 18. The method according to any one of items 1 to 17, wherein the monoclonal antibody that specifically binds to the growth factor is selected from an anti-VEGF antibody, an anti-VEGFR antibody, and an anti-EGFR antibody. (Item 21) 18. The method according to any one of items 1 to 17, wherein the monoclonal antibody is an IgG antibody. (Item 22) 18. The method according to any one of items 1 to 17, wherein the monoclonal antibody is selected from the group consisting of daratumumab, rituximab, cetuximab, and trastuzumab. (Item 23) (a) a long-acting IL-15 receptor agonist having the following structure: [ka] wherein IL-15 is an interleukin-15 moiety, n is an integer from about 150 to about 3,000, m is an integer selected from 2, 3, 4, and 5, n' is 1, and -NH- represents an amino group of the IL-15 moiety; and (b) A monoclonal antibody that specifically binds to a tumor antigen selected from a phosphorylated protein, a transmembrane protein, a glycoprotein, a glycolipid, and a growth factor, the monoclonal antibody having antibody-dependent cellular cytotoxicity (ADCC) as a mechanism of action. 23. A therapeutic combination for use in treating cancer comprising: (Item 24) 24. The therapeutic combination of item 23, wherein the long-acting receptor agonist is a pharma- ceutically acceptable salt. (Item 25) The therapeutic combination of item 23 or 24, wherein the long-acting IL-15 receptor agonist has a structure as described in any one of items 3, 4, or 5. (Item 26) 26. A kit comprising the therapeutic combination according to any one of items 23 to 25, together with instructions for use, wherein the long-acting IL-15 receptor agonist and the monoclonal antibody are each contained within one or more individual unit dosage forms. (Item 27) Use of long-acting receptor agonists to enhance NK cell-mediated antibody-dependent cellular cytotoxicity when administered with an anti-tumor antibody. [Brief description of the drawings]

[0025] [Figure 1] FIG. 1 provides the amino acid sequence of an exemplary recombinant human IL-15 from E. coli (SEQ ID NO:1), a single non-glycosylated polypeptide chain comprising 115 amino acids with a molecular weight of 12.9 kDa. [Diagram 2] FIG. 2 is a graph showing percent survival of mice inoculated with Daudi B lymphoma cells and treated with: (i) isotype control, (ii) 40 mg / kg rituximab, (iii) 0.3 mg / kg long-acting IL-15 receptor agonist, or (iv) a combination of 40 mg / kg rituximab and 0.3 mg / kg long-acting IL-15 receptor agonist, as detailed in Example 1. [Diagram 3] FIG. 3 is a graph showing the number of NK cells in bone marrow tissue of mice inoculated with Daudi B cells as described in Example 2 after treatment with: (i) daratumumab (0.5 mg / kg IP, 14 days after inoculation of Daudi cells) and two doses of a long-acting IL-15 receptor agonist, i.e., mono(methoxyPEG-N-butanamide) interleukin-15 (0.3 mg / kg SC, one dose each at 14 and 21 days after inoculation); (ii) single agent daratumumab (0.5 mg / kg IP, 14 days after inoculation of Daudi cells); (iii) single agent mono(methoxyPEG-N-butanamide) interleukin-15 (0.3 mg / kg SC, 14 and 21 days after inoculation), as compared to (iv) an untreated control group. [Figure 4]FIG. 4 is a graph showing the number of Daudi B cells in bone marrow tissue of mice inoculated with Daudi B cells as described in Example 2 after treatment with: (i) daratumumab (0.5 mg / kg IP, 14 days after inoculation of Daudi cells) and two doses of mono(methoxyPEG-N-butanamide) interleukin-15 (0.3 mg / kg SC, one dose each at 14 and 21 days after inoculation), (ii) single agent daratumumab (0.5 mg / kg IP, 14 days after inoculation of Daudi cells), (iii) single agent mono(methoxyPEG-N-butanamide) interleukin-15 (0.3 mg / kg SC, 14 and 21 days after inoculation), as compared to (iv) an untreated control group. [Diagram 5] FIG. 5 is a graph showing percent survival of mice inoculated with Daudi B lymphoma cells as detailed in Example 3 and subsequently treated with: (i) isotype control, (ii) daratumumab at 0.5 mg / kg IP, (iii) long-acting IL-15 receptor agonist, mono(methoxyPEG-N-butanamide) interleukin-15 at 0.3 mg / kg SC, or (iv) a combination of long-acting IL-15 receptor agonist at 0.3 mg / kg and daratumumab at 0.5 mg / kg, SC. [Figure 6] FIG. 6 is a graph showing granzyme B induction in bone marrow NK cells after treatment of mice inoculated with Daudi B cells with: (i) daratumumab (0.5 mg / kg IP, 14 days after inoculation of Daudi cells) and two doses of mono(methoxyPEG-N-butanamide) interleukin-15 (0.3 mg / kg SC, one dose each at 14 and 21 days after inoculation), (ii) single agent daratumumab (0.5 mg / kg IP, 14 days after inoculation of Daudi cells), (iii) single agent mono(methoxyPEG-N-butanamide) interleukin-15 (0.3 mg / kg SC, 14 and 21 days after inoculation), compared to (iv) an untreated control group. [Figure 7A-7B]7A and 7B are graphs showing the percentage of NK cells in the bone marrow compartment expressing NKG2A (FIG. 7A) or NKG2D (FIG. 7B) on the cell surface after treatment of mice inoculated with Daudi B cells with: (i) daratumumab (0.5 mg / kg IP, 14 days after inoculation of Daudi cells) and two doses of conjugate 1 (0.3 mg / kg or 0.03 mg / kg SC, one dose each at 14 and 21 days after inoculation), (ii) single agent daratumumab (0.5 mg / kg IP, 14 days after inoculation of Daudi cells), (iii) single agent compound 1 (0.3 mg / kg SC, 14 and 21 days after inoculation), when compared to (iv) an untreated control group or (v) an isotype control (0.5 mg / kg). [Figure 8A-8D] 8A-8D are histograms showing % proliferation of human NK cells in human peripheral blood mononuclear cell (PBMC) preparations using (i) Compound 1 alone ( FIG. 8C ), (ii) hIgG alone ( FIG. 8B ), (iii) Compound 1 + hIgG ( FIG. 8D ), or (iv) an untreated control ( FIG. 8A ), as described in detail in Example 5. [Figure 9A] FIG. 9A is a graph showing the median fluorescence intensity (MFI) signal for CD69 detecting antibodies on the surface of CD56+ NK cells in PBMC preparations cultured overnight with (i) hIgG-coated plates, (ii) Compound 1 (1 μg / ml), (iii) hIgG-coated plates + Compound 1 (1 μg / ml), or (ii) a control indicating NK cell activation, as described in detail in Example 6. [Figure 9B] FIG. 9B is a graph showing the median fluorescence intensity (MFI) signal for CD107a detecting the antibody on the surface of CD56+ NK cells in PBMC preparations cultured overnight with (i) hIgG-coated plates, (ii) Compound 1 (1 μg / ml), (iii) hIgG-coated plates + Compound 1 (1 μg / ml), or (ii) a control indicating NK cell activation, as detailed in Example 6. [Figure 9C]9C is a graph showing granzyme B induction in human PBMCs following exposure to: (i) hIgG-coated plates, (ii) Compound 1 (1 μg / ml), hIgG-coated plates + Compound 1 (1 μg / ml), or (ii) control, as detailed in Example 6. The graph shows the concentration of secreted granzyme B (pg / ml) for each of the four treatments. [Figure 10A-10B] Figure 10A is a graph showing the percentage of pSTAT5 positivity in KHYG-1 cells after incubation with IL-15 or a long-acting IL-15 receptor agonist (mono(methoxyPEG-N-butanamide) interleukin-15), as described in detail in Example 7. Figure 10B is a graph of the maximum proliferation (%) of KHYG-1 cells after incubation with IL-15 or a long-acting IL-15 receptor agonist (mono(methoxyPEG-N-butanamide) interleukin-15), as described in detail in Example 7. [Figure 11] FIG. 11 is a graph showing the maximal proliferation (%) of CD56+ human NK cells following incubation with IL-15 or a long-acting IL-15 receptor agonist (mono(methoxyPEG-N-butanamide) interleukin-15), as described in detail in Example 8. [Figure 12] FIG. 12 is a graph showing 7-AAD+ target cells (%) for human multiple myeloma cells pre-coated with daratumumab (+) after stimulation with a long-acting IL-15 receptor agonist (mono(methoxyPEG-N-butanamide) interleukin-15) (+) or left untreated (−), as described in detail in Example 9. [Figure 13A]FIG. 13A is a graph showing the percentage of NK cells in the bone marrow compartment expressing CD16 (FIG. 13A) on the cell surface (% of cells expressing CD16 among all NK cells) after treatment of mice inoculated with Daudi B cells, as detailed in Example 10, with: (i) daratumumab (0.5 mg / kg IP, 14 days after inoculation of Daudi cells) and two doses of conjugate 1 (0.3 mg / kg or 0.03 mg / kg SC, one dose each at 14 and 21 days after inoculation), (ii) single agent daratumumab (0.5 mg / kg IP, 14 days after inoculation of Daudi cells), (iii) single agent compound 1 (0.3 mg / kg SC, 14 and 21 days after inoculation), when compared to (iv) an untreated control group or (v) an isotype control (0.5 mg / kg). [Figure 13B] FIG. 13B is a graph showing CD16 expression changes (increase in Compound 1 treatment group) on a cell-by-cell basis for CD16+ bone marrow NK cells as measured by median fluorescence intensity (MFI) signal following treatment of mice inoculated with Daudi B cells with: (i) daratumumab (0.5 mg / kg IP, 14 days after inoculation of Daudi cells) and two doses of conjugate 1 (0.3 mg / kg or 0.03 mg / kg SC, one dose each at 14 and 21 days after inoculation), (ii) single agent daratumumab (0.5 mg / kg IP, 14 days after inoculation of Daudi cells), (iii) single agent Compound 1 (0.3 mg / kg SC, 14 and 21 days after inoculation), when compared to (iv) untreated control group or (v) isotype control (0.5 mg / kg). [Figure 14]FIG. 14 is a graph showing granzyme B expression in individual bone marrow NK cells as measured by median fluorescence intensity (MFI) signal after treatment of mice inoculated with Daudi B cells, as detailed in Example 11, with: (i) daratumumab (0.5 mg / kg IP, 14 days after inoculation of Daudi cells) and two doses of Compound 1 (0.3 mg / kg or 0.03 mg / kg SC, one dose each at 14 and 21 days after inoculation; (ii) single agent daratumumab (0.5 mg / kg IP, 14 days after inoculation of Daudi cells); (iii) single agent Compound 1 (0.3 mg / kg SC, 14 and 21 days after inoculation), when compared to (iv) an untreated control group or (v) an isotype control (0.5 mg / kg). [Figure 15A-15B] Figures 15A and 15B are graphs showing 7-AAD+ target cells (%) for human multiple myeloma cells pre-coated with daratumumab (+) (Figure 15A) or rituximab (+) (Figure 15B) after stimulation with a long-acting IL-15 receptor agonist (mono(methoxyPEG-N-butanamide) interleukin-15) (+) or left untreated (-), as described in detail in Example 12. [Figure 16] FIG. 16 is a graph showing the percent survival of SCID or SCID beige mice inoculated with Daudi B cell lymphoma cells as detailed in Example 13 and subsequently treated with: (i) untreated control for SCID (□); (ii) untreated control for SCID beige mice (○); (iii) a combination of 0.3 mg / kg of a long-acting IL-15 receptor agonist and 0.5 mg / kg SC daratumumab (■) for SCID mice; and (iv) a combination of 0.3 mg / kg of a long-acting IL-15 receptor agonist and 0.5 mg / kg SC daratumumab (●) for SCID beige mice. [Figure 17A]FIG. 17A is a graph showing the number of Daudi cells in bone marrow tissue after treatment of mice inoculated with Daudi B cells, as described in detail in Example 14, with: (i) high-dose daratumumab (5 mg / kg IP, 14 days after inoculation of Daudi cells) and two low doses of a long-acting IL-15 receptor agonist, i.e., mono(methoxyPEG-N-butanamide) interleukin-15 (Compound 1) (0.03 mg / kg IV, one administration at 14 and 21 days after inoculation, respectively) (◆), (ii) single-agent high-dose daratumumab (5 mg / kg IP, 14 days after inoculation of Daudi cells) (▼), (iii) single-agent low-dose mono(methoxyPEG-N-butanamide) interleukin-15 (Compound 1) (0.03 mg / kg IV, one administration at 14 and 21 days after inoculation, respectively) (◆). IV, 14 and 21 days after inoculation) (▲), when compared with (iv) the untreated control group (●). [Figure 17B] FIG. 17B is a graph showing the number of Daudi cells in bone marrow tissue after treatment of mice inoculated with Daudi B cells, as described in detail in Example 14, with: (i) low-dose daratumumab (0.05 mg / kg IP, 14 days after inoculation of Daudi cells) and two high doses of a long-acting IL-15 receptor agonist, i.e., mono(methoxyPEG-N-butanamide) interleukin-15 (Compound 1) (0.6 mg / kg IV, one administration at 14 and 21 days after inoculation) (◆); (ii) single-agent low-dose daratumumab (0.05 mg / kg IP, 14 days after inoculation of Daudi cells) (▼); (iii) single-agent high-dose mono(methoxyPEG-N-butanamide) interleukin-15 (Compound 1) (0.6 mg / kg IV, one administration at 14 and 21 days after inoculation) (◆); IV, 14 and 21 days after inoculation) (▲), when compared with (iv) the untreated control group (●). [Figures 18A-18D]Figure 18A is a graph showing the intratumoral percentage of NK cells compared to vehicle control on days 3 or 5 after treatment with cetuximab (20 mg / kg, IP) and conjugate 1 (0.3 mg / kg IV) in mice bearing subcutaneous HCT-116 colon-colorectal cell tumors, as described in detail in Example 15. Figure 18B is a graph showing the intratumoral NK cell numbers compared to vehicle control on days 3 or 5 after treatment with cetuximab (20 mg / kg, IP) and conjugate 1 (0.3 mg / kg IV) in mice bearing subcutaneous HCT-116 tumors, as described in detail in Example 15. Figure 18C is a graph showing NK cell proliferation as indicated by Ki67 positivity (%) in blood or tumor cells compared to vehicle control after treatment of mice bearing subcutaneous HCT-116 tumors with cetuximab (20 mg / kg, IP) and conjugate 1 (0.3 mg / kg IV), as described in detail in Example 15. Figure 18D is a graph showing Granzyme B expression (GzmB+ (%)) in blood or tumor cells compared to vehicle control after treatment of mice bearing subcutaneous HCT-116 tumors with cetuximab (20 mg / kg, IP) and conjugate 1 (0.3 mg / kg IV), as described in detail in Example 15. [Fig. 18E-18F] Figure 18E is a graph showing CD16 cell surface expression on NK cells as measured by median fluorescence intensity (MFI) signal, compared to vehicle control, following treatment of mice bearing subcutaneous HCT-116 tumors with cetuximab (20 mg / kg, IP) and conjugate 1 (0.3 mg / kg IV), as described in detail in Example 15. Figure 18F is a graph showing the intratumoral percentage of NK cells expressing NKG2D on their cell surface (NKG2D+ (%)), compared to vehicle control, following treatment of mice bearing subcutaneous HCT-116 tumors with cetuximab (20 mg / kg, IP) and conjugate 1 (0.3 mg / kg IV), as described in detail in Example 15. [Figure 19A-19B]Figure 19A is a graph showing the intratumoral percentage of NK cells compared to vehicle control on days 3 or 5 after treatment with cetuximab (20 mg / kg, IP) and conjugate 1 (0.3 mg / kg IV) in mice bearing subcutaneous FaDu squamous cell carcinoma tumors, as described in detail in Example 16. Figure 19B is a graph showing the intratumoral NK cell numbers compared to vehicle control on days 3 or 5 after treatment with cetuximab (20 mg / kg, IP) and conjugate 1 (0.3 mg / kg IV) in mice bearing subcutaneous FaDu tumors, as described in detail in Example 16. [Fig. 19C-19F] Figure 19C is a graph showing NK cell proliferation as indicated by Ki67 positivity (%) in blood or tumor cells compared to vehicle control after treatment of mice receiving subcutaneous FaDu tumors with cetuximab (20 mg / kg, IP) and conjugate 1 (0.3 mg / kg IV), as described in detail in Example 16. Figure 19D is a graph showing Granzyme B expression (GzmB+ (%)) in blood or tumor cells compared to vehicle control after treatment of mice receiving subcutaneous FaDu tumors with cetuximab (20 mg / kg, IP) and conjugate 1 (0.3 mg / kg IV), as described in detail in Example 16. Figure 19E is a graph showing CD16 cell surface expression on NK cells as measured by median fluorescence intensity (MFI) signal, compared to vehicle control, following treatment of mice bearing subcutaneous FaDu tumors with cetuximab (20 mg / kg, IP) and conjugate 1 (0.3 mg / kg IV), as described in detail in Example 16. Figure 19F is a graph showing the intratumoral percentage of NK cells expressing NKG2D on their cell surface (NKG2D+ (%)), compared to vehicle control, following treatment of mice bearing subcutaneous FaDu tumors with cetuximab (20 mg / kg, IP) and conjugate 1 (0.3 mg / kg IV), as described in detail in Example 16. [Figure 20]FIG. 20 is a graph showing relative tumor volumes for mice inoculated with H1975 lung cancer cells, as detailed in Example 17, compared to vehicle control (●) over days 0-27 following treatment with: (i) cetuximab (0.25 mg / kg, IP) administered on days 9, 12, and 16 following tumor inoculation, and Compound 1 (0.3 mg / kg, IV) administered on days 9, 16, and 23 following inoculation (△); (ii) single agent cetuximab (0.25 mg / kg, IP, BIW×3) (▲); (iii) single agent Compound 1 (0.3 mg / kg, IV, q7d×3) (▼). [Figure 21A] FIG. 21A is a graph showing relative tumor volumes 0-21 days after treatment with the following, compared to vehicle control (●), in mice inoculated with HT-29 colorectal carcinoma cells, as described in detail in Example 18: (i) cetuximab (40 mg / kg, IP, BIW×3) and Compound 1 (0.3 mg / kg, IV, q7d×3) (△), (ii) single-agent cetuximab (40 mg / kg, IP, BIW×3) (▲), (iii) single-agent Compound 1 (0.3 mg / kg, IV, q7d×3) (▼). [Figure 21B] FIG. 21B is a graph showing tumor growth delay (TVQT) from 0 to 23 days after initiation of treatment (mean tumor volume of approximately 150 mm3) as percent survival for mice inoculated with HT-29 colorectal carcinoma cells and treated with: (i) cetuximab (40 mg / kg, IP, BIW x 3) and Compound 1 (0.3 mg / kg, IV, q7d x 3) (▼), (ii) single-agent cetuximab (40 mg / kg, IP, BIW x 3) (■), (iii) single-agent Compound 1 (0.3 mg / kg, IV, q7d x 3) (▲), as detailed in Example 18. [Figure 22A]FIG. 22A is a graph showing relative tumor volumes 0-19 days after treatment with: (i) cetuximab (40 mg / kg, IP, BIW x 3) and Compound 1 (0.3 mg / kg, IV, q7d x 3) (▲), (ii) single-agent cetuximab (40 mg / kg, IP, BIW x 3) (▲), (iii) single-agent Compound 1 (0.3 mg / kg, IV, q7d x 3) (▼), compared to PBS vehicle control (OP, BIW x 3) (●), as described in detail in Example 19. [Figure 22B] FIG. 22B is a graph showing tumor growth delay (TVQT) as percent survival compared to PBS vehicle control (IP, BIW×3) (●) for mice inoculated with HCT-116 colorectal carcinoma cells and treated with: (i) cetuximab (40 mg / kg, IP, BIW×3) and Compound 1 (0.3 mg / kg, IV, q7d×3) (△), (ii) single-agent cetuximab (40 mg / kg, IP, BIW×3) (▲), (iii) single-agent Compound 1 (0.3 mg / kg, IV, q7d×3) (▼), as detailed in Example 19. [Figure 23A] FIG. 23A is a graph showing CD45-EpCAM+7-AAD+ target cells (%) for HCT-116 colorectal carcinoma cells pre-coated with cetuximab (+) alone or after stimulation with Compound 1 (+), isotype control (+), or untreated (-), as described in detail in Example 20. [Figure 23B] FIG. 23B is a graph showing CD45-7-AAD+ target cells (%) for FaDu squamous cell carcinoma cells (HNSCC) pre-coated with cetuximab (+) alone or after stimulation with Compound 1 (+), isotype control (+), or untreated (-), as described in detail in Example 20. [Figure 24]FIG. 24 is a graph showing relative tumor volumes for mice inoculated with SKOV-3 ovarian adenocarcinoma cells, as detailed in Example 21, from 0 to 35 days following treatment with the following, compared to vehicle control (●): (i) trastuzumab (13.5 mg / kg, IV, BIW×3) and Compound 1 (0.3 mg / kg, IV, q7d×3) (△), (ii) single-agent trastuzumab (13.5 mg / kg, IV, BIW×3) (▲), (iii) single-agent Compound 1 (0.3 mg / kg, IV, q7d×3) (▼). [Diagram 25] FIG. 25 is a graph showing relative tumor volumes for mice inoculated with NCI-N87 gastric cancer cells, as detailed in Example 22, from 0 to 35 days following treatment with the following, compared to vehicle control (●): (i) trastuzumab (3 / 1 / 1 mg / kg, IV, q7d x 3) and Compound 1 (0.3 mg / kg, IV, q7d x 3) (△), (ii) single-agent trastuzumab (3 / 1 / 1 mg / kg, IV, q7d x 3) (▲), (iii) single-agent Compound 1 (0.3 mg / kg, IV, q7d x 3) (▼). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Before describing one or more aspects or embodiments of the present disclosure in detail, it should be noted that the present disclosure is not intended to be limited to particular synthesis techniques, IL-15 moieties, or the like, as these may vary as can be understood by those of ordinary skill in the art to which the present disclosure applies.

[0027] In describing and claiming particular features of the present disclosure, the following terminology will be used in accordance with the definitions set out below unless otherwise indicated.

[0028] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0029] "Water-soluble non-peptidic polymer" refers to a polymer that is at least 35% (by weight) soluble in water at room temperature, preferably more than 70% (by weight), more preferably more than 95% (by weight). Typically, an unfiltered aqueous solution of a "water-soluble" polymer transmits at least 75%, more preferably at least 95% of the amount of light that the same solution transmits after filtering. However, it is most preferred that a water-soluble polymer is at least 95% (by weight) soluble in water or completely dissolved in water. With respect to being "non-peptidic", a polymer is non-peptidic when it has less than 35% (by weight) of amino acid residues.

[0030] "PEG" or "polyethylene glycol," as used herein, is intended to encompass any water-soluble poly(ethylene oxide). Unless otherwise indicated, a "PEG polymer" or polyethylene glycol is one in which substantially all (preferably all) of the monomeric subunits are ethylene oxide subunits, but which polymer may contain additional end capping moieties or functional groups, e.g., for conjugation. PEG polymers for use in the present disclosure may have one of the two following structures, depending on whether the terminal oxygen has been replaced, e.g., during synthetic transformations: "-(CH2CHO) n -" or "-(CH2CH2O) n-1 CH2CH2-". As noted above, for PEG polymers, the variable (n) can vary from about 3 to 4000, and the overall PEG end groups and structure can also vary. However, exemplary or preferred PEG-containing molecules can include one or more specific PEG structures and / or linkers, and / or molecular weight ranges.

[0031] Molecular weight in the context of water-soluble polymers such as PEG can be expressed as number average molecular weight or weight average molecular weight. Unless otherwise indicated, all references to molecular weight herein refer to weight average molecular weight. Both number average and weight average molecular weight determinations can be measured using gel permeation chromatography or other liquid chromatography techniques (e.g., gel filtration chromatography). The most commonly used are gel permeation chromatography and gel filtration chromatography. Other methods for determining molecular weight include end group analysis or measurement of colligative properties (e.g., freezing point depression, boiling point elevation, or osmotic pressure) to determine number average molecular weight, or the use of light scattering techniques, ultracentrifugation, MALDI TOF, or viscometers to determine weight average molecular weight. PEG polymers are typically polydisperse (i.e., the number average molecular weight and the weight average molecular weight of the polymer are not equal), and preferably have low polydispersity values ​​of less than about 1.2, more preferably less than about 1.15, even more preferably less than about 1.10, even more preferably less than about 1.05, and most preferably less than about 1.03.

[0032] A "physiologically cleavable" or "hydrolyzable" or "degradable" bond is a relatively unstable bond that reacts with water (i.e., is hydrolyzed) under physiological conditions. The tendency of a bond to be hydrolyzed in water depends not only on the general type of bond connecting two atoms in a given molecule, but may also depend on the substituents attached to those atoms. Suitable hydrolytically unstable or weak bonds can include, but are not limited to, carboxylate esters, phosphate esters, anhydrides, acetals, ketals, acyloxyalkyl ethers, imines, orthoesters, peptides, oligonucleotides, thioesters, and carbonates.

[0033] A "releasable" covalent bond, in the context of polyethylene glycol that can be covalently attached to an active moiety, such as interleukin-15, is one that releases or detaches the polyethylene glycol polymer from the active moiety under physiological conditions, e.g., by any suitable mechanism, at a rate that is clinically useful, including, for example, but not limited to, hydrolyzable bonds and enzymatically degradable bonds.

[0034] "Enzymatically degradable bond" means a bond that is subject to degradation by one or more enzymes.

[0035] A "stable" linkage or bond refers to a chemical bond that is substantially stable in water, i.e., does not undergo hydrolysis under physiological conditions to any appreciable extent over an extended period of time. Examples of hydrolytically stable linkages generally include, but are not limited to: carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, amines, and the like. Generally, a stable linkage is one that exhibits a rate of hydrolysis of less than about 1-2% per day under physiological conditions. Hydrolysis rates for representative chemical bonds can be found in most standard chemistry textbooks.

[0036] "Substantially" or "essentially" means nearly entirely or completely (eg, 95% or more of a given amount).

[0037] Similarly, as used herein, "about" or "approximately" means within ±5% of a given amount.

[0038] "Optional" or "optionally" means that the subsequently described situation may occur, but does not necessarily have to occur, and thus the description includes cases where the situation occurs and cases where it does not occur.

[0039] A "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to a component that can be included in the compositions described herein and that cannot cause significant adverse toxicological effects in a subject.

[0040] The expressions "pharmacologically effective amount" and "therapeutically effective amount" and "physiologically effective amount" are used interchangeably herein and refer to the amount of a long-acting IL-15 receptor agonist provided herein or the amount of a monoclonal antibody provided herein required to provide a desired level of the substance in the bloodstream or target tissue to elicit a desired biological or drug response. For example, such a response may destroy targeted cancer cells in a subject and / or delay or arrest the progression of cancer and / or increase the number of NK cells in the patient. The term also applies to a dose that induces a particular response in a target cell. The exact amount will depend on numerous factors, such as, for example, the particular condition being treated, the intended patient population, individual patient considerations, the components and physical properties of the therapeutic composition and the particular combination being administered, and can be readily determined by one of skill in the art.

[0041] The term "IL-15 moiety" as used herein refers to a peptide or protein moiety that has human IL-15 activity. Moreover, the term "IL-15 moiety" encompasses both pre-conjugated and post-conjugated IL-15 moiety residues. As explained in more detail below, one of skill in the art can determine whether any given moiety has IL-15 activity. A protein that includes an amino acid sequence corresponding to any one of SEQ ID NOs: 1-3 is an IL-15 moiety, as well as any protein or polypeptide substantially homologous thereto. As used herein, the term "IL-15 moiety" includes peptides and proteins that have been modified either deliberately, for example, by site-directed mutagenesis, or accidentally via mutation. These terms also include analogs with 1-6 additional glycosylation sites, analogs with at least one additional amino acid at the carboxy terminus of the peptide or protein (wherein the additional amino acid includes at least one glycosylation site), and analogs with an amino acid sequence that includes at least one glycosylation site. The term includes naturally, recombinantly, and synthetically produced IL-15 moieties. The IL-15 moiety may be produced by any suitable method known in the art. In embodiments, the IL-15 moiety is recombinantly produced in Escherichia coli (E. coli) or Chinese Hamster Ovary (CHO) expression systems. References to long-acting IL-15 receptor agonists described herein are intended to include pharmaceutically acceptable salt forms thereof.

[0042] "Antibody" as used herein is intended to be broad and includes glycoproteins belonging to the immunoglobulin (Ig) superfamily. Antibodies are intended to include polyclonal antibodies, monoclonal antibodies (e.g., murine, human, humanized, humanized, and chimeric), antibody fragments, and single chain antibodies that specifically bind to an antigen (e.g., a tumor antigen). The fragment, the antigen-binding (Fab) region, contains a constant domain and at least one variable domain from each of the heavy and light chains. The antibodies described herein have at least antibody-dependent cellular cytotoxicity (ADCC) as a mechanism of action.

[0043] The term "monoclonal antibody" (mAb) as used herein refers to a non-naturally occurring antibody molecule obtained from a population of substantially homogeneous molecules, such that the antibody molecules have primary sequences that are essentially identical except for naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific for a single binding site or particular epitope. Monoclonal antibodies are examples of isolated antibodies. Monoclonal antibodies can be produced by any means known in the art, including, but not limited to, hybridoma culture techniques, recombinant methods, and gene transfer methods.

[0044] It should be understood that the target binding sequence of an antibody can be altered or modified to, among other things, improve affinity for the target, to humanize the target binding sequence, to improve production of the antibody in cell culture, to reduce the immunogenicity of the antibody in vivo, to create multispecific antibodies, etc. Such altered antibodies are specifically contemplated herein.

[0045] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment.

[0046] As used herein, "binding affinity" refers to the strength of the interaction between the antigen-binding site of an antibody and its binding partner (e.g., an antigen). The affinity of an antibody for its antigen is generally measured by the affinity constant (KA ), the amount of antibody-antigen complex at equilibrium, or the equilibrium dissociation constant (K D Affinity can be measured by any method known in the art, including but not limited to, ELISA, gel shift analysis, pull-down assay, equilibrium dialysis, analytical ultracentrifugation, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and spectroscopic measurements.

[0047] The term "patient" or "subject" as used herein refers to an organism suffering from or susceptible to a condition that can be prevented or treated by administration of a compound or composition provided herein. Subjects include, but are not limited to, mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), and are preferably humans.

[0048] The term "substantially homologous" or "substantially identical" means that a particular subject sequence, e.g., a mutant sequence, differs from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which is no deleterious functional difference between the reference and subject sequences. For purposes of the present invention, sequences having greater than 95 percent homology (identity), equivalent biological activity (equivalent biological activity strength, but not necessarily) to a given sequence, and equivalent expression characteristics are considered to be substantially homologous (identical). For purposes of determining homology, truncations of the native sequence should be ignored. Exemplary IL-15 polypeptides used herein include sequences substantially homologous to SEQ ID NO:1. SEQ ID NO:2 is nearly identical to SEQ ID NO:1, but SEQ ID NO:2 has a methionine at the beginning of the sequence that is required for initiation of translation in E. coli.

[0049] The term "fragment" refers to any protein or polypeptide having the amino acid sequence of a portion or fragment of a protein or polypeptide, e.g., IL-15 portion, and having the biological activity, or substantially the biological activity, of the protein or polypeptide, e.g., IL-15. Fragments include proteins or polypeptides produced by proteolysis and proteins or polypeptides produced by chemical synthesis by methods routine in the art.

[0050] The amino acid residues in the peptides are abbreviated as follows: phenylalanine is Phe or F; leucine is Leu or L; isoleucine is Ile or I; methionine is Met or M; valine is Val or V; serine is Ser or S; proline is Pro or P; threonine is Thr or T; alanine is Ala or A; tyrosine is Tyr or Y; histidine is His or H; glutamine is Gln or Q; asparagine is Asn or N; lysine is Lys or K; aspartic acid is Asp or D; glutamic acid is Glu or E; cysteine ​​is Cys or C; tryptophan is Trp or W; arginine is Arg or R; and glycine is Gly or G.

[0051] overview The present disclosure is directed to providing (among other things) combinations, compositions, methods, and kits relating to the treatment of conditions such as cancer, comprising (a) a long-acting IL-15 receptor agonist and (b) an antibody directed to a tumor antigen, where the tumor-directed antibody comprises antibody-dependent cellular cytotoxicity (ADCC) as a mechanism of action. Such compositions and methods would ideally have several advantageous and unexpected features, such as at least one, and possibly more, of the following: increased tumor clearance; increased patient survival and / or prolonged patient survival; increased activity of either or both of the long-acting IL-15 receptor agonist and antibody when administered in combination, particularly within the target tissue compartment, compared to either active agent administered alone; enhanced NK cell degranulation; increased NK cell proliferation; increased NK cell viability; increased T cell, e.g., CD8+ T cell proliferation, and / or increased T cell, e.g., CD8+ T cell viability. Surprisingly, applicants have arrived at a combination of a long-acting IL-15 receptor agonist and at least one antibody (such as a monoclonal antibody) directed against a tumor antigen, which has a unique combination of advantageous properties, as described in more detail below and illustrated in the supporting examples.

[0052] It has been discovered that the combination of the long-acting IL-15R agonists described herein with targeted antibodies that mediate tumor killing by ADCC exhibits enhanced immunotherapeutic effects. ADCC is an important mechanism in tumor depletion by certain tumor-targeted antibodies, in which receptors on NK cells recognize tumor cell-bound antibodies. Rebinding of the NK cell receptor to the antibody triggers the release of cytotoxic granules and / or cytokines to kill tumor cells. It has been discovered that the long-acting IL-15R agonists described herein are effective in increasing the efficacy of tumor-targeted antibody therapy using the ADCC mechanism of action by increasing NK cells with increased cytotoxicity and / or other functional activation.

[0053] Therapeutic Combinations, Compositions, and Methods of Use In a first aspect, a method for treating a subject suffering from cancer or tumor is described herein. The method includes administering, together or separately, an antibody or antigen-binding portion thereof that specifically binds to a tumor antigen and a long-acting IL-15 receptor agonist. Due to the ability of the long-acting IL-15 receptor agonist to induce the proliferation of NK cells and activate the tumor cell killing ability of NK cells, a combination therapy approach was developed. In this combination therapy approach, a long-acting IL-15 receptor agonist such as mono(methoxyPEG-N-butanamide) interleukin-15 (also referred to as mono(mPEG-butanamide) interleukin-15, mono(mPEG-butanamide) IL-15, or mono-mPEG-SBA-IL15) is combined with a therapeutic monoclonal antibody that recognizes tumor cells, thereby resulting in an increase in the number of cell killing activated NK cells, which can also efficiently bind to the antibody molecule and then be guided to tumor cells by the antibody, promoting a synergistic increase in tumor cell killing. See, for example, the results described in the accompanying Examples herein.

[0054] For the sake of clarity, with regard to the order of administration (wherein the term "administering" is used to refer to the delivery of a long-acting IL-15 receptor agonist or a tumor-directed antibody), the long-acting IL-15 receptor agonist and the tumor-directed antibody can be administered simultaneously or sequentially, and in any order. Furthermore, the treatment of either component of the combination can include a single cycle of therapy or can include multiple cycles. That is, after administration of a long-acting IL-15 agonist and administration of a tumor-directed antibody, additional rounds of therapy can include administration of a long-acting IL-15 receptor agonist in combination with administration of a tumor-directed antibody, administration of a long-acting IL-15 receptor agonist without further administration of a tumor-directed antibody, or administration of a tumor-directed antibody without further administration of a long-acting IL-15 receptor agonist, or any combination of the above administrations.

[0055] In a second aspect, described herein is a composition (or compositions) comprising an antibody, or an antigen-binding portion thereof, that specifically binds to a tumor antigen and a long-acting IL-15 receptor agonist.

[0056] Generally, the antibodies described herein are directed to proteins expressed on the cell surface of cancer or tumor cells, hereinafter referred to as cancer antigens or tumor antigens. Several cancer or tumor antigens are known in the art. Non-limiting examples include phosphorylated proteins, transmembrane proteins, glycoproteins, glycolipids, and growth factors. The assay to determine whether a given compound can act as an antibody against any of the antigens or targets described herein can be determined by one skilled in the art through routine experimentation.

[0057] In some embodiments, the antibody is an immunoglobulin G (IgG) type antibody, which is typically found in the human blood circulation.

[0058] In some embodiments, the antibody is an anti-CD16, anti-CD19, anti-CD20, or anti-CD38 antibody, i.e., an antibody that specifically binds to CD16, CD19, CD20, CD30, CD38, or CD52.

[0059] The human CD19 antigen is a 95 kDa glycoprotein that belongs to the immunoglobulin (Ig) superfamily. CD19 is a biomarker for normal and neoplastic B cells and for follicular dendritic cells. CD19 is expressed from early stages of pre-B cell development until terminal differentiation and regulates B lymphocyte development and function. CD19 expression is highly conserved on most B cell tumors, including B cell lymphomas such as non-Hodgkin's lymphoma. CD19 is also expressed in most types of leukemia, including B cell leukemia, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and Waldenstrom's macroglobulinemia (WM). The majority of B cell malignancies (lymphomas and leukemias) express CD19 at normal to high levels. In an embodiment, the antibody is an anti-CD19 monoclonal antibody. Some exemplary anti-CD19 antibodies contemplated for use in the methods and compositions herein include, but are not limited to, anti-B4-bR, BiTE (bi-specific T-cell engaging antibody), MEDI-551 (MedImmune, LLC), MOR-208 (MorphoSys AG), blinatumomab, bispecific anti-CD19 / CD3 BiTE® antibody (Blincyto®, Amgen), coltuximab ravtansine (ImmunoGen Inc. and Sanofi), denintuzumab mafodotin (Seattle Genetics), taplitumomab paptox (National Cancer Institute), XmAb 5871 (Amgen and Xencor), and combinations thereof. Inc.), MDX-1342 (Medarex), AFM11 (Affimed Therapeutics), and the anti-CD19 antibody (huB4, DI B4, Merck) described in U.S. Pat. No. 8,691,952.In one or more embodiments, a combination of a long-acting IL-15 receptor agonist and an anti-CD19 antibody is used in the treatment of B-cell malignancies, including, but not limited to, non-Hodgkin's lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and Waldenstrom's macroglobulinemia (WM).

[0060] CD20 is a non-glycosylated, phosphorylated protein of approximately 33-37 kD expressed on the surface of almost all normal and malignant B cells. CD20 mAbs can exert anti-tumor effects through Fab-mediated effects, including activation of effector mechanisms (Boross et al., Am J Cancer 2(6):676-690, 2012). In one or more embodiments, the antibody administered with the long-acting IL-15 receptor agonists described herein is an anti-CD20 monoclonal antibody. Some exemplary anti-CD20 antibodies contemplated for use in the methods and compositions herein include rituximab (Rituxan®, Genentech), ofatumumab (Arzerra®, Genmab AC), ocrelizumab (Genentech), veltuzumab (Immunomedics), AME-133V (Eli Lilly), PRO131921 (Genentech), GA101 (Glycart / Roche), ibritumomab tiuxetan (Zevalin), tositumomab (Bexxar), and obinutuzumab (Gazyva®, Genentech). In embodiments, a combination of a long-acting IL-15 receptor agonist, such as those described herein, and an anti-CD20 antibody is used to treat B-cell malignancies, including, but not limited to, non-Hodgkin's lymphoma, CLL, diffuse large B-cell lymphoma (DLBCL), and follicular lymphoma.

[0061] CD38 is a 45 kDa type II transmembrane glycoprotein with receptor and enzymatic functions. CD38 is generally expressed at low levels on various blood and solid tissues, but at high levels by plasma cells (with particularly widespread high expression levels in plasmacytomas, such as multiple myeloma (MM)). CD38 is also expressed in some hematological tumors. In some further embodiments, the combination of the present invention includes administration of a long-acting IL-15 receptor agonist and an anti-CD38 monoclonal antibody. Some exemplary anti-CD38 antibodies contemplated for use in the methods and compositions provided herein include daratumumab (DARZALEX®, Janssen Biotech), isatuximab (SAR650984, Sanofi Oncology), and MOR202 (Morphosys). In some further embodiments, the combination of a long-acting IL-15 receptor agonist and an anti-CD38 antibody is used to treat a condition selected from multiple myeloma, CD38+ non-Hodgkin's lymphoma, CDCLL, Waldenstrom's macroglobulinemia, primary systemic amyloidosis, mantle cell lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, NK cell leukemia, NK / T cell lymphoma, and plasma cell leukemia.

[0062] In some additional embodiments, the cancer immunology-based therapeutic combination comprises a long-acting IL-15 receptor agonist as described herein and an antibody directed to a glycoprotein selected from, but not limited to, SLAMF7, EpCAM, gpA3, or folate binding protein (FBP). In embodiments, the antibody is an anti-SLAMF7 antibody, an anti-EpCAM antibody, an anti-gpA3 antibody, or an anti-FBP antibody.

[0063] Signaling Lymphocytic Activation Molecule Family Member 7 (SLAMF7, formerly known as CS1, CD319, CRACC) is a member of the signaling lymphocyte activation molecule family. SLAMF7 is expressed on immune cells such as B cells, T cells, dendritic cells, NK T cells, and monocytes, as well as on multiple myeloma cells. In an embodiment of the combination of the present invention, the antibody is an anti-SLAMF7 monoclonal antibody. One exemplary anti-SLAMF7 antibody contemplated for use in the methods and compositions herein is elotuzumab (Emplicity™, Bristol-Myers Squibb). In an embodiment, a combination of a long-acting IL-15 receptor agonist and an anti-SLAMF7 antibody is used to treat multiple myeloma.

[0064] Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein expressed by some epithelial cancer cells, including tumors of gastrointestinal origin and some cancers of the urogenital tract. EpCAM is expressed, for example, in human colon cancer, metastatic breast cancer, gallbladder cancer, ovarian cancer, and pancreatic cancer. In an embodiment of the therapeutic combination provided herein, the antibody is an anti-EpCAM monoclonal antibody. Some exemplary anti-EpCAM antibodies contemplated for use in the methods and compositions herein include edrecolomab (Panorex, Creative Biolabs), ING-1 (Xoma), 3622W94 (Creative Biolabs), and adecatumumab (Amgen). In one or more embodiments, a combination of a long-acting IL-15 receptor agonist and an anti-EpCAM antibody is used to treat human colon cancer, metastatic breast cancer, gallbladder cancer, ovarian cancer, adenocarcinoma, and pancreatic cancer.

[0065] In some further embodiments, the antibody is directed to a growth factor selected from, for example but not limited to, vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR), and epidermal growth factor receptor (EGFR). In some embodiments, the antibody is selected from an anti-VEGF antibody, an anti-VEGFR antibody, and an anti-EGFR antibody.

[0066] Vascular endothelial growth factor (VEGF) is a 27 kDa angiogenic signaling protein. VEGF is expressed in most types of non-digestive and digestive cancers, including pancreatic, breast, ovarian, colorectal, and lung cancers. Some exemplary anti-VEGF antibodies contemplated for use in the methods and compositions herein include bevacizumab (Avasatin®, Genentech, Inc.), ranibizumab, 2C3 and r84 (AT001, Affitech AS), and VEGF-Trap (aflibercept, Regeneron Pharmaceuticals, Inc.). In one or more embodiments, a combination of a long-acting IL-15 receptor agonist and an anti-VEGF antibody is used to treat cancer, such as, for example, pancreatic, breast, ovarian, colorectal, metastatic renal cell carcinoma, and non-small cell lung cancer.

[0067] Vascular endothelial growth factor receptors (VEGFRs) are receptor tyrosine kinases (RTKs) that can induce angiogenesis, increase cell growth and metastasis, and the like. The VEGFR family has three main subtypes: VEGFR-1, VEGFR-2, and VEGFR-3. Some exemplary anti-VEGFR antibodies contemplated for use in the methods and compositions herein include MF1 / IMC-18F1 (ImClone Systems); IMC-1121B ImClone Systems), and DC101 / IMC-1C11. In some further embodiments, a combination of a long-acting IL-15 receptor agonist and an anti-VEGFR antibody is used to treat cancer, such as, for example, breast cancer or non-small cell lung cancer. In some embodiments, the anti-VEGFR antibody is used for the indications listed above for anti-VEGF antibodies.

[0068] Epidermal growth factor receptor (EGFR) is a large family of receptor tyrosine kinases expressed in several types of cancer, including breast, lung, esophageal, metastatic colorectal, and head and neck. Some exemplary anti-EGFR antibodies contemplated for use in the methods and compositions herein include cetuximab (Erbitux®, Lilly USA) and panitumumab (Vectibix®, Amgen). In one or more specific embodiments, the antibody is an anti-human epidermal growth factor receptor 2 antibody (anti-HER2). Some exemplary anti-HER2 antibodies contemplated for use in the methods and compositions herein include the humanized monoclonal antibodies trastuzumab (Herceptin®, Genentech, Inc.) and / or pertuzumab (Perjeta®, Genentech, Inc.). In some related embodiments, the combination of a long-acting IL-15 receptor agonist and an anti-EGFR antibody is used to treat cancers, such as breast (e.g., metastatic breast cancer), lung, esophageal, metastatic colorectal, and head and neck cancer.

[0069] Also contemplated for use in the methods and combinations provided herein are anti-BCMA (also called B-cell maturation antigen, CD269) antibodies. BCMA is a member of the tumor necrosis factor receptor (TNFR) superfamily. BCMA binds to B-cell activating factor (BAFF) and proliferation-inducing ligand (APRIL). Among non-malignant cells, BCMA has been reported to be mostly expressed in plasma cells and some mature B cells. BCMA RNA has been detected in multiple myeloma cells, and BCMA protein has been detected on the surface of plasma cells from multiple myeloma patients. BCMA is expressed or overexpressed by a variety of human cancers. Examples of cancers that express or overexpress BCMA include, but are not limited to, Burkitt's lymphoma, diffuse large B-cell lymphoma (DLBCL) lymphoma, acute lymphocytic leukemia (ALL) lymphoma, Hodgkin's lymphoma, and multiple myeloma. Exemplary anti-BCMA antibodies are described, for example, in U.S. Patent Publication Nos. 20120082661, 20170051068, and 20180318435, the contents of which are incorporated by reference in their entirety. Exemplary anti-BCMA antibodies include BCMAxXD3, as described in Pillarisetti, K., et al., Blood, 2016, 128:2116, and the humanized antibody portion of the anti-BCMA antibody drug conjugate, GSK2857916.

[0070] In some embodiments, antibodies are capable of binding two or more specific antigens simultaneously, eg, bispecific antibodies.

[0071] In some embodiments, the antibody cross-competes for binding with any of the antibodies described above. Antibodies that cross-compete with any of the above-mentioned antibodies are expected to have similar or the same functional properties.

[0072] Since it is well known in the art that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody, in some embodiments an antibody for use herein is an antigen-binding portion of any of the antibodies described above.

[0073] It should be noted next that long-acting IL-15 receptor (IL-15R) agonists, and generally preferred long-acting IL-15 receptor agonists or pharma- ceutically acceptable salt forms thereof, comprise a single linear polyalkylene oxide (e.g., polyethylene glycol or "PEG") moiety that is stably covalently attached to an IL-15 amino group via an amide bond. Interposed between the PEG moiety and the stable amide bond to the IL-15 amino group is a linear unsubstituted alkylene group (~CH2~) having 2-5 carbon atoms (i.e., m is 2, 3, 4, or 5). m In one or more preferred embodiments, m is 3, such that the stable amide bond is a butanamide.

[0074] In one or more preferred embodiments, the long-acting IL-15R agonist is (methoxyPEG-N-butanamide) 20-60kD Interleukin-15, more preferably (methoxy PEG-N-butanamide) 20-40kD Interleukin-15, even more preferably (methoxy PEG-N-butanamide) 40kD Interleukin-15. In one or more preferred embodiments, the long-acting IL-15R agonist is mono(methoxyPEG-N-butanamide) 20-60kD Interleukin-15, more preferably mono(methoxyPEG-N-butanamide) 20-40kD Interleukin-15, even more preferably mono(methoxyPEG-N-butanamide) 40kD Interleukin-15. In a preferred embodiment, the long-acting IL-15R agonist is mono(methoxyPEG-N-butanamide). 40kD Interleukin-15.

[0075] IL-15R agonists as described herein having a structure encompassed by formula (I), in particular mono(methoxyPEG-N-butanamide) 40kD Interleukin-15 retains binding affinity for the IL-15 receptor alpha (IL-15Rα) subunit, as well as the beta (β) and gamma (γ) subunits. The long-acting IL-15R agonists described herein are on the same (cis) or adjacent cells (trans) to the IL-2 / IL-15Rβγ complex. Binding of the IL-2 / IL-15Rβγ complex by the long-acting IL-15R agonists described herein can trigger the Janus kinase / signal transducer and activator of transcription-5 (JAK-STAT5) pathway, which increases T cell proliferation, survival, and / or activity. The long-acting IL-15R agonists further significantly enhance the proliferation and activation of CD8+ T cells. The long-acting IL-15R agonists preferably have reduced clearance compared to the corresponding unmodified IL-15R agonists. Without being limited by theory, it is believed that the attachment of PEG increases the hydrodynamic volume of the IL-15 moiety, resulting in a longer effective half-life, lower maximum peak concentration (C) compared to unconjugated rhIL-15. max ) and / or reduced clearance. The long-acting IL-15R agonists described herein provide sustained IL-15 bioactivity, eliminating the need for daily dosing.

[0076] When discussing IL-15 moieties, the term "IL-15 moiety" refers to the IL-15 moiety prior to conjugation and to the IL-15 moiety following attachment to a non-peptidic, water-soluble polymer such as PEG. Although PEG will be specifically referenced hereinafter as the non-peptidic, water-soluble polymer, it will be understood that the present disclosure generally relates to non-peptidic, water-soluble polymers or poly(alkylene glycols). However, it will be understood that when the original IL-15 moiety is attached to a polyethylene glycol moiety, the IL-15 moiety is slightly altered due to the presence of one or more covalent bonds associated with the attachment to the polymer.

[0077] The IL-15 moiety can be obtained from non-recombinant and recombinant methods, and the disclosure is not limited in this respect. In addition, the IL-15 moiety can be derived from human sources, animal sources (including insects), fungal sources (including yeast), and plant sources.

[0078] Alternatively, the IL-15 moiety can be obtained, for example, according to the procedure described by Grabstein et al. (Grabstein et al. (1994) Science 264:965-968). The IL-15 moiety can also be prepared using recombinant methods, such as those described in European Patent No. 0772624 B2 to Immunex Corporation. The IL-15 moiety can be purchased commercially, for example, from GenScript USA Inc. (Piscataway NJ) and Peprotech (Rockyhill, NJ).

[0079] The IL-15 moiety may be derived from bacteria (e.g., E. coli, e.g., Fischer (1995) Biotechnol. Appl. Biotechnol. 21(3):295-311), mammalian (see, e.g., Kronman et al. (1992) Gene 121:295-304), yeast (e.g., Pichia pastoris, see, e.g., Morel et al. (1997) Biochem. J. 328(1):121-129), and plant (see, e.g., Mor et al. (2001) Biotechnol. Bioeng. 75(3):259-266) expression systems. Expression can occur by exogenous expression (where the host cell naturally contains the desired genetic code) or by endogenous expression.

[0080] Further methods for preparation and / or purification of IL-15 moieties are described in PCT Application / US Publication No. 2018 / 032817, which is incorporated by reference in its entirety.

[0081] Depending on the system used to express a protein having IL-15 activity, the IL-15 moiety may be non-glycosylated or glycosylated, either of which may be used, in one or more embodiments, the IL-15 moiety is non-glycosylated.

[0082] The IL-15 moiety can advantageously be modified to include and / or substitute one or more amino acid residues, e.g., lysine, cysteine, and / or arginine, which facilitates attachment of the polymer to an atom in the side chain of that amino acid. Examples of substitutions of the IL-15 moiety are described in U.S. Pat. No. 6,177,079. Additionally, the IL-15 moiety can be modified to include a non-naturally occurring amino acid residue. Methods for adding amino acid residues and non-naturally occurring amino acid residues are known to those of skill in the art. See J. March, Advanced Organic Chemistry: Reactions Mechanisms and Structure, 4th Ed. (New York: Wiley-Interscience, 1992), and Bioinformatics for Geneticists (eds. Michael R. Barnes and Ian C Gray), 2003 John Wiley & Sons, Ltd, Chapter 14, Amino Acid Properties and Consequences of Substitutions, Betts, MJ, and Russell, RB.

[0083] Additional suitable modifications of the IL-15 moiety and methods for such modifications are described in PCT Application / US Patent Publication No. 2018 / 032817, which is incorporated herein by reference in its entirety. Exemplary modifications include attachment of a functional group (other than by addition of an amino acid residue containing a functional group), such as a thiol group, an N-terminal alpha carbon, one or more carbohydrate moieties, an aldehyde group, or a ketone group. In some embodiments, the IL-15 moiety is preferably not modified to include one or more of a thiol group, an N-terminal alpha carbon, a carbohydrate, an aldehyde group, or a ketone group.

[0084] Exemplary IL-15 moieties are described herein, in the literature, and in, for example, U.S. Patent Application Publication No. 2006 / 0104945, Pettit et al. (1997) J. Biol. Chem. 272(4):2312-2318, Wong et al. (2013) OncoImmunology 2(11), e26442:1-3, and PCT Application No. PCT / US2018 / 032817, which are incorporated herein by reference in their entireties. Preferred IL-15 moieties include those having an amino acid sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 1-3, and sequences substantially homologous thereto (it will be understood that SEQ ID NOs: 2 and 3, and sequences substantially homologous thereto, are also understood to be "IL-15 moieties" for purposes of the present invention, even if they do not meet the in vitro activity criteria of an IL-15 moiety provided herein). A preferred IL-15 moiety has an amino acid sequence corresponding to SEQ ID NO: 1. In some embodiments, the IL-15 portion is a functional homolog having at least about 85% or at least about 90% identity to any one of SEQ ID NOs: 1-3. In some embodiments, the IL-15 portion is a functional homolog having at least about 95%, 98% or 99% identity to any one of SEQ ID NOs: 1-3.

[0085] In some cases, the IL-15 moiety may be in a "monomeric" form, where a single expression of the corresponding peptide is organized as an individual unit. In other cases, the IL-15 moiety may be in a "dimeric" form, where two monomeric forms of the protein are associated with each other (e.g., a dimer of recombinant IL-15).

[0086] In addition, a precursor form of IL-15 can be used as the IL-15 moiety. An exemplary precursor form of IL-15 has the sequence of SEQ ID NO:3.

[0087] In some embodiments, the long-acting IL-15R agonist is a PEGylated IL-15 molecule described in US Patent Publication No. 2018 / 0360977, in particular the multi-arm PEG IL-15 described therein.

[0088] In some embodiments, the IL-15 moiety is an IL-15 mutein or other IL-15 related molecule described in US Pat. No. 10,350,270.

[0089] Truncates, hybrid variants, and peptidomimetics of any of the foregoing sequences can also serve as IL-15 moieties. Biologically active fragments, deletion variants, substitution variants, or addition variants of any of the foregoing that maintain at least some IL-15 activity can also serve as IL-15 moieties.

[0090] For a given peptide, protein moiety, or conjugate, it is possible to determine whether the peptide, protein moiety, or conjugate has IL-15 activity. Various methods for determining in vitro IL-15 activity have been described in the art. An exemplary approach is based on the pSTAT assay. Briefly, when IL-15-dependent CTLL-2 cells are exposed to a test agent having IL-15 activity, the result is a signaling cascade that includes phosphorylation of STAT5 at tyrosine residue 694 (Tyr694), which can be quantitatively measured. Assay protocols and kits are known and include, for example, the MSD Phospho(Tyr694) / Total STATa,b whole cell lysate kit (Meso Scal Diagnostics, LLC, Gaithersburg, MD); for example, using this approach, a pSTAT5 EC50 of about 300 ng / mL or less (more preferably about 150 ng / mL or less) at least one of the 5 and 10 minute time points can be determined. 50 Candidate IL-15 moieties exhibiting pSTAT5 EC values ​​of less than 150 ng / mL at at least one of the 5 or 10 minute time points, such as less than about 1 ng / mL at at least one of the 5 or 10 minute time points, and even more preferably less than 0.5 ng / mL, are considered to be "IL-15 moieties" in the context of the present disclosure. 50 It is preferred that the .lambda.-based .DELTA..times ...

[0091] Other methods known in the art, including electrometric, spectroscopic, chromatographic, and radiometric methods, can also be used to assess IL-15 function. For one such alternative type of assay, see, e.g., Ring et al. (2012) Nat. Immunol. 13(12):1187-1195.

[0092] The assays used in connection with measuring the activity of the IL-15 moiety can also be used to measure the activity of the long-acting IL-15 receptor agonists described herein, see, e.g., the supporting examples provided herein.

[0093] A compound is considered a long-acting IL-15 receptor agonist according to the present disclosure as long as the agonist exhibits IL-15 receptor activating activity in vivo for a longer period of time than administration of IL-15 after administration to a subject. Conventional techniques can be used to assess whether a compound proposed as a long-acting IL-15 receptor agonist is "long-acting" (i.e., has a longer clearance than IL-15 administered to the same in vivo system), including, for example, radiolabeling the compound, administering the compound in vivo, and determining its clearance. For purposes of the present invention, the long-acting nature of a long-acting IL-15 receptor agonist can be, or is typically, determined by measuring STAT5 phosphorylation in lymphocytes at various time points after administration of the agonist to be evaluated to mice using flow cytometry. For reference, the signal is lost by IL-15 in approximately 24 hours, whereas in the case of a long-acting IL-15 agonist, it is persistent over a period of time.

[0094] Preferred long-acting IL-15 receptor agonists will generally contain a single linear PEG (polyethylene glycol) moiety stably and covalently attached to an IL-15 amino group via an amide bond. Between the PEG moiety and the stable amide bond to the IL-15 amino group is a linear unsubstituted alkylene group (~CH2~) having 2-5 carbon atoms. m (i.e., m=2, 3, 4, or 5).

[0095] For example, in some embodiments, the unsubstituted alkylene group is (~CH2~)2; or in some additional embodiments, the unsubstituted alkylene group is (~CH2~)3; in some still further embodiments, the unsubstituted alkylene group is (~CH2~)4; in some still further embodiments, the unsubstituted alkylene group is (~CH2~)5.

[0096] For example, in some embodiments, the long-acting IL-15 receptor agonist has the structure: [ka] wherein IL-15 is an interleukin-15 moiety, n is an integer from about 150 to about 3,000; m is an integer from 2 to 5 (e.g., 2, 3, 4, or 5), and n' is 1. In formula I (and similar formulae provided herein), ~NH~ in the structure represents the amino group of the IL-15 moiety. Formula (I) may also be represented as follows: [ka] where the brackets have been moved to reflect the terminal PEG methoxy group, the two formulas may be used interchangeably. Examples of exemplary compounds encompassed by formula (I) include the following: [ka]

[0097] In some preferred embodiments, the long-acting IL-15 receptor agonist corresponds to formula (Ia) or formula (Ib). In some particularly preferred embodiments, the long-acting IL-15 receptor agonist corresponds to formula (Ib).

[0098] In some further embodiments related to the structures and formulas described herein, n is an integer from about 200 to about 2000, or from about 400 to about 1300, or from about 450 to about 1200. That is, in some embodiments, n is an integer from about 200 to about 2000. In some still further embodiments, n is an integer from about 400 to about 1300. In some still further embodiments, n is an integer from about 450 to about 1200.

[0099] PEG having a molecular weight corresponding to any of the ranges of n values ​​given above is generally preferred.

[0100] In one or more additional embodiments, n is an integer having a value corresponding to a polyethylene glycol polymer having a weight average molecular weight selected from the group consisting of about 10,000 daltons (where n is about 227), or about 15,000 daltons (where n is about 340), or about 20,000 daltons (where n is about 454), or about 25,000 daltons (where n is about 568), or about 30,000 daltons (where n is about 681), or about 40,000 daltons (where n is about 907-909, e.g., about 909), or about 50,000 daltons (where n is about 1136), or even about 60,000 daltons (where n is about 1364) or greater.

[0101] Further exemplary weight average molecular weights for the polyethylene glycol portion of the compound include, in addition to those mentioned above, about 11,000 daltons, about 12,000 daltons, about 13,000 daltons, about 14,000 daltons, about 22,500 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 55,000 daltons, about 65,000 daltons, about 70,000 daltons, and about 75,000 daltons. In some embodiments, the weight average molecular weight of the polyethylene glycol portion of the IL-15 conjugate is about 37,000 to about 45,000 daltons.

[0102] In some preferred embodiments, the weight average molecular weight of the polyethylene glycol polymer portion of the IL-15 conjugate is about 40,000 Daltons. For each of formulas (I) and (II) described herein, including any sub-formulas thereto, a weight average molecular weight of PEG of 40,000 Daltons is preferred.

[0103] The PEG moiety is preferably end-capped with a methoxy group, as shown above in formula (I), however, the PEG moiety may also be terminated with any lower C 1-6 It may be capped with an alkoxy group or may be terminated with a hydroxyl group or other suitable end capping group.

[0104] In some embodiments, the long-acting IL-15 receptor agonists have the following formulas, taken collectively: [ka] where the values ​​of n and m are as provided for formula (I) above), includes up to about 20 mole percent (mol %) of a long-acting IL-15 receptor agonist (of the IL-15-containing molecules in the composition). That is, when included in a composition of long-acting IL-15 receptor agonist compounds, for example, a monoPEGylated long-acting IL-15 receptor agonist having a different number of PEG moieties (e.g., dimers, trimers, etc.) covalently attached to an interleukin-15 moiety, but of the same chemical formula, e.g., of any of formulas (I), (Ia), (Ib), (Ic), and (Id), e.g., formula (Ib), includes at most about 20 mole percent of a long-acting IL-15 receptor agonist of formula (II).

[0105] In some additional embodiments, the long-acting IL-15 receptor agonist compositions have the following formulas, when considered collectively: [ka] (wherein the values ​​of n and m are as provided for Formula (I) above. That is, in terms of the long-acting IL-15 receptor agonist component of such compositions, about 15 mol % or less of the long-acting IL-15 receptor agonist included in the composition is of Formula (II). In some embodiments, the long-acting IL-15 receptor agonist composition comprises about 0.1-20 mol % or less of a compound of Formula (II). In embodiments, the composition comprises about 0.1-15, 0.1-10, 0.1-5, 0.1-1, 1-20, 1-15, 1-10, 1-5, 5-20, 5-15, 5-10, 10-20, 10-15, or 15-20 mol % or less of a compound of Formula (II). A preferred embodiment is where "m" in formula (II) is 3.

[0106] In certain particular embodiments related to the foregoing, the long-acting IL-15 receptor agonist compositions related to Formula (Ia) have the following formulas, when considered collectively: [ka] wherein the values ​​of n and m are as provided for formula (Ia) above, and about 15 mole percent (mol %) or less of a long-acting IL-15 receptor agonist (of the IL-15-containing molecules in the composition).

[0107] In some particularly preferred embodiments, the long-acting IL-15 receptor agonist compositions related to formula (Ib) have the following formulas, when considered collectively: [ka] wherein the values ​​of n and m are as provided for formula (Ib) above, and the compound (Ib) includes no more than 15 mole percent (mol %) of a long-acting IL-15 receptor agonist (of the IL-15-containing molecules in the composition).

[0108] In some further embodiments, the long-acting IL-15 receptor agonist compositions related to Formula (Ic) have the following formulas, when considered collectively: [ka] wherein the values ​​of n and m are as provided for formula (Ic) above, and about 15 mole percent (mol %) or less of a long-acting IL-15 receptor agonist (of the IL-15-containing molecules in the composition).

[0109] In some further embodiments, the long-acting IL-15 receptor agonist compositions related to Formula (Id) have the following formulas, when considered collectively: [ka] wherein the values ​​of n and m are as provided for formula (Id) above, and about 15 mole percent (mol %) or less of a long-acting IL-15 receptor agonist (of the IL-15-containing molecules in the composition).

[0110] In some embodiments, the long-acting IL-15 receptor agonist composition comprises about 0.1-20 mol % or less of a compound of formula (II) (including compounds of formulas (IIa), (IIb), (IIc), and (IId)). In some additional embodiments, the composition comprises about 0.1-15, 0.1-10, 0.1-5, 0.1-1, 1-20, 1-15, 1-10, 1-5, 5-20, 5-15, 5-10, 10-20, 10-15, or 15-20 mol % or less of a compound of formula (II) (including compounds of formulas (IIa), (IIb), (IIc), and (IId)). In some embodiments, the composition comprises about 0.1, 1, 5, 10, 15, or 20 mol % or less of a compound of formula (II) (including compounds of formulas (IIa), (IIb), (IIc), and (IId)). It will be appreciated that the composition may be purified by methods known in the art for compounds of formula (I), such that compounds of formula (II) may be absent, present in trace amounts, or substantially absent from the composition.

[0111] For example, in some embodiments, the long-acting IL-15 receptor agonist composition comprises about 12 mole percent or less, or about 10 mole percent or less, of long-acting IL-15 receptor agonists encompassed by Formula (II), which includes compounds of Formulas (IIa), (IIb), (IIc), and (IId), taken collectively.

[0112] In some additional embodiments of the foregoing, the composition comprises about 7 mol % or less of a long-acting IL-15 receptor agonist having an n' equal to 2, 3, or greater than 3 (i.e., higher PEGmers). In still some other embodiments, the composition comprises about 5 mol % or less of a long-acting IL-15 receptor agonist having an n' equal to 2, 3, or greater than 3 (i.e., 2 or greater).

[0113] In some further embodiments, the composition comprises a compound represented by formula (I): [ka] (wherein n and m are as described above, and n' represents the average number of polyethylene glycol moieties covalently attached to IL-15 amino groups (with respect to the composition), and n' with respect to the composition is in the range of 1.0 to about 1.3. For example, the average number of polyethylene glycol moieties per IL-15 moiety is selected from about 1.0, 1.1, 1.2, and about 1.3. That is, preferred long-acting IL-15 receptor agonists according to formula (I) may be referred to herein as "mono-PEGylated", where it should be understood that there is some variability in the degree of PEGylation, as described above. In some preferred embodiments related to the formulas described herein, "m" is equal to 3.

[0114] A composition of long-acting IL-15 receptor agonists may comprise a single species where n' is equal to about 1 and the PEG moiety is attached at the same position for substantially all IL-15 conjugates in the composition, or alternatively, may comprise a mixture of mono-PEGylated conjugate species where attachment of the linear polyethylene glycol moiety occurs at different sites on the interleukin-15 moiety, i.e., the specific attachment site is not the same for all of the mono-PEGylated IL-15 species contained in the composition.) Thus, such compositions are substantially homogeneous in terms of the number of PEG moieties attached to IL-15 (e.g., 1-mer), but heterogeneous in terms of the position of amino group attachment on the IL-15 molecule.

[0115] Additional PEG constructions and conjugation chemistries can be used to arrive at long-acting IL-15 receptor agonists, however, as will become apparent when considered in light of the supporting examples, compounds such as those described above are preferred in one or more embodiments. However, additional long-acting IL-15 receptor agonists having the structures provided herein are also contemplated.

[0116] In some embodiments, the long-acting IL-15 receptor agonist composition comprises at least about 80 mol % of a long-acting IL-15 receptor agonist (of the IL-15-containing molecules in the composition) encompassed by formula (I), including formulae (Ia-d), taken collectively. In one or more embodiments, the long-acting IL-15 receptor agonist composition comprises at least about 85 mol %, 90 mol %, 95 mol %, 98 mol %, or 99 mol % of a long-acting IL-15 receptor agonist of formula (I), where a particularly preferred formula is that of formula (Ib). .

[0117] In this specification, all the above compounds, including long-acting IL-15 receptor agonists, are meant to expressly include such compounds in the form of pharmaceutically acceptable salts. Typically, such salts (e.g., of basic compounds) are formed by reaction with a pharmaceutically acceptable acid or acid equivalent. In this context, the term "pharmaceutically acceptable salts" generally refers to relatively non-toxic inorganic or organic acid addition salts. These salts can be prepared in situ during the administration vehicle or dosage form manufacturing process, or by separately reacting the long-acting interleukin-15 receptor agonists described herein with a suitable organic or inorganic acid and isolating the salt thus formed. Alternative salts include, but are not limited to, hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, oxylate, mesylate, glucoheptonate, lactobionate, and laurylsulfonate (see, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19). Thus, the salts described may be derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric acid, and the like; or prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

[0118] In some embodiments, the long-acting IL-15 receptor agonist composition comprises, collectively, about 1-5 mol% or less of free IL-15 protein (of the IL-15-containing molecules in the composition). In some further embodiments, the long-acting IL-15 agonist composition comprises about 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, or 5 mol% or less of free (i.e., unconjugated) IL-15.

[0119] To prepare a long-acting IL-15 receptor agonist, the IL-15 moiety can be conjugated, for example, at its amino group (e.g., lysine or N-terminus) to a PEG reagent functionalized with a succinimidyl group (or other activated ester group). Using this approach, succinimidyl-activated PEG can be attached to the amino group of the IL-15 moiety in aqueous media at a pH of about 7.0-9.0, although the use of different reaction conditions (e.g., a lower pH such as 6-7 or 7-8, or a different temperature and / or a temperature below 15° C.) can result in attachment of the PEG moiety to a different position on the IL-15 moiety.

[0120] Exemplary methods for preparing long-acting IL-15 receptor agonists are described in PCT Application No. PCT / US2018 / 032817, e.g., Examples 1-2, which are incorporated herein by reference in their entirety. In one exemplary embodiment, a long-acting IL-15 receptor agonist can be prepared as described in Example 1. For example, a long-acting IL-15 R agonist can generally be prepared by reacting interleukin-15, e.g., purified IL-15, such as recombinant IL-15, with an activated PEG reagent, such as an activated ester, methoxy PEG-succinimidyl butanoate, mPEG-SBA, etc. Other suitable activated PEG reagents include methoxy PEG-succinimidyl propionate, methoxy PEG-succinimidyl pentanoate, and methoxy PEG-succinimidyl hexanoate. Although succinimidyl activated groups are typically used, any suitable active ester or activated group can be used, such reactive groups being suitable for the formation of the desired stable amide bond. The PEGylated IL-15 reaction product can then generally be purified by any suitable method, such as ion exchange chromatography, to obtain the desired product. For example, anion exchange chromatography can be used. The chromatography product pool can then be concentrated and diafiltered into a suitable formulation buffer (e.g., sodium acetate buffer containing sucrose), for example, using tangential flow filtration (TFF). Analysis can be performed by any suitable method, such as, for example, SDS-PAGE, reverse phase HPLC, or any other suitable analytical method.

[0121] As previously described, the amino group on the IL-15 moiety provides a site of attachment between the IL-15 moiety and the polyethylene glycol moiety to provide a long-acting IL-15 receptor agonist encompassed by formula (I). Considering the exemplary IL-15 amino acid sequences provided herein, it is apparent that there are seven lysine residues, each with an ε-amino acid that may be available for conjugation for at least SEQ ID NOs: 1 and 2. Additionally, the N-terminal amine of methionine may also serve as an attachment point for the PEG moiety. It will be appreciated that the polyethylene glycol moiety may be attached at any one or more of the lysine or N-terminal amine positions. In some embodiments, the polyethylene glycol moiety attachment site is Lys 10 and Lys 11 (using the numbering shown in SEQ ID NO:2 as an example, or using SEQ ID NO:1, 11 and Lys 12 In some embodiments, the polyethylene glycol moiety is attached at the N-terminal amine. Any of the lysine sites may be used as a site for attachment of a PEG moiety (e.g., Lys of SEQ ID NO:1). 37 Or Lys 42 In some embodiments, the long-acting interleukin-15 receptor agonist may be suitable as a long-acting interleukin-15 receptor agonist. In some embodiments, the long-acting interleukin-15 receptor agonist comprises a mixture of positional isomers in which the covalent attachment of the polyethylene glycol moiety is predominantly at the N-terminus (i.e., of the population of positional isomers, the isomer having the PEG moiety attached at the N-terminus is present in the greatest amount compared to the other positional isomers).

[0122] If desired, the product pool may be further separated into positional isomers by reverse phase chromatography using reverse phase high performance liquid chromatography (RP-HPLC) using a suitable column (e.g., a C18 column or a C3 column commercially available from companies such as Amersham Biosciences or Vydac) or by ion exchange chromatography using an ion exchange column, e.g., a Sepharose™ ion exchange column available from Amersham Biosciences. Either technique can be used to separate PEG-interleukin-15 positional isomers having the same molecular weight (i.e., positional isoforms).

[0123] The long-acting IL-15 receptor agonists of the present invention have been found to have certain notable and advantageous characteristics. The characteristics described below are considered to apply generally to the compounds provided herein and encompassed by formula (I), however, one or more of the following characteristics may be exhibited in particular by compounds according to formula (Ib), and by extension, compounds according to formula (IIb). The long-acting IL-15 receptor agonists may have one or more of the following characteristics. For example, in some embodiments, the long-acting IL-15 receptor agonists exhibit about 7-fold or less reduction in EC50 values ​​(ng / mL, CTLL-2 pSTAT5) when compared to unmodified IL-15. For example, in one or more related embodiments, the long-acting IL-15 receptor agonist exhibits no more than about a 6.5-fold decrease in the EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 6-fold decrease in the EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 5.5-fold decrease in the EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 5-fold decrease in the EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 4.5-fold decrease in the EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 4-fold decrease in the EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 3.5-fold decrease in the EC50 value (ng / mL, CTLL-2 pSTAT5), or even no more than about a 3-fold decrease in the EC50 value (ng / mL, CTLL-2 pSTAT5). Exemplary long-acting IL-15 receptor agonists according to the foregoing features are described herein and in PCT Application No. PCT / US2018 / 032817, which is incorporated by reference in its entirety.

[0124] The amount of long-acting IL-15 receptor agonist in the composition will vary depending on several factors, but will optimally be a therapeutically effective dose when the composition is stored in a unit dose container (e.g., a vial). Furthermore, the pharmaceutical preparation can be contained in a syringe. The therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the long-acting IL-15 receptor agonist to determine the amount that results in the clinically desired endpoints described herein. The amount of any individual excipient in the composition will vary depending on the activity of the excipient and the specific needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing various amounts of excipients (ranging from low to high), examining stability and other parameters, and then determining the range that provides optimal performance without significant adverse effects.

[0125] Each pharmacological component of the method can be administered separately. Alternatively, if simultaneous administration of the two pharmacological components is desired--and the two pharmacological components are compatible together in a given formulation--co-administration can be achieved through administration of a single dosage form / formulation (e.g., intravenous administration of an intravenous formulation containing both pharmacologically active agents). Those skilled in the art can determine through routine testing whether two given pharmacological components are compatible together in a given formulation. It is understood that the antibody and the long-acting IL-15 receptor agonist can be administered in any order. Furthermore, the administration of the antibody and the long-acting IL-15 receptor agonist can be separated by minutes, hours, or days, as needed.

[0126] With regard to the frequency of administration of the long-acting IL-15 receptor agonist, those skilled in the art will be able to determine the appropriate frequency. For example, the clinician may decide to administer the long-acting IL-15 receptor agonist relatively infrequently (e.g., once every 2 or 3 weeks) and gradually shorten the interval between doses if tolerated by the patient. With regard to the frequency of administration of the antibody, the frequency of these drugs can be determined in a similar manner. In addition, some of the long-acting IL-15 receptor agonists and antibodies described herein are undergoing clinical testing or are commercially available, so it is also possible to refer to the literature to obtain the appropriate frequency of administration (it should be noted that some adjustments may be necessary in consideration of the combined effects of the treatment regimen).

[0127] In some treatment regimens, the long-acting IL-15 receptor agonist is administered as a single dose at the beginning of treatment. The antibody is administered simultaneously with the long-acting IL-15 receptor agonist, prior to administration of the long-acting IL-15 receptor agonist, or after administration of the long-acting IL-15 receptor agonist. For example, in some treatment modalities, the antibody is administered within 7-14 days (e.g., on any of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) of (before or after) administration of the long-acting IL-15 receptor agonist (wherein day 1 indicates the beginning of treatment). Based on the long-acting nature of the long-acting IL-15 receptor agonist, such compounds can be administered relatively infrequently (e.g., once every 3 weeks, once every 2 weeks, once every 8-10 days, once a week, once every 3 days, etc.). Additionally, the antibody can be administered according to an approved schedule (e.g., daily, 2 or 3 times a week, weekly, biweekly, etc.). In one exemplary schedule, the long-acting IL-15R agonist is administered on day 1 and every 21 days thereafter. The antibody is administered weekly, biweekly, or monthly, starting 7-14 days after administration of the long-acting IL-15R agonist. It is understood that the dosing schedule for either or both of the long-acting IL-15R agonist and antibody can be adjusted during the treatment period. As an example, the long-acting IL-15R agonist and / or antibody can be administered weekly for a period (e.g., 4-8 weeks), then biweekly or monthly for a subsequent period.

[0128] The therapeutic methods described herein can be continued as long as the clinician overseeing the patient's care deems the therapeutic method effective. Non-limiting parameters that indicate that the therapeutic method is effective include: tumor shrinkage (in terms of weight and / or volume); reduction in the number of individual tumor colonies; tumor disappearance; and progression-free survival.

[0129] Exemplary lengths of time for courses of treatment described herein include about 1 week; 2 weeks; about 3 weeks; about 4 weeks; about 5 weeks; about 6 weeks; about 7 weeks; about 8 weeks; about 9 weeks; about 10 weeks; about 11 weeks; about 12 weeks; about 13 weeks; about 14 weeks; about 15 weeks; about 16 weeks; about 17 weeks; about 18 weeks; about 19 weeks; about 20 weeks; about 21 weeks; about 22 weeks; about 23 weeks; about 24 weeks; about 7 months; about 8 months; about 9 months; about 10 months; about 11 months; about 12 months; about 13 months; about 14 months; about 15 months; about 16 months; about 17 months; about 18 months; about 19 months; about 20 months; about 21 months; about 22 months; about 23 months; about 24 months; about 30 months; about 3 years; about 4 years, and about 5 years. It is understood that the dosing frequency of the antibody and the long-acting IL-15 receptor agonist can be the same or different, and furthermore, the dosing frequency or schedule of the antibody and / or the long-acting IL-15 receptor agonist can be designed to achieve a commensurate duration of receptor occupancy / antigen binding.

[0130] In some cases, the antibody and / or long-acting IL-15 receptor agonist are included in a composition that also includes one or more pharma- ceutically acceptable excipients. Exemplary excipients include, without limitation, those selected from the group consisting of carbohydrates, inorganic salts, antibacterial agents, antioxidants, surfactants, buffers, acids, bases, amino acids, and combinations thereof.

[0131] Carbohydrates such as sugars, derivatized sugars, e.g., alditols, aldonic acids, esterified sugars, and / or sugar polymers may be present as excipients. Specific carbohydrate excipients include, for example: monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myo-inositol, cyclodextrin, etc.

[0132] The excipient may also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, potassium phosphate, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.

[0133] The one or more compositions may also include an antimicrobial agent to prevent or inhibit the growth of microorganisms. Non-limiting examples of antimicrobial agents suitable for one or more embodiments of the present disclosure include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.

[0134] Antioxidants can also be present in the composition.Antioxidants are used to prevent oxidation, thereby preventing the deterioration of conjugate or other components of the preparation.Suitable antioxidants for use in one or more embodiments of the present disclosure include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[0135] Surfactants may be present as excipients. Exemplary surfactants include polysorbates such as "Tween 20" and "Tween 80", and Pluronics such as F68 and F88 (both available from BASF, Mount Olive, New Jersey); sorbitan esters; lipids such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (but preferably not in liposomal form), fatty acids, and fatty acid esters; steroids such as cholesterol; and IL-15 chelators such as EDTA, zinc, and other such suitable cations.

[0136] Acids or bases may be present in the composition as excipients.Non-limiting examples of acids that can be used include acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof.Examples of suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate, and combinations thereof.

[0137] One or more amino acids can be present as excipients in the compositions described herein. Exemplary amino acids in this regard include arginine, lysine and glycine. Suitable additional pharma- ceutically acceptable excipients can be found, for example, in Handbook of Pharmaceutical Excipients, 7th ed. th ed., Rowe, R.C., Ed., Pharmaceutical Press, 2012.

[0138] In some embodiments, the long-acting IL-15 receptor agonist is contained in a composition comprising potassium phosphate and trehalose. In some embodiments, the composition is a liquid formulation comprising sterile water for injection. In some other embodiments, the composition is a lyophilized powder suitable for reconstitution.

[0139] According to the methods described herein, the long-acting IL-15 receptor agonist and the antibody are administered to the patient in a therapeutically effective amount. One skilled in the art can determine how much of a given long-acting IL-15 receptor agonist and / or antibody is sufficient to provide a clinically relevant amount (e.g., antagonizing activity at the IL-15 receptor, or antibody binding to the desired receptor / antigen, respectively). For example, one skilled in the art can consult the literature and / or administer a series of increasing amounts of the long-acting IL-15 receptor agonist to determine the amount or amounts that provide clinically antagonizing activity of IL-15. It is understood that the therapeutic amount of one or both of the long-acting IL-15 receptor agonist and the antibody may be lower than the therapeutically effective amount of either component when administered alone. The synergistic effect of administering a long-acting IL-15 receptor agonist in combination with an antibody described herein may allow lower doses of either (for one or both of the components of the combination) to be administered, or may allow greater efficacy at doses of either component determined separately. In general, a therapeutically effective amount of an antibody will be in the range of about 0.01 to about 100 mg / kg, inclusive.In some specific, but non-limiting, embodiments, the antibody is administered at a concentration of about 0.01-50 mg / kg, about 0.05-50 mg / kg, about 0.1-50 mg / kg, about 0.5-50 mg / kg, about 1-50 mg / kg, about 5-50 mg / kg, about 10-50 mg / kg, about 15-50 mg / kg, about 20-50 mg / kg, about 30-50 mg / kg, about 40-50 mg / kg, about 0.01-40 mg / kg, about 0.05-40 mg / kg, about 0.1-40 mg / kg, about 0.5-40 mg / kg, approximately 1~40mg / kg, approximately 5~40mg / kg, approximately 10~40mg / kg, approximately 15~40mg / kg, approximately 20~40mg / kg, approximately 30~40mg / kg, approximately 0.01~30mg / kg, approximately 0.05~30mg / kg, approximately 0.1~ 30mg / kg, approx. 0.5~30mg / kg, approx. 1~30mg / kg, approx. 5~30mg / kg, approx. 10~30mg / kg, approx. 15~30mg / kg, approx. 20~30mg / kg, approx. 0.05~25mg / kg, approx. 0.1~25mg / kg, approx. 0.5~25mg / kg, approx. 1~25mg / kg, approx. 5~25mg / kg, approx. 10~25mg / kg, approx. 15~25mg / kg, approx. 20~25mg / kg, approx. 0.01~20mg / kg, approx. 0.05~20mg / kg, approx. 0.1~20mg / kg, approximately 0.5~20mg / kg, approximately 1~20mg / kg, approximately 5~20mg / kg, approximately 10~20mg / kg, approximately 15~20mg / kg, approximately 0.01~15mg / kg, approximately 0.05~15mg / kg, approximately 0.1~15mg / kg, approximately 0. It is administered or contained in a composition at a dose of 5 to 15 mg / kg, about 1 to 15 mg / kg, about 5 to 15 mg / kg, about 10 to 15 mg / kg, about 0.01 to 10 mg / kg, about 0.05 to 10 mg / kg, about 0.1 to 10 mg / kg, about 0.5 to 10 mg / kg, about 1 to 10 mg / kg, about 5 to 10 mg / kg, about 0.1 to 5 mg / kg, about 0.5 to 5 mg / kg, about 1 to 5 mg / kg, about 0.1 to 1 mg / kg, about 0.5 to 1 mg / kg, or about 0.1 to 0.05 mg / kg.In some embodiments, the antibody is administered at a dose of about 0.01 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 16 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 40 mg / kg, or about 50 mg / kg. It is understood that a therapeutically effective amount of an antibody can be any dose approved by a government regulatory agency.

[0140] In some embodiments, the antibody is rituximab and the therapeutically effective amount is about 100 mg / m 2 ~750mg / m 2 In embodiments, the therapeutically effective amount is about 100 mg / m 2 ~500mg / m 2 , about 150mg / m 2 ~500mg / m 2 , about 200mg / m 2 ~500mg / m 2 , about 375mg / m 2 ~500mg / m 2 , or about 400 mg / m 2 ~500mg / m 2 In some embodiments, the antibody is administered at a dose of about 100 mg / m 2 , about 150mg / m 2 , about 200mg / m 2 , about 250mg / m 2 , about 375mg / m 2 , about 400mg / m 2 , about 500mg / m 2 , or about 750 mg / m 2 is administered at a dose of

[0141] In some embodiments, the antibody is daratumumab and the therapeutically effective amount is in the range of about 10 mg / kg to 50 mg / kg / administration (including extreme values). In embodiments, the therapeutically effective amount is in the range of about 10-25 mg / kg, about 10-20 mg / kg, about 10-16 mg / kg, about 10-15 mg / kg, about 15-50 mg / kg, about 15-25 mg / kg, about 15-20 mg / kg, about 16-50 mg / kg, about 16-25 mg / kg, or about 16-20 mg / kg / administration. In some embodiments, the antibody is administered at a dose of about 10 mg / kg, about 15 mg / kg, about 16 mg / kg, about 20 mg / kg, about 25 mg / kg, or about 50 mg / kg / administration.

[0142] In some embodiments, the antibody is cetuximab and the therapeutically effective amount is about 100 mg / m 2 ~750mg / m 2 In embodiments, the therapeutically effective amount is about 100 mg / m 2 ~500mg / m 2 , about 150mg / m 2 ~500mg / m 2 , about 200mg / m 2 ~500mg / m 2 , about 250mg / m 2 ~500mg / m 2 , about 400mg / m 2 ~500mg / m 2 , about 200mg / m 2 ~400mg / m 2 , about 250mg / m 2 ~400mg / m 2 , or about 300 mg / m 2 ~400mg / m 2 In some embodiments, the antibody is administered at a dose of about 100 mg / m 2 , about 150mg / m 2 , about 200mg / m 2 , about 250mg / m 2 , about 400mg / m 2 , about 500mg / m 2 , or about 750 mg / m 2 is administered at a dose of

[0143] In some embodiments, the antibody is trastuzumab and the therapeutically effective amount is in the range of about 1 mg / kg to 20 mg / kg per dose. In embodiments, the therapeutically effective amount is in the range of about 2 to 20 mg / kg, about 2 to 15 mg / kg, about 2 to 10 mg / kg, about 2 to 8 mg / kg, about 2 to 6 mg / kg, about 2 to 4 mg / kg, about 4 to 10 mg / kg, about 4 to 8 mg / kg, about 4 to 6 mg / kg, about 6 to 10 mg / kg, about 6 to 8 mg / kg, or about 8 to 10 mg / kg. In some embodiments, the antibody is administered at a dose of about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 15 mg / kg, or about 20 mg / kg.

[0144] It is understood that the dosage can be determined by methods known in the art, and the specific dosage can be set by a regulatory agency. A given dosage can be administered periodically, for example, until a clinician determines that an appropriate endpoint (e.g., cure, regression, partial regression, etc.) has been achieved.

[0145] In general, a therapeutically effective amount of a long-acting IL-15 receptor agonist will be in the range of about 0.0001 mg to about 100 mg, or about 0.001 mg to about 100 mg, preferably about 0.01 mg / day to about 75 mg / day, more preferably about 0.10 mg / day to about 50 mg / day. A given dose can be administered periodically, for example, until a clinician determines that an appropriate endpoint (e.g., cure, regression, partial regression, etc.) has been achieved.

[0146] In some embodiments, the therapeutically effective amount of a long-acting IL-15R agonist ranges from about 0.25 to 25 μg / kg. In other embodiments, a therapeutically effective dose is from about 0.25 μg / kg to about 0.1 mg / kg, from about 0.375 μg / kg to about 20 μg / kg, from about 0.375 μg / kg to about 15 μg / kg, from about 0.375 μg / kg to about 10 μg / kg, from about 0.375 μg / kg to about 5.0 μg / kg, from about 0.375 μg / kg to about 1.5 μg / kg, from about 1.0 μg / kg to about 20 μg / kg, from about 1.0 μg / kg to about 15 μg / kg, from about 1.0 μg / kg to about 10 μg / kg, from about 1.0 μg / kg to about 5.0 μg / kg, from about 1 μg / kg to about 1.5 μg / kg, or from about 1.5 μg / kg. g / kg to about 20 μg / kg, about 1.5 μg / kg to about 15 μg / kg, about 1.5 μg / kg to about 10 μg / kg, about 1.5 μg / kg to about 5.0 μg / kg, about 5.0 μg / kg to about 20 μg / kg, about 5.0 μg / kg to about 15 μg / kg, about 5.0 μg / kg to about 10 μg / kg, about 10 about 1.5 μg / kg to about 20 μg / kg, about 10 μg / kg to about 20 μg / kg, about 10 μg / kg to about 15 μg / kg, about 15 μg / kg to about 20 μg / kg, about 0.01 mg / kg to about 0.1 mg / kg / day, or about 0.03 mg / kg to about 0.1 mg / kg / day. In other embodiments, the therapeutically effective dose ranges from about 1 to 10 μg / kg, about 0.03 mg / kg to about 0.1 mg / kg. In some specific, but non-limiting, embodiments, the therapeutically effective dose is about 0.25 μg / kg, about 0.3 μg / kg, about 0.375 μg / kg, about 0.5 μg / kg, about 0.75 μg / kg, about 1 μg / kg, about 1.5 μg / kg, about 2 μg / kg, about 3 μg / kg, about 4 μg / kg, about 4.5 μg / kg, about 5 μg / kg, about 6 μg / kg, about 7 μg / kg, about 8 μg / kg, about 9 μg / kg, about 10 μg / kg, about 15 μg / kg, about 20 μg / kg, about 25 μg / kg, about 0.01 mg / kg, about 0.03 mg / kg, about 0.05 mg / kg, or about 0.1 mg / kg / day.

[0147] It is understood that the doses of long-acting IL-15R agonists described above can refer to conjugates or protein equivalents. In some preferred embodiments, the doses are in protein equivalents.

[0148] In embodiments, the dose levels of the antibody and / or long-acting IL-15R agonist can be reduced when accompanied by an increased dose of the antibody or long-acting IL-15R agonist, respectively. As shown in Example 14, the reduced dose levels for the antibody therapy or long-acting IL-15R agonist were offset by an associated increased dose level of the IL-15R agonist or antibody, respectively, in the mouse tumor model. Thus, both administration of a low dose of antibody in combination with a high dose of a long-acting IL-15R agonist, as well as administration of a high dose of antibody in combination with a low dose of a long-acting IL-15R agonist, reduce the number of Daudi cells in the bone marrow of the mouse tumor model.

[0149] For the doses referred to in the Examples herein, one of skill in the art would be able to convert animal doses (e.g., mice) into doses using conversions known in the art (e.g., Nair et al., J. Basic and Clin. Pharmacy (2016) 7:27-31) can be used to convert to the corresponding dose in humans.

[0150] The unit dosage of any antibody and / or conjugate (again, preferably provided as part of a pharmaceutical preparation) can be administered in a variety of dosing schedules depending on the clinician's judgment, the patient's needs, and so forth. Specific dosing schedules will be known by those skilled in the art and can be determined experimentally using routine methods. Exemplary dosing schedules include, but are not limited to, once daily, three times a week, twice a week, once a week, twice a month (e.g., q / 14 days), once a month (e.g., q / 30 or 31 days, q / 28 days, or q / 21 days) administration, and any combination thereof. It is understood that the dosing schedule can be adjusted as needed, for example, weekly administration for a period of time, and then adjusted to a shorter or longer schedule as needed. Once the desired clinical endpoint is achieved, dosing of the composition is stopped or reduced. In some embodiments, the unit dosage of any conjugate can be administered once to provide sustained effect.

[0151] The actual dose of antibody and / or long-acting IL-15 receptor agonist to be administered will vary depending on the age, weight, and general condition of the subject, as well as the severity of the condition being treated, the judgment of the health care professional, and the conjugate being administered. Therapeutically effective amounts are known to those skilled in the art and / or are described in the pertinent reference texts and literature.

[0152] The combination of the antibody and the long-acting IL-15 receptor agonist is suitable for administration to a patient suffering from a cancer or tumor. The method comprises administering to the patient (preferably provided as part of the same or different pharmaceutical compositions) a therapeutically effective amount of an antibody directed to a tumor antigen and, generally parenterally, a therapeutically effective amount of a long-acting IL-15 receptor agonist.

[0153] The antibody and / or long-acting IL-15 receptor agonist may be administered by any suitable means known in the art. In some embodiments, one or both of the antibody and long-acting IL-15 receptor agonist may be administered parenterally, including subcutaneous, intravenous, intraarterial, intraperitoneal, intracardiac, intrathecal, and intramuscular injections, as well as infusion injections. Suitable formulation types for parenteral administration include, among others, ready-to-inject injection solutions, dry powders that are combined with a solvent before use, ready-to-inject suspensions, dry insoluble compositions that are combined with a vehicle before use, and emulsions and liquid concentrates that are diluted before administration. In some particular embodiments, the antibody and / or long-acting IL-15 receptor agonist is provided in a formulation suitable for intravenous administration and is administered intravenously. In some other embodiments, the antibody and / or long-acting IL-15 receptor agonist is provided in a formulation suitable for subcutaneous administration and is administered subcutaneously. In some additional embodiments, one or both of the antibody and the long-acting IL-15 receptor agonist may be administered intratumorally. Other modes of administration, such as pulmonary, nasal, buccal, rectal, sublingual, and transdermal, are also contemplated.

[0154] The administration method of the long-acting IL-15 receptor agonist (e.g., provided as part of a pharmaceutical composition) can be optionally performed to localize to a specific area. For example, liquid, gel and solid formulations containing the agonist can also be surgically implanted within a disease area (e.g., within a tumor, near a tumor, within an area of ​​inflammation, and near an area of ​​inflammation, etc.). Organs and tissues can also be conveniently imaged to ensure that the desired location is well exposed by the conjugate.

[0155] As described herein, one aspect of the disclosure provides a method that is useful (among other things) for treating a patient suffering from a condition that is responsive to treatment with one or both of these compounds. For example, a patient may be responsive to each of the individual drugs alone as well as the combination, but is more responsive to the combination. As a further example, a patient may not be responsive to one of the individual drugs, but is responsive to the combination. As a further example, a patient may not be responsive to either of the individual drugs alone, but is responsive to the combination.

[0156] As used herein in connection with the treatment of a subject with cancer, the terms "treatment," "treat," and "treating" are intended to include the full spectrum of interventions, e.g., administration of combinations, for the cancer suffered by the subject to reduce, slow, stop, or reverse one or more symptoms of the cancer, or to slow the progression of the cancer, even if the cancer is not actually eliminated. Treatment can include, for example, reducing the severity of symptoms, the number of symptoms, or the frequency of recurrence, e.g., inhibiting tumor growth, arresting tumor growth, or causing regression of an existing tumor. Furthermore, the term "treating cancer" is not intended to be an absolute term and can include, for example, reducing the size of a tumor or the number of cancer cells, putting the cancer into remission, or preventing the growth of the size or number of cancer cells, etc. In some circumstances, treatment according to the present disclosure results in an improved prognosis.

[0157] For example, improvement of cancer or cancer-related disease can be characterized as a complete or partial response. A "complete response" refers to the absence of clinically detectable disease, with normalization of any previously abnormal radiographic tests, bone marrow, and cerebrospinal fluid (CSF) or abnormal monoclonal protein measurements. A "partial response" refers to at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in all measurable tumor burden (i.e., the number of malignant cells present in the subject, or the volume of the measured tumor mass, or the amount of abnormal monoclonal protein) in the absence of new lesions. The term "treatment" contemplates both complete and partial responses.

[0158] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.

[0159] As used herein, "tumor" and "solid tumor" refer to all lesions and neoplastic cell growth and proliferation, either malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0160] As used herein, the term "enhanced" or "enhancing", e.g., in the context of enhanced response, refers to an improvement in the ability of a subject or tumor cells to respond to a treatment, e.g., as disclosed herein, compared to a given baseline or reference treatment. For example, enhanced response can include an increase in responsiveness of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% or more, based on any one or more indicators of responsiveness to a treatment. As used herein, "enhanced" or "enhancing" can also refer to increasing the number of subjects that respond favorably to a treatment, e.g., compared to a given standard for such comparison.

[0161] Exemplary conditions include, for example, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, colon cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, head and neck cancer, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, , renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, colorectal cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma (including, for example, uveal melanoma, mucosal melanoma, and leptomeningeal melanoma), neuroblastoma, retinoblastoma, squamous cell carcinoma of the head and neck, leukemia, and lymphoma. Further exemplary conditions are hematopoietic tumors, including, but not limited to, non-Hodgkin's lymphoma and multiple myeloma.

[0162] In one particular method, the monoclonal antibody and the long-acting IL-15 receptor agonist are used to treat hematological malignancies such as leukemia or lymphoma. In yet another method, the monoclonal antibody and the long-acting IL-15 receptor agonist are used to treat solid cancers. In some embodiments, the solid cancers are selected from, but not limited to, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, renal cell carcinoma, cervical cancer, and small cell lung cancer, non-small cell lung cancer.

[0163] In some embodiments, a combination of an antibody described herein and a long-acting IL-15 receptor agonist (or related composition), when administered to a subject at a therapeutically effective dose, is effective to stimulate NK activation and / or proliferation to a greater extent than observed for either agent administered alone and at a comparable dose, e.g., in a suitable animal model. In some embodiments, a combination of a monoclonal antibody described herein and a long-acting IL-15 receptor agonist or composition, when administered to a subject at a therapeutically effective dose, is effective to provide increased tumor clearance than observed for either agent administered alone and at a comparable dose, e.g., when evaluated in a suitable animal model. In some embodiments, a combination of a monoclonal antibody described herein and a long-acting IL-15 receptor agonist or composition, when administered to a subject at a therapeutically effective dose, is effective to provide increased and / or prolonged subject survival, e.g., increased survival rate, or a greater extent of tumor regression, than observed for either agent administered alone and at a comparable dose, e.g., when evaluated in a suitable animal model. In some embodiments, the combination of an antibody and a long-acting IL-15 receptor agonist or composition described herein, when administered to a subject at a therapeutically effective dose, is effective to provide increased activity of one or both of the long-acting IL-15 receptor agonist and the antibody, particularly in tissue compartments such as, for example, the bone marrow. In some embodiments, the combination of an antibody and a long-acting IL-15 receptor agonist or composition described herein, when administered to a subject at a therapeutically effective dose, is effective to provide enhanced NK cell degranulation and / or increased NK cell viability. In embodiments, administration of an antibody and a long-acting IL-15 receptor agonist or composition described herein, when administered to a subject at a therapeutically effective dose, is effective to treat cancers that are resistant to treatment with a monoclonal antibody alone.

[0164] Notable findings regarding the combination therapy described herein are set forth below and further detailed in the Examples contained herein. In the exemplary Daudi B cell lymphoma mouse model described in Example 1, administration of an exemplary long-acting IL-15 receptor agonist and an anti-CD20 monoclonal antibody, rituximab, was effective in extending survival when compared to administration of a long-acting IL-15 receptor agonist or antibody alone. As shown in Figure 1, mice treated with a long-acting IL-15 receptor agonist and an anti-CD20 antibody, rituximab, had a 100% survival rate after about 45 days. Mice treated with an isotype control or rituximab alone had a 0% survival rate after about 30 days. Mice treated with an isotype control and a long-acting IL-15 receptor agonist had a 0% survival rate after about 35 days. In a further exemplary mouse model described in Example 2, administration of a long-acting IL-15R agonist (e.g., mono(methoxyPEG-N-butanamide) interleukin-15) and an anti-CD38 monoclonal antibody, daratumumab, was effective in extending survival when compared to administration of a long-acting IL-15 receptor agonist or antibody alone. As shown in FIG. 5, mice treated with an illustrative long-acting IL-15 receptor agonist (mono(methoxyPEG-N-butanamide) interleukin-15) and an anti-CD38 antibody, daratumumab, had a median survival of 55 days (after tumor injection) compared to 28.5 days for the antibody alone. As described in Example 13, administration of a long-acting IL-15R agonist (e.g., mono(methoxyPEG-N-butanamide) interleukin-15) and an anti-CD38 monoclonal antibody, daratumumab, to SCID mice resulted in a greater than two-fold increase in survival. In contrast, administration of a long-acting IL-15R agonist and the anti-CD38 monoclonal antibody daratumumab to SCID beige mice, which have reduced NK cell functionality, resulted in reduced increased survival. These results indicate that the effect of administration of a long-acting IL-15R agonist and the anti-CD38 monoclonal antibody daratumumab on NK cell functionality results in longer survival.Examples 18 and 19 demonstrate increased survival upon administration of a long-acting IL-15R agonist (e.g., mono(methoxyPEG-N-butanamide) interleukin-15) and the anti-EGFR monoclonal antibody, cetuximab, in two exemplary colorectal carcinoma mouse models: the HT-29 colorectal carcinoma cell model and the HCT-116 colorectal carcinoma xenograft model.

[0165] In some embodiments, administration of a long-acting IL-15 receptor agonist and an antibody described herein is effective to provide an increase in the subject's survival time relative to treatment with the long-acting IL-15 receptor agonist and / or the antibody alone. In some other embodiments, administration of a long-acting IL-15 receptor agonist and an antibody described herein is effective to provide an increase in the subject's survival time relative to conventional treatment. In one or more embodiments, survival time is increased by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24 months or more when compared to a subject treated with only one of the components of the combination described herein or a conventional therapy for the same indication. In some embodiments, survival time is at least about 1, 2, 3, 4, 5 years or more.

[0166] In some embodiments, the combination therapy of the present invention is effective in increasing the progression-free survival (survival without substantial progression of the disease being treated) of a subject. In embodiments, the progression-free survival is increased by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24 months or more when compared to a subject treated with only one of the components of the combination described herein or with a conventional therapy for the same indication. In some embodiments, the progression-free survival is at least about 1, 2, 3, 4, 5 years or more.

[0167] In certain embodiments, the combination therapy of the present invention is effective to increase the response rate of a subject treated with the combination, hi embodiments, the combination of the present invention is effective to increase the response of a treated subject by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 100% when compared to a subject treated with only one of the components of the combination described herein or a conventional therapy for the same indication.

[0168] This combination therapy is also effective in promoting NK cell expansion in subjects to a degree that is significantly enhanced compared to either agent administered alone or to untreated subjects. In an exemplary Daudi B cell lymphoma xenograft mouse model as described in Example 2, administration of a long-acting IL-15 receptor agonist, more specifically mono(methoxy PEG-N-butanamide) interleukin-15, and an anti-CD38 monoclonal antibody, daratumumab, resulted in a significant increase in NK cells in the bone marrow of treated mice. As seen in Figure 3, mice treated with a long-acting IL-15 receptor agonist and an anti-CD38 antibody, daratumumab, exhibited a significant increase in NK cell proliferation (cell number) in the bone marrow when compared to untreated mice or mice treated with either agent alone. As seen in Figure 4, mice treated with a long-acting IL-15 receptor agonist and an anti-CD38 antibody, daratumumab, exhibited a significant decrease in the number of B cell lymphoma cells in the bone marrow when compared to untreated mice or mice treated with either agent alone. Example 16 shows increased proliferation of NK cells in tumor tissue following administration of an exemplary long-acting IL-15R agonist (e.g., mono(methoxyPEG-N-butanamide) interleukin-15) and an anti-EGFR monoclonal antibody, cetuximab, in an exemplary FaDu mouse model for human head and neck squamous cell carcinoma.

[0169] In some embodiments, administration of a long-acting IL-15 receptor agonist and an antibody described herein is effective to provide increased NK cell expansion and proliferation. In some further embodiments, the combination of the present invention is effective to increase NK cell expansion or proliferation in a treated subject by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 100% when compared to a subject treated with only one of the components of the combination described herein or a conventional therapy for the same indication. In embodiments, the combination of the present invention is effective to increase NK cell expansion or proliferation in a treated subject by at least about 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, or more when compared to a subject treated with only one of the components of the combination described herein or a conventional therapy for the same indication.

[0170] In some embodiments, administration of a long-acting IL-15 receptor agonist and an antibody described herein is effective to provide increased cancer cell elimination / reduction in cancer cell count. In some embodiments, the combination of the present invention is effective to increase the elimination of cancer cells in a treated subject by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 100% when compared to a subject treated with only one of the components of the combination described herein or a conventional therapy for the same indication. In embodiments, the combination of the present invention is effective to increase the elimination of cancer cells in a treated subject by at least about 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, or more when compared to a subject treated with only one of the components of the combination described herein or a conventional therapy for the same indication.

[0171] In an exemplary Daudi B cell lymphoma xenograft mouse model as described in Example 2, administration of an exemplary long-acting IL-15 receptor agonist (e.g., mono(methoxyPEG-N-butanamide) interleukin-15) and an anti-CD38 monoclonal antibody, daratumumab, resulted in a significant increase in granzyme B expression in bone marrow NK cells of treated mice. As seen in FIG. 6, the majority of mice treated with an exemplary long-acting IL-15 receptor agonist (e.g., mono(methoxyPEG-N-butanamide) interleukin-15) and an anti-CD38 antibody, daratumumab, exhibited increased granzyme B expression in NK cells in the bone marrow when compared to untreated mice or mice treated with the antibodies alone. As further seen in FIG. 6, this combination treatment activated the function of the majority of NK cells as indicated by an increase in the granzyme B (cytotoxic protease) marker. As shown in FIG. 14 and described in Example 11, administration of an exemplary long-acting IL-15 receptor agonist (e.g., mono(methoxyPEG-N-butanamide) interleukin-15) with an anti-CD38 monoclonal antibody, daratumumab, is effective to increase granzyme B expression on NK cells in bone marrow on a cell-by-cell basis when compared to untreated mice or mice treated with the antibody alone. In an exemplary FaDu human head and neck squamous cell carcinoma mouse model, as described in Example 16, administration of an exemplary long-acting IL-15 receptor agonist (e.g., mono(methoxyPEG-N-butanamide) interleukin-15) with an anti-EGFR monoclonal antibody, cetuximab, is effective to increase granzyme B expression in NK cells present in blood and tumor samples when compared to untreated mice.

[0172] In some embodiments, administration of a long-acting IL-15 receptor agonist and an antibody described herein is effective to provide increased Granzyme B expression in NK cells. In one or more embodiments, the combination of the present invention is effective to increase Granzyme B expression in NK cells of a treated subject by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 100% when compared to a subject treated with only one of the components of the combination described herein or a conventional therapy for the same indication. In embodiments, the combination of the present invention is effective to increase Granzyme B expression in NK cells of a treated subject by at least about 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, or more when compared to a subject treated with only one of the components of the combination described herein or a conventional therapy for the same indication.

[0173] In an exemplary mouse model, as described in Example 10, administration of an exemplary long-acting IL-15 receptor agonist (mono(methoxyPEG-N-butanamide) interleukin-15) with an anti-CD38 monoclonal antibody, daratumumab, is effective to increase the percentage of NK cells expressing cell surface CD16 receptors when compared to administration of the long-acting IL-15 receptor agonist or the antibody alone. In a further exemplary colorectal carcinoma mouse model, as described in Example 15, administration of a long-acting IL-15 receptor agonist with the anti-EGFR monoclonal antibody cetuximab is effective to increase CD16 cell surface expression in blood and tumor samples when compared to untreated mice. In some embodiments, the long-acting IL-15R agonist, when used in combination with an antibody directed against CD16 and having an ADCC mechanism of action, provides enhanced surface expression of the low affinity Fc receptor CD16, providing an additional immunotherapeutic effect.

[0174] In a third aspect, there is provided herein a kit comprising an antibody and a long-acting IL-15 receptor agonist as described above. The kit may further comprise instructions for use and, optionally, any medical supplies or devices for administration of the antibody and long-acting IL-15 receptor agonist, as required.

[0175] While the methods, combinations, kits, etc. have been described in connection with preferred specific embodiments thereof, it should be understood that the foregoing description, as well as the following examples, are intended to be illustrative and are not intended to limit the scope of the disclosed methods, combinations, kits, etc. Other aspects, advantages and modifications within the scope of the present disclosure will be apparent to those skilled in the art.

[0176] All articles, works, patents and other publications referred to in this specification are incorporated herein by reference in their entirety. In the event of any discrepancy between the teachings of this specification and the art incorporated by reference, the meanings of the teachings and definitions in this specification shall prevail (especially with respect to the terms used in the claims appended hereto). For example, if this application and the publications incorporated by reference define the same term differently, the definition of the term shall be maintained within the teachings of the document in which the definition is located. EXAMPLES

[0177] It should be understood that the foregoing description and following examples are illustrative of the methods, combinations, kits, and the like provided herein, but are not limiting in scope. Other aspects, advantages, and modifications will be apparent to those skilled in the art to which this disclosure pertains.

[0178] In the following examples, efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, temperatures are in degrees Celsius and pressures are at or near marine pressure. Each of the following examples is considered an instruction for one of ordinary skill in the art to practice one or more of the embodiments described herein.

[0179] Materials and Methods Exemplary long-acting IL-15 receptor agonists used in the examples below, unless otherwise indicated, generally have the following structure: [ka] (where n has a value of about 909 (so that the polyethylene glycol portion of the molecule has a weight average molecular weight of about 40 kilodaltons). As explained by [mPEG 40kD Mono(methoxyPEG-N-butanamide), also referred to as -CH2CH2CH2C(O)NH]-IL-15 40kD Interleukin-15. This exemplary long-acting IL-15 receptor agonist is described herein as conjugate 1 or compound 1 (these terms are used interchangeably in the following examples). The preparation of mono(methoxyPEG-N-butanamide) interleukin-15 is described in Example 1 of PCT Application / US Patent Publication No. 2018 / 032817 (WO 2018 / 213341). The doses and concentrations in these examples are based on IL-15 protein content.

[0180] SDS-PAGE analysis Samples may be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using an Invitrogen gel electrophoresis system (XCell SureLock Mini-Cell). Samples were mixed with sample buffer and the prepared samples were loaded onto NuPAGE Novex precast gels and run for approximately 30 minutes.

[0181] RP-HPLC analysis Reverse phase chromatography (RP HPLC) analysis may be performed on an Agilent 1200 HPLC system (Agilent). Samples may be analyzed using a Poroshell 300SB-C3 column (2.1x75mm, Agilent) at 60°C. The mobile phases used may be 0.1% TFA / H2O (A) and 0.1% TFA / CH3CN (B). The column flow rate may be 0.5ml / min. Eluted proteins and PEG-protein conjugates are detected using UV at 280nm.

[0182] Bioassay Potency assay based on STAT5 phosphorylation in CTLL-2 cells (mouse T cells) In the phospho-STAT5 assay following receptor binding, downstream cell signaling can then activate signal transducer and activator of transcription 5 (STAT5) via phosphorylation to promote gene expression and induce cell proliferation. As described in PCT Application No. PCT / US2018 / 032817, which is incorporated herein by reference, activation of phospho-STAT5 is measured in appropriate cell lines using a phospho-STAT5 / total STAT5 multiplex assay (Meso Scale Discovery, MD) in response to sample and reference treatments over a period of approximately 10 minutes.

[0183] HuPBMC-pStat5 assay The potency of IL-15 or long-acting IL-15 receptor agonists on various cells can be determined by a phospho-STAT5 (Y694) dose response assay, as further described in PCT Application No. PCT / US2018 / 032817.

[0184] Example 1 Evaluating survival in the DAUDI B-cell lymphoma xenograft model following administration of a long-acting IL-15 receptor agonist and an exemplary anti-CD20 monoclonal antibody, rituximab On day 0, SCID mice were injected with 1 × 10 7Daudi cells were injected intravenously. The mice were divided into four groups. On days 14 and 17 after Daudi cell injection, the groups were treated by injection as follows: Group 1: isotype control antibody, Group 2: 40 mg / kg IP Rituximab, Group 3: 0.3 mg / kg mono(methoxyPEG-N-butanamide). 40kD Interleukin-15 (long-acting IL-15 receptor agonist), Group 4: 40 mg / kg IP rituximab and 0.3 mg / kg SC mono(methoxyPEG-N-butanamide) 40kD Interleukin-15 (combination).

[0185] Survival was determined by measuring the onset of hind limb paralysis as a surrogate parameter, and the results are shown in FIG.

[0186] As can be seen in Figure 2, the combined administration of rituximab and mono(methoxyPEG-N-butanamide) interleukin-15 (shown as "IL-15 receptor agonist" in Figure 2) provided a significant increase in long-term survival when compared to administration of either component when administered as monotherapy (see horizontal line marked "Combination"). The results demonstrate the ability of a long-acting receptor agonist, mPEG-BA-IL-15, when administered in combination with an illustrative anti-CD20 monoclonal antibody, such as rituximab, to significantly enhance survival time in mice when evaluated in an in vivo lymphoma model.

[0187] Example 2 Assessment of cell numbers and granzyme B expression in bone marrow in the DAUDI B cell lymphoma xenograft model following administration of a long-acting IL-15 receptor agonist and an exemplary anti-CD38 monoclonal antibody, daratumumab On day 0, SCID mice (N=6 / group) were administered 1×10 7Daudi cells were inoculated intravenously and treated with a single dose of the anti-human CD38 antibody, daratumumab (0.5 mg / kg IP, 14 days after Daudi cell inoculation), and two doses of mono(methoxyPEG-N-butanamide) interleukin-15 (0.3 mg / kg SC, one dose each at 14 and 21 days after inoculation). Mice were also treated with monotherapy: daratumumab (0.5 mg / kg IP, 14 days after Daudi cell inoculation), or two doses of mono(methoxyPEG-N-butanamide) interleukin-15 (0.3 mg / kg SC, one dose each at 14 and 21 days after inoculation). 40kD Interleukin-15 (0.3 mg / kg SC, 14 and 21 days after inoculation).

[0188] Increase and activation of NK cells in bone marrow and decrease of lymphoma were observed with mono(methoxyPEG-N-butanamide) 40kD Three days after the second dose of interleukin-15 (day 24), the tumor was assessed by flow cytometry as shown in Figures 3 and 4, respectively. As shown in Figure 3, the combination of mono(methoxyPEG-N-butanamide) interleukin-15 with daratumumab was significantly higher than that of mono(methoxyPEG-N-butanamide) as monotherapy. 40kD This resulted in a significant increase in NK cells in the bone marrow of treated mice compared to mice treated with interleukin-15 and daratumumab (p=0.0026 and p<0.0001, respectively) and compared to untreated controls (p<0.0001). (One-way ANOVA, Tukey's multiple comparison test). Mono(methoxyPEG-N-butanamide) 40kD A 19-fold increase in NK cell numbers was observed for the combination therapy versus a 10-fold increase for interleukin-15 monotherapy, demonstrating synergy between the exemplary monoclonal antibody and mono(methoxyPEG-N-butanamide) interleukin-15 within bone marrow tissue.

[0189] As shown in Figure 4, mono(methoxyPEG-N-butanamide)interleukin-15 in combination with daratumumab significantly improved B-cell lymphoma reduction compared to mono(methoxyPEG-N-butanamide)interleukin-15 and daratumumab monotherapy (p=0.02 and p=0.001, respectively) and compared to untreated controls (p<0.0001) (One-way ANOVA, Tukey's multiple comparison test).

[0190] Granzyme B expression was measured in bone marrow NK cells as shown in Figure 6. As shown in Figure 6, mono(methoxy PEG-N-butanamide) interleukin-15 is effective in controlling NK cell dynamics, even in highly specialized immune compartments, and is effective in increasing granzyme B expression in the bone marrow of treated mice, either alone or in combination with a monoclonal antibody such as daratumumab. Thus, the combination of mono(methoxy PEG-N-butanamide) interleukin-15 with a tumor-binding antibody such as daratumumab not only significantly increases the number of NK cells in the bone marrow as shown by the granzyme B (cytotoxic protease) marker in Figure 6, but is also effective in activating the lethal function of the majority (>70%) of these NK cells.

[0191] These results further demonstrate that mono(methoxyPEG-N-butanamide) interleukin-15 is effective in substantially enhancing NK cell-mediated ADCC when administered together with an anti-tumor antibody.

[0192] Example 3 Assessment of survival in the DAUDI B-cell lymphoma xenograft model following treatment with a long-acting IL-15 receptor agonist and the anti-CD38 monoclonal antibody, daratumumab 1×10 7 SCID mice (N=8 / group) inoculated intravenously with Daudi B-cell lymphoma cells were treated with a single dose of daratumumab (0.5 mg / kg, IP, 14 days after inoculation) and a total of three doses of mono(methoxyPEG-N-butanamide). 40kDInterleukin-15 (0.3 mg / kg, SC, administered 14, 21, and 28 days after tumor inoculation). Untreated mice as controls and mice treated with daratumumab as monotherapy were also evaluated. Survival of tumor-inoculated mice was measured by body condition scoring as an endpoint marker. Results are presented graphically in FIG. 5.

[0193] As shown in Figure 5, the combination of mono(methoxy PEG-N-butanamide) interleukin-15 with daratumumab significantly increases median survival compared to daratumumab monotherapy (p<0.05, log-rank test). The median survival for the group treated with the combination therapy was 55 days after tumor injection, whereas the median survival for the daratumumab monotherapy group was 28.5 days.

[0194] Example 4 Modulation of NK cell activation and inhibitory receptor cell surface expression following treatment with tumor-targeted therapeutic antibodies and long-acting IL-15 receptor agonists Balb / c mice (n=6 / group) bearing Daudi B-cell lymphoma tumors in the bone marrow were administered a single 0.5 mg / kg dose of the clinical antibody daratumumab (DARZALEX®, Janssen Biotech) targeting the human CD38 protein or a non-specific control antibody with a matching isotype intraperitoneally on day 14 after intravenous inoculation with Daudi cells. Additional groups received mono(methoxyPEG-N-butanamide) at a dose of 0.3 mg / kg as a single agent on days 14 and 21 after inoculation with Daudi cells. 40kD Interleukin-15 (conjugate 1) was administered subcutaneously or combination treatment with conjugate 1 administered subcutaneously at a dose of 0.03 mg / kg or 0.3 mg / kg on days 14 and 21 after Daudi cell inoculation and daratumumab administered intraperitoneally at a dose of 0.5 mg / kg on day 14 after Daudi cell inoculation was administered.

[0195] Three days after the second Conjugate 1 administration (day 24 post-Daudi inoculation), bone marrow was harvested and the percentage of NK cells was measured in whole bone marrow by flow cytometry. Expression of the cell surface activation receptor NKG2D and the inhibitory checkpoint receptor NKG2A are shown in Figures 7A and 7B, respectively.

[0196] The results are provided in Table 1 and Figures 7A-7B. All groups receiving therapeutic doses of conjugate 1 as a single agent or in combination with daratumumab showed an increased percentage of NK cells expressing NKG2A on the cell surface (Figure 7A) and a decreased percentage of NK cells expressing NKG2D on the cell surface (Figure 7B) when compared to all treatments without conjugate 1. The increased inhibitory checkpoint receptor activation and decreased NK cells expressing activating receptors, respectively, suggested NK cell activation by conjugate 1 in the presence of a therapeutic tumor-targeting antibody, exemplified in this example by daratumumab.

[0197] [Table 1]

[0198] Example 5 Induction of human NK cell proliferation following treatment with a long-acting IL-15 receptor agonist and immunoglobulin NK cell proliferation was induced in vitro in human peripheral blood mononuclear cells (PBMCs) labeled with CFSE preparations. CFSE-labeled PBMCs were treated with (i) mono(methoxyPEG-N-butanamide) at a concentration of 100 ng / ml; 40kDThe cells were incubated in vitro for 5 days in the presence of interleukin-15 (conjugate 1); (ii) on cell culture plastic previously coated overnight with a solution of human whole immunoglobulin G (hIgG) at a concentration of 0.1 mg / ml, (iii) on cell culture plastic previously coated overnight with a solution of human whole immunoglobulin G (hIgG) at a concentration of 0.1 mg / ml and in the presence of conjugate 1 at a concentration of 100 ng / ml; or (iv) on regular cell culture plastic. After the incubation period, cell proliferation, as indicated by a decrease in CFSE in cells that had undergone cell doubling, was measured using flow cytometry to measure CFSE levels in CD56+ human NK cells within the PBMC preparations in each of the four incubation conditions (Figures 8A-8D).

[0199] The results are provided in Table 2 and Figures 8A-8D. The data show that when PBMCs were exposed to neither conjugate 1 nor hIgG, only 2.25% of NK cells proliferated. In the presence of hIgG alone, 3.92% of NK cells proliferated. Conjugate 1 exposure resulted in 49.7% proliferation of NK cells. However, when PBMCs were exposed to both hIgG and conjugate 1, 72% of NK cells proliferated, indicating that the combination treatment of hIgG and conjugate 1 proliferated significantly more cells than the sum of hIgG and conjugate 1 monotherapy alone.

[0200] [Table 2]

[0201] Example 6 Functional activation of human NK cells following administration of a long-acting IL-15 receptor agonist and immunoglobulin Functional activation of human NK cells was induced in vitro in human peripheral blood mononuclear cell (PBMC) preparations. PBMCs from two donors were treated with (i) mono(methoxyPEG-N-butanamide) at a concentration of 1 μg / ml; 40kDIncubated overnight in the presence of interleukin-15 (conjugate 1); (ii) on cell culture plastic previously coated overnight with a solution of human whole immunoglobulin G (hIgG) at a concentration of 0.1 mg / ml, (iii) on cell culture plastic previously coated overnight with a solution of human whole immunoglobulin G (hIgG) at a concentration of 0.1 mg / ml and in the presence of conjugate 1 at a concentration of 1 μg / ml; or (iv) on regular cell culture plastic.

[0202] After the incubation period, NK cell activation was measured using flow cytometry by measuring the increase in median fluorescence intensity (MFI) signal of CD69 (Figure 9A) and CD107a (Figure 9B) detection antibodies on the surface of CD56+ human NK cells in PBMCs. Functional activation of NK cells was assessed by calculating the fold change in MFI compared to control conditions in which cells were not exposed to either hIgG or conjugate 1. An increase in the fold change in activation marker MFI indicated an increase in these marker proteins on individual NK cells, indicating functional activation of NK cells. Secreted granzyme B concentrations, also indicative of NK cell activation, were measured by ELISA in the culture medium at the end of the experiment (Figure 9C).

[0203] The results are provided in Table 3 and Figures 9A-9C. The data show that CD69 MFI on NK cells increased 1.4-fold in the presence of hIgG. Exposure to conjugate 1 resulted in a 1.8-fold increase in CD69 MFI. However, when PBMCs were exposed to both hIgG and conjugate 1, CD69 MFI increased 3.2-fold on NK cells, greater than each individual treatment. CD107a MFI on NK cells increased 1.8-fold in the presence of hIgG. Exposure to conjugate 1 resulted in a 2.6-fold increase in CD107a MFI. However, when PBMCs were exposed to both hIgG and conjugate 1, CD107a MFI increased 4.3-fold on NK cells, greater than each individual treatment. Secreted granzyme B concentration did not increase in the presence of hIgG compared to control conditions. Exposure to conjugate 1 resulted in a 4.1-fold increase in granzyme B concentration in the culture medium. However, when PBMCs were exposed to both hIgG and conjugate 1, granzyme B concentrations increased 18.7-fold in the culture medium, significantly greater than either treatment individually.

[0204] [Table 3]

[0205] Example 7 Effects of long-acting IL-15 receptor agonists on signal transduction and proliferation in vitro KHYG-1 cells were cultured in 20% FBS, 2 mM L-glutamine, and 20 ng / mL The cells were cultured in RPMI-1640 medium supplemented with IL-2 (complete growth medium) in a humidified incubator at 37° C. and 5% CO 2 .

[0206] One day prior to treatment with test compounds, cells were seeded at a density of 500,000 viable cells / ml in complete growth medium and incubated overnight under normal growth conditions. On the day of the assay, KHYG-1 cells were washed three times with DPBS, resuspended in assay medium (RPMI-1640 medium supplemented with 20% FBS, 2 mM L-glutamine) and incubated for 4-5 hours at 37°C, 5% CO2. Cells were cultured at the appropriate density (8 × 10 5 The concentration was adjusted to 100,000 cells / mL and 50,000 cells / well were dispensed into wells of a 96-well plate (View-Plate 96, Perkin-Elmer).

[0207] Assessment of STAT5 phosphorylation KHYG-1 cells were stimulated with IL-15 or a long-acting IL-15 receptor agonist (mono(mPEG-butanamide)IL-15) followed by incubation for 10 min at 37° C., 5% CO. Treated cells were lysed on ice.

[0208] Detection of phospho-STAT5 protein levels in cell lysates was performed using a Phospho(Tyr694) / Total STAT5a,b Assay Whole Cell Lysate kit (MSD product #K15163D). Relative light unit (RLU) signals corresponding to phosphor-STAT5 were collected using a Sector® Imager 2400 plate reader (MSD), and the results are provided in FIG. 10A. Both IL-15 and long-acting IL-15 receptor agonists led to dose-dependent phosphorylation of STAT5 in the human NK cell line KHYG-1, indicating that these compounds were effective in signal transduction in vitro. Long-acting IL-15 receptor agonists induced phosphorylation of STAT5, leading to a maximum phosphorylation (percentage) of STAT5 that was similar to that of IL-15, albeit with approximately 3-fold lower potency. This data suggests that a long-acting IL-15 receptor agonist can efficiently engage and activate downstream signaling in the KHYG-1 cell line.

[0209] Assessment of cell proliferation Stimulation of KHYG-1 cells was initiated by transfer of IL-15 or a long-acting IL-15 receptor agonist (mono(mPEG-butanamide)IL-15) to duplicate wells containing the cells, followed by incubation for 48 hours at 37°C, 5% CO2.

[0210] Cell growth in each well was measured by measuring ATP levels using CellTiter-Glo 2.0 according to the manufacturer's instructions. The effect of long-acting IL-15 receptor agonists on KHYG-1 cell proliferation was compared to IL-15 after 48 hours of treatment using CellTiter-Glo 2.0, and the results are shown in Figure 10B. As can be seen in this figure, the maximum proliferation response for the long-acting IL-15 receptor agonists was comparable to the response induced by IL-15.

[0211] Example 8 In vitro induction of human NK cell proliferation following treatment with a long-acting IL-15 receptor agonist Human peripheral blood mononuclear cells (PBMCs) labeled with CFSE preparations were incubated in vitro for 5 days in the presence of increasing doses of rhIL-15 (dose range 1 ng / ml to 100 ng / ml, 5 points, 3-fold dilutions) or a long-acting IL-15 receptor agonist (mono(mPEG-butanamide)IL-15) (dose range 10 ng / ml to 1000 ng / ml, 5 points, 3-fold dilutions). After the incubation period, cell proliferation, indicated by a decrease in CFSE in cells that had undergone cell doublings, was measured using flow cytometry to measure CFSE levels in CD56+ human NK cells within the PBMC preparations. The results are provided in Figure 11 and Table 4 below.

[0212] [Table 4]

[0213] Both rhIL-15 and the long-acting IL-15 receptor agonist induced human NK cell proliferation in a dose-dependent manner. The long-acting IL-15 receptor agonist was approximately 17-fold less potent than rhIL-15 in inducing human NK cell proliferation. However, the maximal proliferative responses induced by rhIL-15 and the long-acting IL-15 receptor agonist were comparable.

[0214] Example 9 Assessment of NK cell cytotoxicity after treatment with long-acting IL-15 receptor agonists and immunoglobulins Human NK cells were magnetically sorted from normal donor PBMCs using negative selection. NK cells were seeded in 96-well U-bottom plates at a density of 400,000 cells / well. NK cells were stimulated (+) with 1000 ng / mL of a long-acting IL-15 receptor agonist (mono(mPEG-butanamide)IL-15) or left unstimulated (-) overnight at 37°C in 5% CO2 in RPMI-1640 complete medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic / antimycotic.

[0215] Human multiple myeloma RPMI-8226 cells (ATCC #CCL-155) were washed with PBS and stained with 0.15 μM CFSE for 5 min at 37° C. Where indicated, the tumor cells were pre-coated with 100 ng / mL daratumumab (+) for 1 h at 37° C. Stimulated NK cells (effector cells) were co-cultured with pre-coated CFSE-labeled tumor cells (target cells) at an effector (E):target (T) ratio of 10:1 for 3 h at 37° C. in 5% CO2. After incubation, cells were washed with flow staining buffer, Fc blocked for 15 min at 4° C., and surface stained (CD45, CD56, CD3-fluorescent conjugated antibodies). The cells were washed with PBS and incubated in 7-aminoactinomycin (7-AAD) viability stain for 30 minutes at 4° C., followed by flow cytometry analysis using a Fortessa (BD). The percentage of 7-AAD+CFSE+ target cells from the total cell population is the percentage for dead tumor cells.

[0216] The results are presented in Figure 12. The data show that human NK cells stimulated with a long-acting IL-15 receptor agonist enhance ADCC of multiple myeloma cells pre-coated with daratumumab. NK cells stimulated with a long-acting IL-15 receptor agonist were more cytotoxic to target cells than untreated NK cells or NK cells treated with daratumumab alone.

[0217] Example 10 Modulation of NK cell low-affinity immunoglobulin receptor (CD16) cell surface expression following treatment with tumor-targeted therapeutic antibodies and long-acting IL-15 receptor agonists Balb / c mice (n=6 / group) bearing Daudi B-cell lymphoma tumors in the bone marrow were administered a single 0.5 mg / kg dose of the clinical antibody daratumumab (DARZALEX®, Janssen Biotech) targeting the human CD38 protein or a non-specific control antibody with a matching isotype intraperitoneally on day 14 after intravenous inoculation of Daudi cells. Additional groups were treated with (i) mono(mPEG-N-butanamide) at a dose of 0.3 mg / kg as a single agent on days 14 and 21 after inoculation of Daudi cells. 40kD Interleukin-15 (Conjugate 1) administered subcutaneously, or (ii) treated with a combination therapy of Conjugate 1 administered subcutaneously at a dose of 0.03 mg / kg or 0.3 mg / kg on days 14 and 21 after Daudi cell inoculation and daratumumab administered intraperitoneally at a dose of 0.5 mg / kg on day 14 after Daudi cell inoculation.

[0218] Three days after the second Conjugate 1 administration (day 24 post-Daudi inoculation), bone marrow was harvested and the percentage of NK cells was measured in whole bone marrow by flow cytometry. The percentage of NK cells expressing the cell surface CD16 receptor, known to mediate antibody-dependent cellular cytotoxicity mechanisms in the presence of tumor-targeting antibodies, and the change in CD16 expression on a cell-by-cell basis on NK cells are shown in Figures 13A and 13B, respectively.

[0219] The results are provided in Tables 5 and 6 and Figures 13A-13B. All groups receiving therapeutic doses of conjugate 1 as a single agent or in combination with daratumumab showed an increased percentage of NK cells expressing CD16 on the cell surface when compared to all treatments without conjugate 1 (Figure 13A), and there was also an increase in CD16 expression on NK cells on a cell-by-cell basis (Figure 13B). The respective increase in NK cells expressing the CD16 receptor, as well as the increase in CD16 expression on individual NK cells, suggested NK cell activation by conjugate 1 in the presence of a therapeutic tumor-targeting antibody, exemplified in this example by daratumumab.

[0220] [Table 5]

[0221] [Table 6]

[0222] Example 11 Modulation of NK cell cytotoxicity protein granzyme B expression following treatment with tumor-targeted therapeutic antibodies and long-acting IL-15 receptor agonists Balb / c mice (n=6 / group) bearing Daudi B-cell lymphoma tumors in the bone marrow were administered a single 0.5 mg / kg dose of the clinical antibody daratumumab (DARZALEX®, Janssen Biotech) targeting the human CD38 protein or a non-specific control antibody with a matching isotype intraperitoneally on day 14 after intravenous inoculation of Daudi cells. Additional groups were treated with (i) mono(mPEG-N-butanamide) at a dose of 0.3 mg / kg as a single agent on days 14 and 21 after inoculation of Daudi cells. 40kD Interleukin-15 (Conjugate 1) administered subcutaneously, or (ii) treated with a combination therapy of Conjugate 1 administered subcutaneously at a dose of 0.03 mg / kg or 0.3 mg / kg on days 14 and 21 after Daudi cell inoculation and daratumumab administered intraperitoneally at a dose of 0.5 mg / kg on day 14 after Daudi cell inoculation.

[0223] Three days after the second Conjugate 1 administration (day 24 after Daudi inoculation), bone marrow was harvested and the percentage of NK cells was measured in whole bone marrow by flow cytometry. The change in intracellular granzyme B expression on a cell-by-cell basis on NK cells is shown in FIG.

[0224] The results are provided in Table 7 and Figure 14. Groups receiving therapeutic doses of conjugate 1 as a single agent or in combination with daratumumab showed increased expression of Granzyme B on a cell-by-cell basis in NK cells when compared to all treatments without conjugate 1 (Figure 14). The respective increases in Granzyme B expression in individual NK cells suggested NK cell activation by conjugate 1 in the presence of a therapeutic tumor-targeting antibody, exemplified in this example by daratumumab.

[0225] [Table 7]

[0226] Example 12 Evaluation of antibody-mediated antibody-dependent cellular cytotoxicity (ADCC) against B-cell lymphoma cells in vitro following treatment with a long-acting IL-15 receptor agonist Human NK cells were magnetically sorted from normal donor PBMCs using negative selection. NK cells were seeded in 96-well U-bottom plates at a density of 80,000 cells / well (FIG. 15A) or 400,000 cells / well (FIG. 15B). NK cells were stimulated (+) with 300 ng / mL of a long-acting IL-15 receptor agonist (mono(mPEG-butanamide)IL-15) or left unstimulated (-) overnight at 37° C. in 5% CO2 in RPMI-1640 complete medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic / antimycotic.

[0227] Human multiple myeloma RPMI-8226 cells (ATCC #CCL-155) were washed with PBS and stained with 0.15 μM CFSE for 5 min at 37°C. Stimulated NK cells (effector cells) and daratumumab-coated (+1 μg / mL) CFSE-labeled tumor cells (target cells) were simultaneously co-cultured at an E:T ratio of 10:1 for 3 h at 37°C in 5% CO2. After incubation, cells were washed with flow staining buffer, Fc blocked for 15 min at 4°C, and surface stained (CD45, CD56, CD3-fluorescent conjugated antibodies). The cells were washed with PBS and incubated in 7-aminoactinomycin (7-AAD) viability stain for 30 min at 4°C, followed by flow cytometry analysis using Fortessa (BD). The percentage of 7-AAD+CFSE+ target cells from the total cell population is the percentage for dead tumor cells. The results are presented in Figure 15A.

[0228] Alternatively, human B-cell lymphoma (Daudi) cells were washed with PBS and stained with 0.3 μM CFSE for 5 min at 37° C. Rituximab-coated (+10 ng / mL) CFSE-labeled tumor cells (target cells) were simultaneously co-cultured with NK cells (effector cells) at an E:T ratio of 2:1 for 3 h at 37° C. in 5% CO2. After incubation, cells were washed with flow staining buffer, Fc-blocked for 15 min at 4° C., and surface stained (CD45, CD56, CD3-fluorescent conjugated antibodies). The cells were washed with PBS and incubated in 7-aminoactinomycin (7-AAD) viability stain for 30 min at 4° C., and then subjected to flow cytometry analysis using Fortessa (BD). The percentage of 7-AAD+CFSE+ target cells from the total cell population is the percentage for dead tumor cells. The results are provided in FIG. 15B.

[0229] The data show that human NK cells stimulated with a long-acting IL-15 receptor agonist enhance ADCC of daratumumab-coated and rituximab-coated B cell lymphoma cells. NK cells stimulated with a long-acting IL-15 receptor agonist are more cytotoxic to target cells than untreated NK cells.

[0230] Example 13 Assessment of survival in the DAUDI B-cell lymphoma xenograft model following treatment with a long-acting IL-15 receptor agonist and the anti-CD38 monoclonal antibody, daratumumab SCID or SCID-beige mice (N=8 / group) inoculated intravenously with 10 million Daudi B-cell lymphoma cells were treated with a single dose of daratumumab (0.5 mg / kg, IP, 14 days after inoculation) and a total of three doses of mono(methoxyPEG-N-butanamide). 40kD Interleukin-15 (0.3 mg / kg, intravenously, administered 14, 21, and 28 days after tumor inoculation). Untreated SCID / beige or CB17 SCID mice as controls were also evaluated. Survival of tumor-inoculated mice was measured by body condition scoring and onset of hind limb paralysis as endpoint markers. Results are presented graphically in Figure 16.

[0231] As shown in Figure 16, the combination of mono(methoxyPEG-N-butanamide) interleukin-15 with daratumumab treatment resulted in a more than two-fold increase in survival (33 days), from a median survival of 27 days in untreated control SCID mice to 60 days in combination-treated mice. In contrast, the combination of mono(methoxyPEG-N-butanamide) interleukin-15 with daratumumab treatment in SCID mice carrying a beige mutation that reduces NK cell functionality (SCID beige) resulted in only a 7-day increase in survival (from 23 days in untreated SCID beige mice to 30 days in combination-treated SCID beige mice). These results indicate that NK cell activity is required for the full therapeutic effect of the combination of mono(methoxyPEG-N-butanamide) interleukin-15 with daratumumab treatment.

[0232] Example 14 Assessment of cell numbers in the DAUDI B-cell lymphoma xenograft model following administration of a long-acting IL-15 receptor agonist and an exemplary anti-CD38 monoclonal antibody, daratumumab On day 0, SCID mice (N=6 / group) were inoculated intravenously with 10 million Daudi cells and administered a single dose of low or high dose daratumumab (0.05 mg / kg or 5 mg / kg Mice were treated with monotherapy: daratumumab (0.05 mg / kg or 5 mg / kg IP, 14 days after Daudi cell inoculation), or two doses of low or high dose mono(methoxyPEG-N-butanamide) interleukin-15 (compound 1) (0.03 mg / kg or 0.6 mg / kg, intravenously, one dose each at 14 and 21 days after inoculation). 40kD The mice were treated with interleukin-15 (0.03 mg / kg or 0.6 mg / kg, intravenously, 14 and 21 days after inoculation). The number of Daudi cells in the bone marrow was measured using mono(methoxy PEG-N-butanamide) 40kDThree days after the second dose of interleukin-15 (day 24), cells were assessed by flow cytometry as shown in Figures 17A and 17B.

[0233] As shown in Figures 17A-17B, the combination of mono(methoxyPEG-N-butanamide) interleukin-15 (compound 1) with daratumumab was equally effective in eliminating intramedullary tumor cells when comparing high daratumumab dose levels (5 mg / kg) combined with low dose mono(methoxyPEG-N-butanamide) interleukin-15 (0.03 mg / kg) which eliminated 96% of the tumor cells (Figure 17A) to low dose daratumumab (0.05 mg / kg) combined with high dose mono(methoxyPEG-N-butanamide) interleukin-15 (0.6 mg / kg) which eliminated 92% of the tumor cells (Figure 17B). As monotherapy, low dose daratumumab and mono(methoxyPEG-N-butanamide) interleukin-15 eliminated only 30% and 42% of the tumor cells, respectively. As monotherapy, high-dose daratumumab and mono(methoxyPEG-N-butanamide)interleukin-15 eliminated only 58% and 69%, respectively, of tumor cells. These results indicate that lower dose levels or tissue concentrations of daratumumab treatment can be offset by increased dose levels or tissue concentrations of mono(methoxyPEG-N-butanamide)interleukin-15, maintaining the high efficacy of the combination treatment in killing tumor cells.

[0234] Example 15 Evaluation of the HCT-116 colorectal carcinoma cell model following administration of a long-acting IL-15 receptor agonist and an exemplary anti-EGFR monoclonal antibody, cetuximab HCT-116 cells were cultured in RPMI1640 containing 5% FBS for 2 weeks prior to inoculation. Balb / c SCID mice (N=4 / group) were subcutaneously inoculated with 5 million HCT-116 cells. Seven days after tumor cell inoculation, the average tumor volume was approximately 150 mm 3On day 0, mice were treated with a single dose of the anti-human EGFR antibody, cetuximab (20 mg / kg IP), and a single dose of mono(methoxyPEG-N-butanamide) interleukin-15 (Compound 1) (0.3 mg / kg IV).

[0235] After 3 and 5 days of treatment, blood and tumor tissues were collected and the percentage of NK cells was measured by flow cytometry, as shown in Figure 18A. As shown in Figure 18A, an increase in the percentage of NK cells among the total viable cells in the tumor was induced by this treatment. After 3 and 5 days of treatment, blood and tumor tissues were collected and the number of NK cells was measured by flow cytometry, as shown in Figure 18B. As shown in Figure 18B, an increase in the number of NK cells in the tumor tissue was induced by this treatment.

[0236] Samples were immunophenotyped for Ki-67 (%) by flow cytometry on day 3 after treatment (see FIG. 18C). FIG. 18C is a plot of NK cell proliferation as measured by Ki-67 positivity (%), used as a marker for cell proliferation over time. *Ki67 marker induction was observed in the majority of NK cells, indicating an effective proliferative response after treatment.

[0237] Granzyme B expression was measured in blood or tumor NK cells by flow cytometry on day 3 after treatment, as shown in Figure 18D. As shown in Figure 18D, increased GzmB expression was observed in both blood and tumor NK cells after treatment, indicating cytotoxic activation of NK cells by treatment.

[0238] The percentage of blood or tumor NK cells expressing cell surface CD16 receptor was measured using flow cytometry by measuring the increase in CD16 median fluorescence intensity (MFI) signal, as shown in Figure 18E. As shown in Figure 18E, an increase in cell surface CD16 expression was induced by treatment, indicating NK cell activation.

[0239] Three days after treatment, blood and tumor cells were collected and the expression of the cell surface activation receptor NKG2D was measured by flow cytometry, and the results are shown in Figure 18 F. As shown in Figure 18 F, NKG2D surface expression was decreased, consistent with functional activation of NK cells by treatment.

[0240] These results further suggest that mono(methoxyPEG-N-butanamide) interleukin-15 is effective in substantially enhancing NK cell-mediated ADCC when administered together with an anti-tumor antibody.

[0241] Example 16 Evaluation of the FaDu human head and neck squamous cell carcinoma model following administration of a long-acting IL-15 receptor agonist and an exemplary anti-EGFR monoclonal antibody, cetuximab Balb / c SCID mice (N=4 / group) were inoculated intravenously with 3 million FaDu cells. Seven days after tumor cell inoculation, the average tumor volume was approximately 150 mm 3 On day 0, mice were treated with a single dose of the anti-human EGFR antibody, cetuximab (20 mg / kg IP) and a single dose of mono(methoxyPEG-N-butanamide) interleukin-15 (Compound 1) (0.3 mg / kg IV) (approximately 150 mm Hg). 3 ) for treatment.

[0242] After 3 and 5 days of treatment, blood and tumor tissues were collected and the percentage of NK cells was measured by flow cytometry, as shown in Figure 19A. As shown in Figure 19A, the treatment induced an increase in the percentage of NK cells among the total viable cells in the tumor. After 3 and 5 days of treatment, blood and tumor tissues were collected and the number of NK cells was measured by flow cytometry, as shown in Figure 19B. As shown in Figure 19B, the treatment induced an increase in the number of NK cells in the tumor tissue.

[0243] Samples were subjected to immunophenotyping for Ki-67 (%) on day 3 after treatment (see FIG. 19C). FIG. 19C is a plot of NK cell proliferation as measured by Ki-67 positivity (%), used as a marker for cell proliferation over time. Ki67 marker induction was observed in the majority of NK cells, indicating an effective proliferative response after treatment.

[0244] Granzyme B expression was measured in blood or tumor NK cells by flow cytometry on day 3 after treatment, as shown in Figure 19D. As shown in Figure 19D, increased GzmB expression was observed in both blood and tumor NK cells after treatment, indicating cytotoxic activation of NK cells by treatment.

[0245] The percentage of blood or tumor NK cells expressing cell surface CD16 receptor was measured using flow cytometry by measuring the increase in CD16 median fluorescence intensity (MFI) signal, as shown in Figure 19E. As shown in Figure 19E, an increase in cell surface CD16 expression was induced by treatment, indicating NK cell activation.

[0246] Three days after treatment, blood and tumor cells were collected and the expression of the cell surface activation receptor NKG2D was measured by flow cytometry, and the results are shown in Figure 19F. As shown in Figure 19F, NKG2D surface expression was decreased, consistent with functional activation of NK cells by treatment.

[0247] These results further suggest that mono(methoxyPEG-N-butanamide) interleukin-15 is effective in substantially enhancing NK cell-mediated ADCC when administered together with an anti-tumor antibody.

[0248] Example 17 Evaluation of tumor growth inhibition in the H1975 lung tumor model following administration of a long-acting IL-15 receptor agonist and an exemplary anti-EGFR monoclonal antibody, cetuximab Balb / c nude mice (N=10 / group) inoculated with 5 million H1975 cells subcutaneously in the flank were treated with a total of three doses of cetuximab (0.25 mg / kg, IP, administered 9, 12, and 16 days after inoculation) and a total of three doses of mono(methoxyPEG-N-butanamide). 40kD The mice were treated with interleukin-15 (compound 1) (0.3 mg / kg, intravenously) administered on days 9, 16, and 23 after tumor inoculation. The delay in loss of tumor growth control was evaluated on day 27 after the start of treatment. The results are presented graphically in FIG. 20.

[0249] As shown in Figure 20, mono(methoxyPEG-N-butanamide) interleukin-15 treatment did not result in tumor growth inhibition in treated mice. Cetuximab-treated mice showed continued tumor growth after the cetuximab treatment schedule was completed. In contrast, the combination of mono(methoxyPEG-N-butanamide) interleukin-15 with cetuximab treatment resulted in a delay in tumor growth recurrence until day 27 after the start of treatment, which indicated significantly slower tumor growth (p=0.02, Mann-Whitney test) in the combination of mono(methoxyPEG-N-butanamide) interleukin-15 with cetuximab treatment compared to cetuximab monotherapy. These data indicate that the combination of mono(methoxyPEG-N-butanamide) interleukin-15 with cetuximab results in superior tumor growth control compared to cetuximab monotherapy in this tumor model.

[0250] Example 18 Evaluation of Tumor Growth Inhibition and Retardation in an HT-29 Colorectal Carcinoma Cell Model Following Administration of a Long-Acting IL-15 Receptor Agonist and an Exemplary Anti-EGFR Monoclonal Antibody, Cetuximab SCID mice (N=8 / group) inoculated with 5 million HT-29 cells subcutaneously in the flank were treated with a total of six doses of cetuximab (40 mg / kg, IP, administered 7, 11, 14, 18, 21, and 25 days after inoculation) and a total of three doses of mono(methoxyPEG-N-butanamide). 40kDThe mice were treated with interleukin-15 (compound 1) (0.3 mg / kg, intravenously) administered 7, 14, and 21 days after tumor inoculation. Tumor growth inhibition was evaluated 21 days after the start of treatment. The results are presented graphically in FIG. 21A.

[0251] As shown in Figure 21A, mono(methoxyPEG-N-butanamide) interleukin-15 or cetuximab monotherapy did not result in tumor growth inhibition in treated mice. In contrast, the combination of mono(methoxyPEG-N-butanamide) interleukin-15 with cetuximab treatment resulted in significant tumor growth inhibition. These data indicate that the combination of mono(methoxyPEG-N-butanamide) interleukin-15 with cetuximab results in tumor growth inhibition in a cetuximab-resistant tumor model.

[0252] Tumor growth delay (TVQT) was evaluated using an endpoint set as 400% tumor growth from baseline. Figure 21B shows that a 38% tumor growth delay was induced by measuring the tumor volume quadrupling time (TVQT) from the baseline tumor volume on the day of treatment initiation.

[0253] Example 19 Evaluation of Tumor Growth Inhibition and Retardation in the HCT-116 Colorectal Carcinoma Xenograft Model Following Administration of a Long-Acting IL-15 Receptor Agonist and an Exemplary Anti-EGFR Monoclonal Antibody, Cetuximab SCID mice (N=8 / group) inoculated with 5 million HCT-116 cells subcutaneously in the flank were treated with a total of six doses of cetuximab (40 mg / kg, IP, administered 7, 11, 14, 18, 21, and 25 days after inoculation) and a total of three doses of mono(methoxyPEG-N-butanamide). 40kD The mice were treated with interleukin-15 (compound 1) (0.3 mg / kg, intravenously, administered 7, 14, and 21 days after tumor inoculation). Tumor growth inhibition was evaluated 19 days after the start of treatment. The results are presented graphically in FIG. 22A.

[0254] As shown in Figure 22A, cetuximab monotherapy did not result in tumor growth inhibition in treated mice. Mono(methoxyPEG-N-butanamide) interleukin-15 monotherapy resulted in 31% tumor growth inhibition. In contrast, the combination of mono(methoxyPEG-N-butanamide) interleukin-15 with cetuximab treatment resulted in 42% tumor growth inhibition. These data indicate that the combination of mono(methoxyPEG-N-butanamide) interleukin-15 with cetuximab results in tumor growth inhibition in a cetuximab-resistant tumor model.

[0255] Tumor growth delay was assessed by measuring the tumor volume quadruple doubling time (TVQT) from the baseline tumor volume on the day treatment began. Figure 22B shows that the combination treatment induced a 42% tumor growth delay, with mono(methoxyPEG-N-butanamide) 40kD It is shown that interleukin-15 (compound 1) monotherapy induced a 26% tumor growth delay, whereas cetuximab monotherapy did not result in tumor growth delay.

[0256] Example 20 Evaluation of antibody-mediated cell-mediated cytotoxicity (ADCC) in the HCT-116 colorectal carcinoma in vitro model following administration of a long-acting IL-15 receptor agonist and an exemplary anti-EGFR monoclonal antibody, cetuximab Human NK cells were magnetically sorted from normal donor PBMCs using negative selection. NK cells were seeded in 96-well U-bottom plates at a density of 400,000 cells / well. NK cells were incubated overnight at 37°C in 5% CO2 with mono(methoxyPEG-N-butanamide) in RPMI-1640 complete medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic / antimycotic. 40kD They were stimulated (+) with interleukin-15 (compound 1) (300 ng / mL) or left unstimulated (-).

[0257] Colonic human HCT-116 cells (ATCC # CCL-247) were cultured in McCoy's 5A medium (ATCC # 5A) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic / antimycotic. HCT-116 cells were cultured in 0.25% trypsin-0.53 mM EDTA (30-2007) at 37°C in 5% CO2. HCT-116 cells were subcultured by incubation in 0.25% trypsin-0.53 mM EDTA for 5 min at 37°C, washed with sterile PBS, resuspended in culture medium, counted, and co-cultured with NK cells using effector:target (E:T) of 10:1. At the same time, cetuximab (McKesson Medical Surgical #66733-948-23 lot# C1800115) and IgG1 isotype (Biolegend; LEAF purified IgG1) were added (+) to corresponding wells at a concentration of 30 μg / mL, followed by incubation at 37°C in 5% CO2 for 3 h. After incubation, cells were washed with flow staining buffer, Fc blocked for 15 min at 4°C, and surface stained (EpCAM, CD45, CD56, CD3-fluorescent conjugated antibodies) in the dark for 20 min at 4°C. The cells were washed with PBS, incubated in 7-aminoactinomycin (7-AAD) viability stain for 30 min at 4°C, and then subjected to flow cytometry analysis using Fortessa (BD). The percentage of 7-AAD+CD45-EpCAM+ target cells from the total cell population is the percentage for dead tumor cells. The results are provided in Figure 23A.

[0258] Epithelial / pharyngeal head and neck human FaDu cells (ATCC # HTB-43) were cultured in Eagle's Minimum Essential Medium (EMEM; ATCC+ 30-2003) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic / antimycotic at 37° C. in 5% CO2. FaDu cells were subcultured by incubation in 0.25% trypsin-0.53 mM EDTA for 5 min at 37° C., washed with sterile PBS, resuspended in culture medium, counted, and co-cultured with NK cells using an effector:target (E:T) of 10:1. At the same time, cetuximab (McKesson Medical Surgical #66733-948-23 lot# C1800115) and IgG1 isotype (Biolegend; LEAF purified IgG1) were added (+) to corresponding wells at a concentration of 300ng / mL and then incubated at 37°C in 5% CO2 for 3 hours. After incubation, cells were washed with flow staining buffer, Fc blocked for 15 minutes at 4°C, and surface stained (CD45, CD56, CD3-fluorescent conjugated antibodies) for 20 minutes at 4°C in the dark. The cells were washed with PBS and incubated in 7-aminoactinomycin (7-AAD) viability stain for 30 minutes at 4°C, and then subjected to flow cytometry analysis using Fortessa (BD). The percentage of 7-AAD+CD45- target cells from the total cell population is the percentage for dead tumor cells. The results are provided in Figure 23B.

[0259] As can be seen in Figures 23A and 23B, mono(methoxyPEG-N-butanamide) 40kD Interleukin-15 stimulated human NK cells enhance ADCC of cetuximab-coated human solid tumor cells.

[0260] Example 21 Evaluation of tumor growth inhibition in the SKOV-3 ovarian carcinoma cell model following administration of a long-acting IL-15 receptor agonist and an exemplary anti-HER monoclonal antibody, trastuzumab Balb / c nude mice (N=10 / group) inoculated subcutaneously in the flank with 10 million SKOV-3 cells mixed 1:1 with Matrigel were treated with a total of six doses of trastuzumab (13.5 mg / kg, IV, administered twice weekly for three weeks starting on day 6 post-inoculation) and three doses of mono(methoxyPEG-N-butanamide). 40kD The mice were treated with interleukin-15 (compound 1) (0.3 mg / kg, intravenously) (administered 6, 13, and 20 days after tumor inoculation). Tumor growth inhibition in the inoculated mice was measured 35 days after the start of treatment. The results are presented graphically in FIG. 24.

[0261] As shown in Figure 24, mono(methoxy PEG-N-butanamide) interleukin-15 combination treatment did not result in tumor growth inhibition in treated mice. Trastuzumab-treated mice showed 61% tumor growth inhibition, but trastuzumab treatment did not result in tumor-free animals. In contrast, combination of mono(methoxy PEG-N-butanamide) interleukin-15 with trastuzumab treatment resulted in complete loss of tumors in all treated animals, and the animals remained tumor-free. These data indicate that combination of mono(methoxy PEG-N-butanamide) interleukin-15 with trastuzumab results in superior tumor growth inhibition compared to trastuzumab monotherapy in this tumor model.

[0262] Example 22 Evaluation of tumor growth inhibition in the NCI-N87 gastric cancer cell model following administration of a long-acting IL-15 receptor agonist and an exemplary anti-HER monoclonal antibody, trastuzumab Balb / c nude mice (N=10 / group) inoculated subcutaneously in the flank with 10 million NCI-N87 cells mixed 1:1 with Matrigel were treated with a total of three doses of trastuzumab (initial dose of 3 mg / kg and two subsequent doses of 1 mg / kg, IV, administered 5, 12, and 20 days after inoculation) and a total of three doses of mono(methoxyPEG-N-butanamide). 40kDThe mice were treated with interleukin-15 (compound 1) (0.3 mg / kg, intravenously) (administered 5, 12, and 19 days after tumor inoculation). Tumor growth inhibition in the inoculated mice was measured 25 days after the start of treatment. The results are presented graphically in FIG. 25.

[0263] As shown in Figure 25, mono(methoxy PEG-N-butanamide) interleukin-15 treatment did not result in tumor growth inhibition in treated mice. Mice treated with trastuzumab showed 29% tumor growth inhibition. In contrast, the combination of mono(methoxy PEG-N-butanamide) interleukin-15 with trastuzumab treatment resulted in a higher tumor growth inhibition of 41% in treated mice. These data indicate that the combination of mono(methoxy PEG-N-butanamide) interleukin-15 with trastuzumab results in superior tumor growth inhibition compared to trastuzumab monotherapy in this tumor model.

[0264] Embodiments of the methods, therapeutic combinations, and kits of the present invention include, but are not limited to, the following.

[0265] Embodiment 1. A method of treating a subject having cancer, comprising: (a) a long-acting IL-15 receptor agonist having the following structure: [ka] wherein IL-15 is an interleukin-15 moiety, n is an integer from about 150 to about 3,000, m is an integer selected from 2, 3, 4, and 5, n' is 1, and -NH- represents an amino group of the IL-15 moiety; and (b) a tumor-directed antibody that specifically binds to a tumor antigen selected from a phosphorylated protein, a transmembrane protein, a glycoprotein, a glycolipid, and a growth factor, the antibody having antibody-dependent cellular cytotoxicity (ADCC) as a mechanism of action; (wherein steps (a) and (b) are performed simultaneously or sequentially, in any order) The method comprises administering to a subject.

[0266] Embodiment 2. The method of embodiment 1, wherein the long-acting IL-15 receptor agonist is a pharma- ceutically acceptable salt.

[0267] Embodiment 3. The method of any of embodiments 1-2, combined or separate, wherein m in formula (I) is 2 or 3.

[0268] Embodiment 4. The method of any of embodiments 1 to 3, combined or separate, wherein m in formula (I) is 3.

[0269] Embodiment 5. The method of any of embodiments 1 to 4, combined or separate, wherein n in formula (I) has a value of about 909.

[0270] Embodiment 6. The long-acting IL-15 receptor agonist is (methoxy PEG-N-butanamide) 20-60kD Interleukin-15, (Methoxy PEG-N-butanamide) 20-40kD Interleukin-15, (Methoxy PEG-N-butanamide) 40kD Interleukin-15, mono(methoxy PEG-N-butanamide) 20-60kD Interleukin-15, mono(methoxy PEG-N-butanamide) 20-40kD Interleukin-15, and mono(methoxyPEG-N-butanamide) 40kD The method of any one of the preceding embodiments, combined or separate, wherein the anti-inflammatory agent is selected from interleukin-15.

[0271] Embodiment 7. The long-acting IL-15 receptor agonist is mono(methoxyPEG-N-butanamide). 40kD The method of any one of the preceding combined or separate embodiments, wherein the inhibitor is interleukin-15.

[0272] Embodiment 8. The method of any of the combined or separate embodiments 1 to 7, wherein the cancer is a solid tumor.

[0273] Embodiment 9. The method of any of the combined or individual embodiments 1-8, wherein the solid cancer is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, colorectal cancer, gastric cancer, malignant melanoma, liver cancer, small cell lung cancer, non-small cell lung cancer, thyroid cancer, kidney cancer, cancer of the bile duct, brain cancer, cervical cancer, maxillary sinus cancer, bladder cancer, esophageal cancer, Hodgkin's disease, and adrenocortical carcinoma (including metastatic forms of any of the foregoing).

[0274] Embodiment 10. The method of any of the combined or separate embodiments 1 to 7, wherein the cancer is a hematopoietic tumor.

[0275] Embodiment 11. The method of embodiments 1-7 and 10, combined or separately, wherein the hematopoietic malignancy is selected from the group consisting of multiple myeloma, non-Hodgkin's lymphoma, leukemia, and lymphoma.

[0276] Embodiment 12. The method of embodiments 1-7 and 10-11, combined or separately, wherein the cancer is a B-cell malignancy.

[0277] Embodiment 13. The method of any one of embodiments 1 to 12, combined or separate, wherein step (a) is carried out before step (b).

[0278] Embodiment 14. The method of any one of embodiments 1 to 12, combined or separate, wherein step (b) is carried out before step (a).

[0279] Embodiment 15. The method of any one of embodiments 1 to 12, wherein step (a) and step (b) are performed simultaneously or substantially simultaneously, either combined or separate.

[0280] Embodiment 16. The method of any of combined or separate embodiments 1-15, wherein said administering is effective to stimulate NK activation, as measured in a suitable animal model, to a degree greater than that observed when the long-acting IL-15 receptor agonist or tumor-directed antibody is administered as a single agent.

[0281] Embodiment 17. The method of any of combined or separate embodiments 1-16, wherein said administering is effective to stimulate NK proliferation, as measured in a suitable animal model, to a greater extent than is observed when the long-acting IL-15 receptor agonist or tumor-directed antibody is administered as a single agent.

[0282] Embodiment 18. The method of any of combined or separate embodiments 1-17, wherein said administering is effective to support CD8 T cell survival and memory formation to a greater extent than is observed when the long-acting IL-15 receptor agonist or tumor-directed antibody is administered as a single agent, as measured in a suitable animal model.

[0283] Embodiment 19. The method of any of embodiments 1 to 18, combined or separate, wherein a long-acting IL-15 receptor agonist is administered intravenously or subcutaneously.

[0284] Embodiment 20. The method of any one of embodiments 1 to 19, combined or separate, wherein the long-acting IL-15 receptor agonist is administered q7d, q14d, q21d, or monthly, or any combination thereof.

[0285] Embodiment 21. The method of any of embodiments 1 to 20, combined or separate, wherein the tumor-directed antibody is administered intravenously or intraperitoneally.

[0286] Embodiment 22. The method of any one of embodiments 1 to 21, combined or separate, wherein the tumor-directed antibody is administered q7d, q14d, q21d, or monthly, or any combination thereof.

[0287] Embodiment 23. The method of any of the combined or separate embodiments 1 to 22, wherein the tumor-directed antibody is a monoclonal antibody.

[0288] Embodiment 24. The method of any of the combined or separate embodiments 1 to 23, wherein the tumor-directed antibody is selected from an anti-CD19 antibody, an anti-CD20 antibody, and an anti-CD38 antibody.

[0289] Embodiment 25. The method of any of the combined or separate embodiments 1 to 23, wherein the tumor-directed antibody that specifically binds to the glycoprotein is selected from an anti-SLAMF7 antibody, an anti-EpCAM antibody, an anti-gpA3 antibody3, and an anti-FBP antibody.

[0290] Embodiment 26. The method of any of embodiments 1 to 23, combined or separate, wherein the tumor-directed antibody that specifically binds to the growth factor is selected from an anti-VEGF antibody, an anti-VEGFR antibody, and an anti-EGFR antibody.

[0291] Embodiment 27. The method of any of the combined or separate embodiments 1 to 23, wherein the tumor-directed antibody is an IgG antibody.

[0292] Embodiment 28. The method of any of the combined or individual embodiments 1 to 23, wherein the tumor-directed antibody is selected from the group consisting of daratumumab, rituximab, cetuximab, and trastuzumab.

[0293] Embodiment 29. (a) A long-acting IL-15 receptor agonist having the following structure: [ka] wherein IL-15 is an interleukin-15 moiety, n is an integer from about 150 to about 3,000, m is an integer selected from 2, 3, 4, and 5, n' is 1, and -NH- represents an amino group of the IL-15 moiety; and (b) a tumor-directed antibody that specifically binds to a tumor antigen selected from a phosphorylated protein, a transmembrane protein, a glycoprotein, a glycolipid, and a growth factor, wherein the tumor-directed antibody comprises antibody-dependent cellular cytotoxicity (ADCC) as a mechanism of action. 23. A therapeutic combination for use in treating cancer comprising:

[0294] Embodiment 30. The therapeutic combination of embodiment 29, wherein the long-acting receptor agonist is a pharma- ceutically acceptable salt.

[0295] Embodiment 31. A therapeutic combination of any of embodiments 29 to 30, combined or separately, wherein m in formula (I) is 2 or 3.

[0296] Embodiment 32. The therapeutic combination of any of the combined or separate embodiments 29 to 31, wherein m in formula (I) is 3.

[0297] Embodiment 33. The therapeutic combination of embodiments 29 to 32, combined or separate, wherein n in formula (I) has a value of about 909.

[0298] Embodiment 34. The long-acting IL-15 receptor agonist is (methoxyPEG-N-butanamide). 20-60kD Interleukin-15, (Methoxy PEG-N-butanamide) 20-40kD Interleukin-15, (Methoxy PEG-N-butanamide) 40kD Interleukin-15, mono(methoxy PEG-N-butanamide) 20-60kD Interleukin-15, mono(methoxy PEG-N-butanamide) 20-40kD Interleukin-15, and mono(methoxyPEG-N-butanamide) 40kD The therapeutic combination of embodiments 29 to 33, combined or separate, is selected from interleukin-15.

[0299] Embodiment 35. The long-acting IL-15 receptor agonist is mono(methoxyPEG-N-butanamide). 40kD The therapeutic combination of any of the combined or separate embodiments 29 to 34, wherein the therapeutic agent is interleukin-15.

[0300] Embodiment 36. The therapeutic combination of embodiments 29 to 35, combined or separate, wherein the long-acting IL-15 receptor agonist is formulated for intravenous or subcutaneous administration.

[0301] Embodiment 37. The therapeutic combination of embodiments 29 to 36, combined or separate, wherein the tumor-directed antibody is formulated for intravenous or intraperitoneal administration.

[0302] Embodiment 38. The therapeutic combination of embodiments 29 to 37, combined or separate, wherein the long-acting IL-15 receptor agonist is formulated for intravenous or subcutaneous administration.

[0303] Embodiment 39. The therapeutic combination of embodiments 29 to 38, combined or separate, wherein the tumor-directed antibody is formulated for intravenous or intraperitoneal administration.

[0304] Embodiment 40. The therapeutic combination of embodiments 29 to 39, combined or separately, wherein the tumor-directed antibody is a monoclonal antibody.

[0305] Embodiment 41. The therapeutic combination of embodiments 29 to 40, combined or separate, wherein the tumor-directed antibodies are selected from anti-CD19 antibodies, anti-CD20 antibodies, and anti-CD38 antibodies.

[0306] Embodiment 42. The therapeutic combination of embodiments 29 to 40, combined or separate, wherein the tumor-directed antibodies that specifically bind to the glycoprotein are selected from anti-SLAMF7 antibodies, anti-EpCAM antibodies, anti-gpA3 antibodies 3, and anti-FBP antibodies.

[0307] Embodiment 43. The therapeutic combination of embodiments 29 to 40, combined or separate, wherein the tumor-directed antibodies that specifically bind to the growth factor are selected from anti-VEGF antibodies, anti-VEGFR antibodies, and anti-EGFR antibodies.

[0308] Embodiment 44. The therapeutic combination of embodiments 29 to 40, combined or separately, wherein the tumor-directed antibody is an IgG antibody.

[0309] Embodiment 45. The therapeutic combination of embodiments 29 to 40, combined or separate, wherein the tumor-directed antibody is selected from the group consisting of daratumumab, rituximab, cetuximab, and trastuzumab.

[0310] Embodiment 46. The therapeutic combination of embodiments 29 to 45, combined or separately, wherein the cancer is a solid tumor.

[0311] Embodiment 47. The therapeutic combination of embodiments 29 to 46, combined or separate, wherein the solid cancer is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, colorectal cancer, gastric cancer, malignant melanoma, liver cancer, small cell lung cancer, non-small cell lung cancer, thyroid cancer, kidney cancer, cancer of the bile duct, brain cancer, cervical cancer, maxillary sinus cancer, bladder cancer, esophageal cancer, Hodgkin's disease, and adrenocortical carcinoma (including metastatic forms of any of the foregoing).

[0312] Embodiment 48. The therapeutic combination of embodiments 29 to 45, combined or separate, wherein the cancer is a hematopoietic tumor.

[0313] Embodiment 49. The therapeutic combination of embodiments 29 to 45 and 48, combined or separately, wherein the hematopoietic malignancies are selected from the group consisting of multiple myeloma, non-Hodgkin's lymphoma, leukemia, and lymphoma.

[0314] Embodiment 50. The therapeutic combination of embodiments 29 to 45 and 48 to 49, combined or separately, wherein the cancer is a B-cell malignancy.

[0315] Embodiment 51. A kit comprising the therapeutic combination of embodiments 29 to 50, combined or separate, with instructions for use.

[0316] Embodiment 52 The kit of embodiment 51, wherein the long-acting IL-15 receptor agonist and the monoclonal antibody are each contained within one or more individual unit dosage forms.

[0317] Embodiment 53 The kit of embodiment 51, wherein the long-acting IL-15 receptor agonist and the monoclonal antibody are each contained within the same unit dosage form.

[0318] Sequence Listing SEQ ID NO:1 (rhIL-15) [ka] SEQ ID NO:2 [ka] SEQ ID NO:3 [ka]

Claims

[Claim 1] The invention described in this specification.

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