Long-acting interleukin-15 receptor agonists and related immunotherapy compositions and methods

Abstract

The present disclosure provides long-acting IL-15 receptor agonists, related compositions and methods of preparation, and uses thereof, for example, in treating conditions responsive to therapies that effectively provide, for example, sustained immune activation and / or anti-tumor activity.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 506,494, filed on May 15, 2017; and U.S. Provisional Patent Application No. 62 / 536,966, filed on July 25, 2017; and U.S. Provisional Patent Application No. 62 / 582,186, filed on November 6, 2017; and U.S. Provisional Patent Application No. 62 / 648,240, filed on March 26, 2018, the disclosures of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates, inter alia, to long-acting interleukin-15 ("IL-15") receptor agonists, related compositions and methods of preparation, and their use, for example, in treating conditions responsive to therapies that effectively provide, for example, sustained immune activation and anti-tumor activity. Background Art

[0004] Interleukin-15 ("IL-15") is a pleiotropic cytokine first reported by Grabstein et al. (Grabstein et al. (1994) Science 264 :965-968). Secreted as a 162 amino acid precursor, human IL-15 comprises a 29 amino acid leader sequence and a 19 amino acid presequence; the mature protein is therefore 114 amino acids in length. IL-15 belongs to a family of cytokines with four α-helical bundles, binding to a heterotrimeric receptor in which a unique α subunit (IL-15Rα) confers specificity to the receptor for IL-15, and the β and γ subunits of the receptor share commonalities with one or more other cytokine receptors. Giri et al. (1995) EMBO J. [European Molecular Biology Journal] 14 :3654-3663.

[0005] As a cytokine, IL-15 has an impact on both the innate and adaptive immune systems (DiSabitino et al. (2011) Cytokine Growth Factor Rev. 22: 19-33). With regard to the innate immune system (which generally defends the host from foreign invaders), among other properties, IL-15 leads to the development of natural killer cells ("NK cells") and natural killer-T cells ("NK-T cells") and maintains their survival. Consistent with their role in the innate immune system, NK cells do not specifically attack invading pathogens; instead, these cells destroy damaged host cells (such as tumor cells or virus-infected cells). NK-T cells produce immunomodulatory cytokines, particularly interferon-γ, which leads to general activation of the immune response.

[0006] In the adaptive immune system (which defends the host against specific foreign invaders after an initial encounter with that pathogen), IL-15 is necessary for the maintenance of helper T cells that produce immunomodulatory cytokines. Importantly, IL-15 also supports the long-term maintenance of "antigen-experienced" memory T cells, which have the ability to rapidly multiply, thereby generating a faster and stronger immune response upon re-exposure to the specific foreign pathogen that has invaded the host.

[0007] Finally, although IL-15 has specific roles in both the innate and adaptive immune systems, it has significant and widespread effects in both classes of the immune system. Specifically, IL-15 inhibits or reduces apoptosis (or cell death) of many cell types associated with both classes of the immune system, including dendritic cells, neutrophils, eosinophils, mast cells, CD4+ T cells, and B cells.

[0008] Because it stimulates the proliferation and maintenance of many cells within the immune system that can fight cells that appear foreign (or "non-self") to the host, IL-15 has been proposed for use in the treatment of individuals with cancer (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 individuals with viral infections (such as HIV infection).

[0009] Although IL-15-based therapeutic approaches have the potential to be used to treat individuals suffering from a variety of diseases, they face many challenges. For example, IL-15 is rapidly cleared from plasma and is relatively unstable under physiological conditions. In addition, the in vivo signaling activity of IL-15 is also short-lived, and the molecule disadvantageously requires daily dosing or multi-day continuous infusion to achieve optimal activity. Certain approaches attempt to overcome these limitations by complexing IL-15 with the IL-15 receptor α subunit. However, this approach may eliminate the desired signaling, which occurs uniquely through the IL-15 receptor α expressed on a variety of cell types. Non-releasable PEGylation with a succinimidyl carbonate-terminated polymer with a relatively small molecular weight (5 kDa) has been reported, but this resulted in significant changes in the biological activity of IL-15. Pettit et al. (1997) J. Biol. Chem. [Journal of Biological Chemistry] 272 (4):2312-2318.

[0010] Despite the foregoing approaches, however, there remains a need for new IL-15 receptor agonists with improved characteristics and profiles, such as, for example, potent immunostimulatory effects, low systemic toxicity, stability, and / or improved pharmacokinetics. Accordingly, the present disclosure provides, inter alia, long-acting IL-15 receptor agonists having a number of advantageous features, as described in greater detail below, as well as compositions and kits comprising such agonists, and related preparation methods and uses as described herein, which are believed to be novel and completely unproven in the art. Summary of the Invention

[0011] In a first aspect, provided herein are long-acting IL-15 receptor agonists, including pharmaceutically acceptable salt forms thereof. The long-acting IL-15 receptor (IL-15R) agonist comprises at least a single linear PEG (polyethylene glycol) moiety stably covalently linked to an IL-15 amino group via an amide linkage. Interposed between the linear PEG chain and the stable amide linkage to the IL-15 amino group may be a linear unsubstituted alkylene group (-CH2-) having from 2 to 5 carbon atoms. m (ie, m=2, 3, 4, or 5).

[0012] For example, in some embodiments, the unsubstituted alkylene group is (~CH2~)2; or, in some other embodiments, the unsubstituted alkylene group is (~CH2~)3; in some other embodiments, the unsubstituted alkylene group is (~CH2~)4; in some other embodiments, the unsubstituted alkylene group is (~CH2~)5.

[0013] For example, in some embodiments, the long-acting IL-15 receptor agonist has the following structure:

[0014]

[0015] wherein IL-15 is an interleukin-15 moiety, n is an integer from about 150 to about 3,000; m is an integer from 2-5 (e.g., 2, 3, 4, or 5) and n' is 1. Formula (I) can also be depicted as [CH3O-(CH2CH2O) n (CH2) m C(O)-NH-] n’ -IL15, and the two formulas can be used interchangeably. In Formula I (and in similar formulas provided herein), the ~NH~ in the structure represents the amino group of the IL-15 moiety.

[0016] In some other embodiments, n is an integer from about 200 to about 2000, or from about 400 to about 1300, or from about 450 to about 1200.

[0017] In yet one or more additional embodiments, m is 2 or 3, such that the linear alkylene group separating the PEG moiety from the stabilizing amide linkage to IL-15 is ˜(CH 2 ) 2 ˜ or (CH 2)3 In some preferred embodiments, m is 3.

[0018] In one or more 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 10,000 Daltons (e.g., n is about 227), 15,000 Daltons (e.g., n is about 340), 20,000 Daltons (e.g., n is about 454), 25,000 Daltons (e.g., n is about 568), 30,000 Daltons (e.g., n is about 681), 40,000 Daltons (e.g., n is about 909), 50,000 Daltons (e.g., n is about 1136), and 60,000 Daltons (e.g., n is about 1364).

[0019] In one or more illustrative embodiments, compositions are provided comprising a long-acting IL-15 receptor agonist according to Formula (I), including but not limited to each of the related embodiments thereof as provided herein.

[0020] In some embodiments, the long-acting IL-15 receptor agonist composition comprises no more than about 15 mole % of a long-acting IL-15 receptor agonist encompassed by the formula:

[0021]

[0022] wherein the values ​​of n and m are as provided above for formula (I).That is, with respect to the long-acting IL-15 receptor agonist component of such compositions, the composition comprises no more than about 15 mol% of the long-acting IL-15 receptor agonist having formula (II).

[0023] For example, in some embodiments, the long-acting IL-15 receptor agonist composition comprises no more than about 10 mol% of a long-acting IL-15 receptor agonist encompassed by Formula (II), when considered together.

[0024] In some additional embodiments of the foregoing, the composition comprises no more than about 7 mol% of a long-acting IL-15 receptor agonist having n' equal to 2, 3, or greater than 3 (i.e., a higher order PEG polymer, also referred to as a "higher polymer"). In still other embodiments, the composition comprises no more than about 5 mol%, 6 mol%, 9 mol%, or 10 mol% of a long-acting IL-15 receptor agonist having n' equal to 2, 3, or greater than 3 (i.e., 2 or greater).

[0025] In some other embodiments, the composition comprises a long-acting IL-15 receptor agonist according to formula (I),

[0026] wherein n and m are as described above, and n' represents the average number of polyethylene glycol moieties covalently attached to the amino groups of IL-15 (for the composition), and n' for the composition ranges from 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.

[0027] In yet another aspect, provided herein are methods for preparing long-acting interleukin-15 receptor agonists such as those described in Formula (I) (e.g., Formula (Ia), (Ib), (Ic), (Id)) and Formula (II) (e.g., (IIa), (IIb), (IIc), and (IId)). In the methods, interleukin-15 (typically dissolved in a buffer, such as phosphate-buffered saline or any other suitable buffer, at a pH of about 7 (e.g., 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6)) is reacted with an activated PEG reagent (such as methoxy PEG-succinimidyl alkanoate) according to the following structure (wherein n is an integer from about 150 to about 3,000):

[0028] Lasts for a period of time sufficient to form the following, wherein n' is 1. Exemplary methoxy PEG-succinimidyl alkanoate reagents for reaction with interleukin-15 include the following:

[0029]

[0030] In some preferred embodiments, the methoxy PEG-succinimidyl alkanoate reagent is mPEG-succinimidylbutyrate.

[0031] In some embodiments, the methoxy PEG-succinimidyl alkanoate agent has a weight average molecular weight selected from the group consisting of about 10,000 Daltons (e.g., n is about 227), about 15,000 Daltons (e.g., n is about 340), about 20,000 Daltons (e.g., n is about 454), about 25,000 Daltons (e.g., n is about 568), about 30,000 Daltons (e.g., n is about 681), about 40,000 Daltons (e.g., n is about 909), about 50,000 Daltons (e.g., n is about 1136), and about 60,000 Daltons (e.g., n is about 1364).

[0032] In one or more embodiments of the methods, the methoxy PEG-succinimidyl alkanoate reagent and interleukin-15 are added in equimolar amounts (ie, in an equimolar ratio).

[0033] In one or more alternative embodiments, the methoxy PEG-succinimidyl alkanoate reagent is added in a molar excess relative to interleukin-15. In some specific embodiments, the methoxy PEG-succinimidyl alkanoate reagent is present in a 2-fold molar excess, or a 5-fold molar excess, or a 7-fold molar excess, or a 10-fold molar excess, or even a 12-fold molar excess or more. In some embodiments, the methoxy PEG-succinimidyl alkanoate reagent is added in a 5- to 10-fold molar excess.

[0034] In some embodiments, the methoxy PEG-succinimidyl alkanoate reagent is added as a solid.

[0035] In some other embodiments, the methoxy PEG-succinimidyl alkanoate reagent is dissolved in a suitable solvent. In certain embodiments, the methoxy PEG-succinimidyl alkanoate reagent is dissolved in an aqueous acid solution, such as, for example, dilute hydrochloric acid, although any suitable acid may be used.

[0036] In some other embodiments of the method, interleukin-15 is initially (i.e., prior to mixing with the methoxy PEG-succinimidyl alkanoate reagent) present in solution at a concentration of about 0.5 mg / mL to about 10 mg / mL. Additional illustrative concentration ranges include, for example, from about 0.5 mg / mL to about 5 mg / mL interleukin-15, from about 0.5 mg / mL to about 3 mg / mL, and from about 1.0 mg / mL to about 4 mg / mL interleukin-15.

[0037] In some other embodiments, the pH of the interleukin-15 solution is adjusted to about 8.0 prior to the addition of the methoxy PEG-succinimidyl alkanoate reagent.

[0038] In some other embodiments, the pH of the reaction mixture is adjusted to about 8.0 after addition of the methoxy PEG-succinimidyl alkanoate reagent.

[0039] In some other embodiments, the resulting reaction mixture is stirred (or mixed) for a time period sufficient to allow the reactants to react. In some embodiments, the reactants are mixed for up to and including from about 15 minutes to about 10 hours. In some other embodiments, the reactants are mixed for from about 30 minutes to about 5 hours, or from about 30 minutes to about 2 hours.

[0040] In some embodiments, the reaction is carried out under ambient conditions, such as at room temperature, i.e., in the absence of added heat. Illustrative temperature ranges for carrying out the reaction include, for example, from about 5°C to about 50°C, or from about 10°C to about 40°C, or from about 15°C to about 30°C. In some other embodiments, the reaction is carried out at a temperature of from about 20°C to about 25°C.

[0041] In some embodiments of the methods, the reaction is quenched by adding an amino acid. In some related embodiments, the reaction is quenched by adding glycine.

[0042] In some other embodiments of the methods, the conjugation product, ie, the methoxy PEG-alkanoate-interleukin-15 conjugate, is isolated from the reaction mixture.

[0043] In some additional embodiments, the reaction mixture containing the methoxy PEG-alkanoate-interleukin-15 conjugate is purified.

[0044] In some specific embodiments, the reaction results in the formation of where n' is 1.

[0045] In some other embodiments, the reaction is effective to form a composition comprising, when taken together, no more than about 15 mole percent (mol %) of a long-acting IL-15 receptor agonist encompassed by the formula (IL-15-containing molecule in the composition):

[0046]

[0047] wherein the values ​​of n and m are as provided above for formula (I).

[0048] In yet other embodiments, the reaction is effective to produce less than 20%-35%, or less than about 25% deamidated PEGylated interleukin-15.

[0049] In some embodiments, the long-acting IL-15 receptor agonist exhibits no more than about a 7-fold decrease in EC50 value (ng / mL, CTLL-2 pSTAT5) when compared to unmodified (ie, unconjugated) 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 EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 6-fold decrease in EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 5.5-fold decrease in EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 5-fold decrease in EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 4.5-fold decrease in EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 4-fold decrease in EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 3.5-fold decrease in EC50 value (ng / mL, CTLL-2 pSTAT5), or even no more than about a 3-fold decrease in EC50 value (ng / mL, CTLL-2 pSTAT5) when compared to IL-15.

[0050] In some other embodiments, the long-acting IL-15 receptor agonist exhibits receptor α binding (K) when compared to unconjugated IL-15, for example, using a technique suitable for measuring receptor α binding, such as, for example, surface plasmon resonance (SPR). D In some related embodiments, the long-acting IL-15 receptor agonist exhibits receptor α binding (K D , pM), or exhibit no more than about 45% reduction in receptor α binding (K D , pM), or exhibit no more than about 40% reduction in receptor α binding (K D, pM), or even exhibit no more than about 35% reduction in receptor α binding (K D , pM) is not more than about 30% reduction.

[0051] In yet other embodiments, the long-acting IL-15 receptor agonist exhibits no more than about a 7-fold decrease in EC50 value (ng / mL, CTLL-2pSTAT5) compared to unmodified IL-15 and exhibits receptor α binding (K D , pM) by no more than about 50%, including the above EC50 values ​​or K D Any specific combination of one or more values ​​that decreases.

[0052] In yet one or more embodiments, a composition is provided, comprising a long-acting IL-15R agonist as described herein, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

[0053] In yet other embodiments, the long-acting IL-15R agonist or composition is effective in stimulating NK activation and / or proliferation when administered to a subject at a therapeutically effective dose.

[0054] In yet one or more other embodiments, the long-acting IL-15R agonist or composition is effective in supporting CD8 T cell survival and / or memory formation when administered to a subject at a therapeutically effective dose.

[0055] In another aspect, provided herein are methods of treating a condition responsive to treatment with IL-15 by administering to a subject suffering from the condition a therapeutically effective dose of a long-acting IL-15R agonist as provided herein, or a composition comprising such an agonist.

[0056] In yet another aspect, provided are methods for treating cancer by administering to a subject having cancer a therapeutically effective dose of a long-acting IL-15R agonist or composition as provided herein.

[0057] Additional aspects and embodiments are set forth in the following description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 The amino acid sequence of an exemplary recombinant human IL-15 from Escherichia coli is provided (SEQ ID NO: 1), which is a single non-glycosylated polypeptide chain containing 115 amino acids and a molecular weight of 12.9 kDa.

[0059] Figure 2 is a chromatogram illustrating RP-HPLC analysis of an exemplary conjugation reaction mixture as described in Example 1.

[0060] Figure 3 is the FPLC purification profile of the anion exchange chromatography column as described in Example 1.

[0061] Figure 4 Figure 1 is an SDS-PAGE of an exemplary, purified long-acting IL-15 receptor agonist, mono-mPEG-butyramide-IL-15, as described in Example 1. Lane 1 provides molecular weight markers as indicated; lane 2 is unconjugated parent molecule IL-15, and lane 3 is mono-mPEG-butyramide-IL-15.

[0062] Figure 5 is RP-HPLC analysis of purified mono-mPEG-butyramide-IL-15 as described in Example 1.

[0063] Figure 6 . is a plot of plasma concentration of the test article (IL-15, filled circles; or mPEG2-CAC-FMOC-20K-NHS-IL-15, also known as N-(2-methoxyPEG-ethyl)-7-(4-((2-methoxyPEG-ethyl)amino)-4-oxobutyl)-9-ethyl-9H-fluorene-4-carboxamidecarbamate-IL-15, or Conjugate 2, filled squares) over time following administration of a single intravenous dose of the test article in mice as described in Example 6.

[0064] Figure 7 Figure 7 is a graph of the mean plasma concentration of mPEG2-CAC-FMOC-20K-NHS-IL-15 (also known as N-(2-methoxyPEG-ethyl)-7-(4-((2-methoxyPEG-ethyl)amino)-4-oxobutyl)-9-ethyl-9H-fluorene-4-carboxamidecarbamate-IL-15, or Conjugate 2) over time following administration of a single intravenous dose of Conjugate 2 at doses of 0.3 (■), 0.15 (▲), and 0.075 mg / kg (●), or a single subcutaneous dose of Conjugate 2 at 0.15 mg / kg (◆) in rats as described in Example 7.

[0065] Figure 8A and 8B As described in Example 8, a single intravenous dose of IL-15 ( Figure 8A ) or conjugate 2 (0.3 mg / kg, Figure 8B ) after STAT5 phosphorylation in various lymphocytes (i.e., CD4 (■), CD8 (▲)) and NK cells (▼).

[0066] Figure 9A 、 9B9A and 9C are graphs showing the degree of STAT5 phosphorylation in various lymphocytes (ie, CD4, CD8, and NK cells) following administration of a single intravenous dose (0.5 mg / kg) of Conjugate 2 in cynomolgus monkeys as described in Example 9.

[0067] Figure 10A and 10B The following curves are shown: As described in detail in Example 10, the NK subsets of human PBMCs: CD56 bright ( Figure 10A ) and CD56 dim ( Figure 10B In vitro activity of exemplary long-acting IL-15 receptor agonists (Conjugates 1, 3, and 5) as measured by signaling in NK cells.

[0068] Figure 11A and 11B Figure 5 is a graph illustrating NK cell proliferation in mice following intravenous administration of a single mPEG-SBA40K-IL-15 (also referred to herein as Conjugate 1) at a dose of 0.03 mg / kg (low dose, open squares), 0.3 mg / kg (medium dose, filled circles, solid line), or 1 mg / kg (high dose, diamonds) as described in Example 11, when compared to vehicle. Figure 11A illustrates Ki67 expression over time (expressed as a percentage), while Figure 11B NK cell counts (cells / ul) relative to time after administration of each of the sample groups are illustrated.

[0069] Figures 12A-12D Figure 5 is a graph illustrating the increased number of NK cells of all maturation levels in mice following intravenous administration of Conjugate 1 at doses of 0.03 mg / kg (low dose, open squares), 0.3 mg / kg (medium dose, filled circles, solid line), or 1.0 mg / kg (high dose, diamonds) compared to vehicle (filled circles, dashed line) as described in Example 11. Subpopulations of NK cells were defined by CD11b and CD27 expression. Figure 12A demonstrated an increase in the number of terminal effector cells, expressed as cells / μL, from 24 to 120 hours after administration; Figure 12B demonstrated an increase in the number of precursor NK cells, expressed as cells / μL, from 24 to 120 hours after administration; Figure 12C demonstrated an increase in the number of high effector cells, expressed as cells / μL, from 24 to 120 hours after administration, and Figure 12D An early increase in the number of NK cells expressed as cells / μL was demonstrated from 24 to 120 hours after administration.

[0070] Figure 13A and 13Bare curves illustrating the effect of NK cell responses to NKG2D ( Figure 13A ) and granzyme B ( Figure 13B ) expression levels.

[0071] Figure 14

[00145] Figure 1 is a graph illustrating the number of CD8 T cells in cells / μl before and from 24 hours to 120 hours after intravenous administration of Conjugate 1 in mice at a dose of 0.03 mg / kg (low dose), 0.3 mg / kg (medium dose), or 1 mg / kg (high dose) as described in Example 11.

[0072] Figure 15A and 15B

[00145] Figures 2A and 2B are graphs illustrating the expression levels of Ki67 on T effector memory cells and T central memory cells, respectively, over time following intravenous administration of Conjugate 1 at a dose of 0.3 mg / kg or 1.0 mg / kg in mice, relative to time post-administration, as described in Example 11, compared to vehicle and IL-15.

[0073] Figure 16A and 16B is a graph illustrating NK cell proliferation in cynomolgus monkeys following intravenous administration of Conjugate 2 at a dose of 500 μg / kg as described in Example 12. Figure 16A The Ki67 expression in NK cells from before administration to 15 days after administration is shown (expressed as a percentage). Figure 16B NK cell counts from before dosing to 15 days after administration are shown.

[0074] Figure 17 is a graph illustrating CD8 T cell counts in cynomolgus monkeys following intravenous administration of Conjugate 2 at a dose of 500 μg / kg as described in Example 12, from pre-dose to 14 days post-administration (shown for each animal).

[0075] Figure 18A and 18B are curves illustrating the following: after intravenous administration of Conjugate 2 at a dose of 500 μg / kg as described in Example 12, the number of CD8 T effector memory cells (T EM cells) and CD8 T central memory cells (T CM ) number.

[0076] Figure 19 Illustrated are the total number of lesions per mouse in the lungs of female Balb / c mice inoculated with mouse CT-26 colon cancer cells, as described in detail in Example 13, following treatment with one of the following test articles: vehicle, phosphate-buffered saline (Group A); naive IL-15 alone (Group B); Conjugate 2 at a dose of 0.03 mg / kg (Group C); Conjugate 2 at a dose of 0.1 mg / kg (Group D); Conjugate 2 at a dose of 0.3 mg / kg (Group E); Conjugate 2 at a dose of 1.0 mg / kg (Group F); and Conjugate 2 at a dose of 3.0 mg / kg (Group G).

[0077] Figure 20 Illustrated are the total number of lesions per mouse in the lungs of female Balb / c mice inoculated with mouse CT-26 colon cancer cells, as described in detail in Example 13, following treatment with one of the following test articles: vehicle, phosphate-buffered saline (Group A); Conjugate 1 at a dose of 0.03 mg / kg (Group H); and Conjugate 1 at a dose of 0.3 mg / kg.

[0078] Figure 21 Illustrated are the percentage of specific lysis as a function of E:T (effector:target) ratio at 1000 ng / mL (squares), 3000 ng / mL (circles), 300 ng / mL (triangles), 30 ng / mL (upper X), 3 ng / mL (diamonds), and unstimulated (lower X), as described in Example 14. The data demonstrate dose-dependent increased cytotoxicity of NK cells in vitro following culture with Conjugate 1.

[0079] Figures 22A-22D is a graph illustrating the increased number of NK cells of all maturation levels in mice following intravenous administration of Conjugate 1 at doses of 0.01 mg / kg, 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg and 1.5 mg / kg as described in Example 11. Figure 22A demonstrated an increase in the number of terminal effector cells, expressed as cells / μL, from 24 to 120 hours after administration; Figure 22B demonstrated an increase in the number of precursor NK cells, expressed as cells / μL, from 24 to 120 hours after administration; Figure 22C demonstrated an increase in the number of high effector cells, expressed as cells / μL, from 24 to 120 hours after administration, and Figure 22D An early increase in the number of NK cells expressed as cells / μL was demonstrated from 24 to 120 hours after administration.

[0080] Figure 23is a graph illustrating granzyme B expression over time following treatment with Conjugate 1 at doses of 0.01 mg / kg, 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, and 1.5 mg / kg, as described in Example 15. This data demonstrates that treatment with Conjugate 1 increases granzyme B expression by NK cells.

[0081] Figure 24 Figure 5 is a graph illustrating the number of CD8 T cells isolated from the spleen of mice, expressed in cells / μl, before and 24 to 96 hours after intravenous administration of Conjugate 1 at doses of 0.03 mg / kg (filled circles, dashed line) and 0.3 mg / kg (filled circles, solid line) and vehicle IL-15 buffer (open circles, dashed line) in mice as described in Example 11.

[0082] Figure 25 is a graph illustrating the Ki67 expression level (expressed as a percentage) of CD49b cells relative to time after administration following intravenous administration of Conjugate 1 at a dose of 0.03 mg / kg or 0.3 mg / kg in mice, as described in Example 11, compared to vehicle IL-15 buffer.

[0083] Figure 26 is a graph illustrating the level of granzyme B expression by CD49b cells over time (expressed as a percentage) following intravenous administration of Conjugate 1 at a dose of 0.03 mg / kg or 0.3 mg / kg in mice, relative to the time after administration, as described in Example 11.

[0084] Figure 27 is a graph illustrating the in vivo cytotoxicity results following treatment with Conjugate 1 at 0.3 mg / kg as described in Example 14. Cytotoxicity was assessed 24 hours, 48 ​​hours, and 72 hours after treatment.

[0085] Figures 28A-28D are curves illustrating the increased number of NK cells of all maturation levels in mice following intravenous administration of Conjugate 1 at doses of 0.03 mg / kg and 0.3 mg / kg as a single dose or after the third dose in a q7dx3 schedule as described in Example 11. Figure 28A demonstrated an increase in the percentage of Ki67CD49b cells from 24 to 240 hours after administration; Figure 22B demonstrated an increase in the percentage of granzyme B CD49b cells from 24 to 240 hours after administration; Figure 28C demonstrates an increase in the number of CD49b cells, expressed as cells / μL, from 24 hours to 240 hours after administration; and Figure 28D Demonstrated is an increase in the percentage of granzyme B (granB)+ MFI CD49b cells from 24 to 240 hours after administration.

[0086] Figures 29A-29C is a curve associated with the study described in Example 16. Figure 29A

[00145] Figures 144 and 144 are plasma concentrations of the test article (IL-15 or Conjugate 1) following a single intravenous dose of 0.5 and 0.3 mg / kg, respectively, in balb / c mice. Conjugate 1 exhibited a half-life of approximately 12 hours, while IL-15 was rapidly cleared from plasma with a half-life of less than 1 hour. Figure 29B Figure 2 is a graph showing the percentage of pSTAT5 positive cells in CD8 T cells in mice following a single injection of Conjugate 1 at 0.03 and 0.3 mg / kg. Conjugate 1 induced sustained pSTAT5 signaling in CD8 T cells at both dose levels. A 120-hour time course is shown, including pre-dose administration. Figure 29C is a graph of the percentage of pSTAT5 positivity in murine NK cells following a single injection of 0.03 and 0.3 mg / kg of Conjugate 1. Conjugate 1 induced robust and sustained pSTAT5 signaling in NK cells at both dose levels.

[0087] Figures 30A-30C It is a curve of total CD8, CD8 central memory (Tcm) and CD8 effector memory (Tem) cell numbers after a single administration of conjugate 1 at 0.01, 0.03, 0.1, 0.3, 1 or 1.5 mg / kg as described in Example 17. As described in Example 17, conjugate 1 at a dose level equal to or greater than 0.03 induces a significant increase in total CD8 T cells in the blood. The lowest dose of 0.01 mg / kg increases CD8 Tcm and CD8 Tem. At 0.3 mg / kg, conjugate 1 increases CD8, CD8 Tcm and CD8 Tem by 6.4X, 37.9X and 14.5X, respectively. It is noteworthy that when conjugate 1 is administered at 0.3-1.5 mg / kg, CD8 and CD8 memory T cell numbers do not return to baseline 240 hours after injection, which demonstrates the sustained PD effect of conjugate 1.

[0088] Figure 30D 、 30E 30F and 30F are plots of the percentage of Ki-67 positivity within the total CD8, CD8 Tcm, and CD8 Tem populations, respectively, in mice as described in Example 17. A single dose of Conjugate 1 increased Ki-67 positivity in all CD8 and CD8 subset populations at all dose levels.

[0089] Figure 31A 、 31B 31C and 31C are plots of CD8 and CD8 memory subset T cell numbers following single (dashed line) or Q7dx3 (solid line) administration of Conjugate 1 at 0.03 and 0.3 mg / kg, as described in Example 17. Repeated dosing further increased these populations, with CD8, CD8 Tcm, and CD8 Tem increasing by 35.3X, 183X, and 73.8X, respectively. At the end of the time course (240 hours after the first or last dose at 0.3 mg / kg), cell numbers in mice had not returned to baseline.

[0090] Figure 32A and 32B Figure 1 is a graph of NK cell numbers and Ki-67 positive percentages following single doses of Conjugate 1 from 0.01 to 1.5 mg / kg in mice as described in Example 17. At all dose levels, NK cell numbers increased significantly above the dose control and returned to baseline by 240 hours post-dose. All dose levels induced a robust increase in the percentage of Ki-67 positive NK cells.

[0091] Figure 32C Figure 1 is a graph of murine NK cell numbers following single (solid line) or Q7dx3 (dashed line) administration of Conjugate 1 at 0.03 and 0.3 mg / kg, as described in Example 17. Repeated administration of Conjugate 1 at 0.3 mg / kg induced slightly fewer, but still significant, NK cell numbers compared to a single dose. Similar NK cell numbers were achieved with single versus repeated administration at 0.03 mg / kg.

[0092] Figure 33A The in vitro NK cell toxicity assay as described in 18 in the example is described, which measures the changes in NK-mediated target cell lysis after test article treatment in mice. The time course under the indicated hours of the specific lysis percentage of YAC-1 cells by spleen NK cells isolated from balb / c mice treated with 0.006, 0.03 or 0.3 mg / kg conjugate 1 or 1 mg / kg IL-15 is shown. The spleen NK cells from mice administered with vehicle serve as a control. Compared with the NK cells from mice receiving a single injection of IL-15 at 1 mg / kg, the amplitude and duration of the NK cell toxicity induced by conjugate 1 administered at 0.3 mg / kg are more excellent.

[0093] Figure 33B yes Figure 33AFigure 1 is a graph showing the percentage of granzyme B positive cells in blood from the same mice used in a dedicated NK in vitro cytotoxicity assay. See Example 18. Conjugate 1 induced a significant increase in NK granzyme B expression at 0.03 and 0.3 mg / kg, with a robust and sustained increase seen at 0.3 mg / kg.

[0094] Figure 34A and 34B The percentage inhibition of lung nodules in balb / c mice treated with conjugate 1 at 0.03 or 0.3 mg / kg, administered twice a week, as described in Example 19, is shown. Injections of conjugate 1 at 0.03 and 0.3 mg / kg inhibited lung nodule formation by 40% and 80%, respectively. The same mice dosed at 0.3 mg / kg were followed up for 32 days after tumor cell injection to assess survival. Treatment with conjugate 1 significantly increased survival compared to mice injected with tumors that received vehicle control.

[0095] Figure 35 Figure 2 is a graph of the percent inhibition of lung nodules in CT-26 injected mice treated with Conjugate 2 that received antibody-mediated NK cell depletion (olive green), IgG control (blue), or PBS (orange) as described in Example 20. Data are presented as percent inhibition of lung nodules relative to CT-26 injected mice that were not subjected to NK cell depletion and treated with vehicle control (black). Conjugate 2 efficacy in this tumor model was abolished when mice lacked NK cells.

[0096] Figure 36A and 36B Figure 21 is a graph illustrating the two-week time course of CD8 cell number and percent Ki-67 positivity as a measure of proliferation in one male (dashed line) and one female (solid line) cynos following intravenous administration of Conjugate 1 at a dose of 0.1 mg / kg as described in Example 21. Conjugate 1 induced a significant increase in CD8 T cells in the cynos, with a 7-10X increase in cell number after a single dose.

[0097] Figure 36C and 36D Demonstrated is the increase in cyno CD8 Tcm and CD8 Tem cell numbers following a single injection of Conjugate 1 as described in Example 21. CD8 Tcm and Tem numbers increased 27-30X and 21-33X, respectively.

[0098] Figure 37A and 37Bis a graph of the number of NK cells and the percentage of Ki-67 positivity in cynos following a single 0.1 mg / kg dose of Conjugate 1. See Example 21. Following treatment with Conjugate 1, NK cells increased 9-10X.

[0099] Figure 38A and 38B EC50 curves for IL-15 (red, filled circles) versus Conjugate 1 (green, solid squares) treatment of human PBMCs and subsequent measurement of the percentage of pSTAT5 positivity in CD8 and CD56 bright NK cells as described in Example 22. Conjugate 1 was 5.5 and 15X less potent than IL-15 in engaging CD8 and CD56 bright NK cells, respectively. However, Conjugate 1 achieved the same maximal response as regular IL-15.

[0100] Figure 39 Figure 23 is the plasma concentration profile over time of IL-15 at 500 μg / kg (green, filled circles) or Conjugate 1 at 10 μg / kg (pink, filled squares), 30 μg / kg (purple, filled upward facing triangles), 100 μg / kg (red, filled downward facing triangles), 300 μg / kg (orange, filled diamonds), or 1000 μg / kg (dark red, open hexagons) following a single intravenous dose in mice as described in Example 23.

[0101] Figure 40 Figure 24 is the plasma concentration profile of Conjugate 1 over time at 10 μg / kg (pink, solid squares), 75 μg / kg (purple, solid upward facing triangles), or 150 μg / kg (orange, solid downward facing triangles) following a single intravenous dose in rats as described in Example 24.

[0102] Figure 41 Figure 25 is the plasma concentration profile over time of IL-15 at 50 μg / kg (green, filled circles) or Conjugate 1 at 10 μg / kg (red, filled downward facing triangles), 50 μg / kg (blue, filled diamonds), or 100 μg / kg (orange, filled squares) following administration of a single intravenous dose of the test article in cynomolgus monkeys as described in Example 25.

[0103] Figure 42A and 42BFigure 26 shows a plot of CD4 T cell counts and Ki-67 positive percentages after a single dose of 0.03 mg / kg (blue, solid circles) or 0.3 mg / kg (orange, solid circles) of Conjugate 1 in mice, respectively. Vehicle (black) and pre-dose (open circles) cell levels are also shown. All dose levels returned to baseline by 240 hours post-dose. The 0.3 mg / kg dose level induced a robust increase in the percentage of Ki-67 positive CD4 T cells.

[0104] Figure 43 Figure 26 is a graph of the percentage of pSTAT5 positivity in CD4 T cells in mice following a single injection of Conjugate 1 at 0.03 mg / kg (orange, filled circles) or 0.3 mg / kg (blue, filled squares), as described in Example 26. Conjugate 1 at both dose levels induced an increase in pSTAT5 signaling in CD4 T cells, with 0.3 mg / kg inducing a greater increase. A 120-hour time course is shown, including vehicle (black) and pre-dose (open circles).

[0105] Figure 44A 、 44B and 44C are curves illustrating the effect of intravenous administration of vehicle (black) or Conjugate 1 at doses of 0.003 mg / kg (blue, downward-facing triangles), 0.01 mg / kg (green, diamonds), or 0.1 mg / kg (orange, solid squares) against NK cells ( Figure 44A ), CD8 T cells ( Figure 44B ) and CD4 T cells ( Figure 44C ) relative to the time after administration.

[0106] Figure 45A 、 45B 45C and 45C are curves illustrating the percentage of Ki-67 positivity following a single dose of conjugate 1 at a dose of 0.001 mg / kg (purple, solid squares), 0.003 mg / kg (blue, downward-facing triangles), or 0.1 mg / kg (orange, solid squares) in cynomolgus monkeys as described in Example 27. Vehicle (black) levels are also shown. All dose levels returned to baseline by at least 17 days after dosing. The 0.1 mg / kg and 0.003 mg / kg dose levels induced a robust increase in the percentage of Ki-67 positivity in NK cells and CD8 T cells. The 0.1 mg / kg dose level induced an increase in the percentage of Ki-67 positivity in all cell types tested.

[0107] Figure 46A 、 46Band 46C illustrate the NK cell ( Figure 46A ), CD8 T cells ( Figure 46B ) and CD4 T cells ( Figure 46C ) in the STAT5 phosphorylation level.

[0108] Figures 47A-47D are graphs illustrating the percentage of Ki-67 positivity following a single dose of Conjugate 1 at a dose of 0.001 mg / kg (purple, solid squares), 0.01 mg / kg (green, diamonds), or 0.1 mg / kg (orange, solid squares) in cynomolgus monkeys as described in Example 27. Vehicle (black) levels are also shown. Figure 47A Shown is the CD8 T 初始 The results of cells, Figure 47B Shown is the CD8 T scm Cell results. Figure 47C Shown is the CD8 T cm Cell results. Figure 47D Shown is the CD8 T em Cell results.

[0109] Figure 48A 、 48B and 48C are curves illustrating the following: As described in Example 28, in the presence of Conjugate 1 at 0.001 mg / kg ( Figure 48A )、0.01mg / kg( Figure 48B ) and 0.1mg / kg( Figure 48C ) over time following treatment with a dose of . The data demonstrate that treatment with Conjugate 1 increases granzyme B levels in NK cells in non-human primates (NHPs).

[0110] Figure 49A 、 49B and 49C are curves illustrating the following: As described in Example 28, in the presence of Conjugate 1 at 0.001 mg / kg ( Figure 49A )、0.01mg / kg( Figure 49B ) and 0.1mg / kg( Figure 49C ) over time after treatment with a dose of . This data indicates that treatment with Conjugate 1 increases perforin levels in NK cells in NHPs. DETAILED DESCRIPTION

[0111] 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 specific synthetic techniques, IL-15 moieties, etc., and as such may vary as understood by one of ordinary skill in the art to which the present disclosure applies.

[0112] In describing and claiming certain features of the present disclosure, the following terminology will be used in accordance with the definitions set forth below, unless otherwise indicated.

[0113] It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0114] In describing and claiming one or more embodiments, the following terminology will be used in accordance with the definitions set forth below.

[0115] A "physiologically cleavable" or "hydrolyzable" or "degradable" bond is a relatively unstable bond that reacts (i.e., is hydrolyzed) with water under physiological conditions. The tendency of a bond to hydrolyze in water may depend not only on the general type of linkage connecting two atoms within a given molecule, but also on the substituents attached to those atoms. Suitable hydrolytically unstable or weak linkages may include, but are not limited to, carboxylates, phosphates, anhydrides, acetals, ketals, acyloxyalkyl ethers, imines, orthoesters, peptides, oligonucleotides, thioesters, and carbonates.

[0116] "Enzymatically degradable linkage" means a linkage that is subject to degradation by one or more enzymes.

[0117] A "stable" linkage or bond refers to a chemical bond that is substantially stable in water, that is, does not undergo any appreciable degree of hydrolysis under physiological conditions over an extended period of time. Examples of hydrolytically stable linkages generally include, but are not limited to, the following: carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, amines, and the like. Generally speaking, a stable linkage is one that exhibits a daily hydrolysis rate of less than about 1%-2% under physiological conditions. The hydrolysis rates of representative chemical bonds can be found in most standard chemistry textbooks.

[0118] For example, in the context of polyethylene glycol covalently attached to an active moiety (such as interleukin-15), a covalent "releasable" linkage is one that releases or separates the polyethylene glycol polymer from the active moiety under physiological conditions, e.g., by any suitable mechanism, at a clinically useful rate, and includes, for example, and without limitation, hydrolyzable linkages and enzymatically degradable linkages.

[0119] "Substantially" or "essentially" means almost entirely or completely, for example 95% or greater of a given amount.

[0120] Similarly, "about" or "approximately" as used herein means within plus or minus 5% of a given amount.

[0121] "Optional" or "optionally" means that the subsequently described circumstance may but need not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0122] A "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to a component that can be included in the compositions as described herein and does not cause significant adverse toxicological effects in a subject.

[0123] The phrases "pharmaceutically effective amount," "pharmacologically effective amount," "therapeutically effective amount," and "physiologically effective amount" are used interchangeably herein and refer to the amount of a long-acting IL-15R agonist, as provided herein, required to provide the desired level of the substance in the bloodstream or in the target tissue to produce the desired biological or pharmaceutical response. For example, such a response may destroy target cancer cells or slow or arrest cancer progression in a subject. The term also applies to a dose that will induce a specific response in the target cells. The exact amount will depend on many factors, such as, for example, the specific condition being treated, the intended patient population, individual patient considerations, the components and physical properties of the therapeutic composition to be administered, and the like.

[0124] References to long-acting IL-15R agonists as described herein are meant to encompass pharmaceutically acceptable salt forms thereof.

[0125] As used herein, the term "patient" or "subject" refers to a living organism suffering from or susceptible to a condition that can be prevented or treated by administering a compound or composition as provided herein. Subjects include, but are not limited to, mammals (e.g., murine, simian, equine, bovine, porcine, canine, feline, etc.), and are preferably humans.

[0126] In the context of water-soluble polymers such as PEG, molecular weight 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 molecular weight determinations (number average and weight average molecular weight) can be measured using gel permeation chromatography or other liquid chromatography techniques (e.g., gel filtration chromatography). Gel permeation chromatography and gel filtration chromatography are the most commonly used. Other methods for determining molecular weight include end-group analysis or colligative properties (e.g., freezing point depression, boiling point elevation, or osmotic pressure) measurements to determine the number average molecular weight; or light scattering techniques, ultracentrifugation, MALDI TOF, or viscometry to determine the weight average molecular weight. PEG polymers are typically polydisperse (i.e., the number average molecular weight of these polymers is not equal to the weight average molecular weight), with a low polydispersity value of preferably less than about 1.2, more preferably less than about 1.15, still more preferably less than about 1.10, yet more preferably less than about 1.05, and most preferably less than about 1.03.

[0127] The terms "active," "reactive," or "activated," when used in conjunction with a specific functional group, refer to a reactive functional group that readily reacts with an electrophile or nucleophile on another molecule. This is in contrast to those groups that require strong catalysts or highly impractical reaction conditions in order to react (i.e., "non-reactive" or "inert" groups).

[0128] As used herein, the term "functional group" or any synonym thereof is meant to encompass protected as well as unprotected forms thereof.

[0129] The terms "spacer moiety," "linkage," and "linker" may be used herein to refer to a bond or an atom or collection of atoms that is optionally used to connect interconnected moieties (such as a terminus of a polymeric reagent and an IL-15 moiety). The spacer moiety may be hydrolytically stable or may include a physiologically hydrolyzable, enzymatically degradable, or otherwise releasable linkage. Unless the context clearly dictates otherwise, a spacer moiety is optionally present between any two elements of a compound (e.g., an IL-15 moiety and a water-soluble polymer (such as PEG) may be linked directly or indirectly via a spacer moiety).

[0130] "Alkyl" refers to hydrocarbon chains typically ranging from about 1 to 15 atoms in length. Such hydrocarbon chains are preferably, but not necessarily, saturated and can be branched or straight, though straight chains are typically preferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 3-methylpentyl, and the like.

[0131] "Lower alkyl" refers to an alkyl group containing from 1 to 6 carbon atoms and which may be straight or branched, as exemplified by methyl, ethyl, n-butyl, isobutyl, and tert-butyl.

[0132] "Alkoxy" refers to a -OR group where R is alkyl or substituted alkyl, preferably C 1-6 Alkyl groups (eg, methoxy, ethoxy, propoxy, etc.).

[0133] The term "substituted", as in, for example, "substituted alkyl", refers to a moiety (e.g., an alkyl group) that is substituted with one or more non-interfering substituents such as, but not limited to, alkyl, C 3-8 Cycloalkyl, for example, cyclopropyl, cyclobutyl, etc.; halo, for example, fluorine, chlorine, bromine, and iodine; cyano; alkoxy, lower phenyl; substituted phenyl; etc. "Substituted aryl" is an aryl group having one or more non-interfering groups as substituents. For substitutions on the phenyl ring, the substituents can be in any orientation (i.e., ortho, meta, or para).

[0134] "Non-interfering substituents" are those groups that, when present in a molecule, typically do not react with other functional groups contained within that molecule.

[0135] "Aryl" means one or more aromatic rings, each having 5 or 6 core carbon atoms. Aryl includes multiple aryl rings, which may be fused (as in naphthyl) or unfused (as in biphenyl). Aryl rings may also be fused or unfused to one or more cyclic hydrocarbons, heteroaryls, or heterocycles. As used herein, "aryl" includes heteroaryl.

[0136] "Heteroaryl" is an aryl group containing from one to four heteroatoms (preferably sulfur, oxygen, or nitrogen, or a combination thereof). The heteroaryl ring may also be fused to one or more cyclic hydrocarbon rings, heterocyclic rings, aryl rings, or heteroaryl rings.

[0137] "Heterocycle" or "heterocyclic" means one or more rings having 5 to 12 atoms, preferably 5 to 7 atoms, with or without unsaturation or aromatic character and having at least one ring atom that is not carbon. Preferred heteroatoms include sulfur, oxygen, and nitrogen.

[0138] A "substituted heteroaryl" is a heteroaryl group having one or more non-interfering groups as substituents.

[0139] A "substituted heterocycle" is a heterocycle having one or more side chains formed from non-interfering substituents.

[0140] As used herein, "organic group" shall include alkyl, substituted alkyl, aryl, and substituted aryl groups.

[0141] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the composition of the present invention and that does not cause significant adverse toxicological effects to the patient.

[0142] As used herein, the term "IL-15 portion" refers to a peptide or protein portion that has human IL-15 activity. Furthermore, the term "IL-15 portion" encompasses the IL-15 portion before conjugation as well as the IL-15 portion residue after conjugation. As explained in further detail below, one of ordinary skill in the art can determine whether any given portion has IL-15 activity. Proteins comprising an amino acid sequence corresponding to any one of SEQ ID NOs: 1 to 3, as well as any proteins or polypeptides substantially homologous thereto, are IL-15 portions. As used herein, the term "IL-15 portion" includes such peptides and proteins that have been intentionally modified, for example, by site-directed mutagenesis, or accidentally modified by mutation. These terms also include analogs having from one to six additional glycosylation sites, analogs having at least one additional amino acid at the carboxyl terminus of the peptide or protein, wherein the one or more additional amino acids comprise at least one glycosylation site, and analogs having an amino acid sequence comprising at least one glycosylation site. The term includes naturally, recombinantly, and synthetically produced moieties.

[0143] The terms "substantially homologous" or "substantially identical" mean 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 does not result in an undesirable functional difference between the reference sequence and the subject sequence. For purposes herein, sequences having greater than 95% homology (identity), equivalent biological activity (but not necessarily of equivalent strength), and equivalent expression characteristics to a given sequence are considered substantially homologous (identical). For purposes of determining homology, truncation of the mature sequence should be disregarded. Exemplary IL-15 polypeptides used herein include those that are substantially homologous to SEQ ID NO: 1. SEQ ID NO: 2 is nearly identical to SEQ ID NO: 1, except that SEQ ID NO: 2 has a methionine at the beginning of the sequence, which is required for translation initiation in E. coli.

[0144] The term "fragment" refers to any protein or polypeptide having the amino acid sequence of a portion or fragment of an IL-15 portion and having, or substantially having, the biological activity of IL-15. Fragments include proteins or polypeptides produced by proteolytic degradation of an IL-15 portion, as well as proteins or polypeptides produced by chemical synthesis using methods conventional in the art.

[0145] The amino acid residues in the peptides are abbreviated as follows: Phe or F for phenylalanine; Leu or L for leucine; Ile or I for isoleucine; Met or M for methionine; Val or V for valine; Ser or S for serine; Pro or P for proline; Thr or T for threonine; Ala or A for alanine; Tyr or Y for tyrosine; His or H for histidine; Gln or Q for glutamine; Asn or N for asparagine; Lys or K for lysine; Asp or D for aspartic acid; Glu or E for glutamic acid; Cys or C for cysteine; Trp or W for tryptophan; Arg or R for arginine; and Gly or G for glycine.

[0146] Overview

[0147] The present disclosure relates to providing a long-acting IL-15 receptor agonist. Such an agonist would ideally have several advantageous and unpredictable features, such as at least one (if not more) of the following: (i) delivering sustained IL-15 activity without the need for daily dosing by providing a measurable pharmacodynamic effect, (ii) retaining binding to IL-15 receptor alpha to a large extent (i.e., compared to IL-15), (iii) stimulating NK cell activation and / or proliferation, and / or (iv) supporting CD8 T cell survival and / or memory formation, and (v) providing inhibition of tumor growth. Unexpectedly, the applicant has achieved a long-acting IL-15R agonist with a unique combination of advantageous properties, which will be described in detail below.

[0148] Long-acting IL-15R agonists and related compositions

[0149] Generally, a long-acting IL-15 receptor agonist or a pharmaceutically acceptable salt thereof comprises a single linear PEG (polyethylene glycol) moiety stably covalently linked to an IL-15 amino group via an amide linkage. Between the PEG moiety and the stable amide linkage to the IL-15 amino group is a linear unsubstituted alkylene group (~CH2~) having from 2 to 5 carbon atoms. m (ie, m=2, 3, 4, or 5).

[0150] In the context of an IL-15 moiety, the term "IL-15 moiety" refers to the IL-15 moiety prior to conjugation as well as the IL-15 moiety after attachment to a non-peptide water-soluble polymer, such as a poly(alkylene oxide) (e.g., poly(ethylene glycol) or PEG). Although PEG is specifically referred to below as a non-peptide water-soluble polymer, it should be understood that the present disclosure generally relates to non-peptide water-soluble polymers or poly(alkylene glycol). However, it should be understood that when the original IL-15 moiety is attached to the polyethylene glycol moiety, the IL-15 moiety is slightly altered due to the presence of one or more covalent bonds associated with the polymer linkage(s).

[0151] The IL-15 portion can be derived from non-recombinant and recombinant methods, and the present disclosure is not limited in this regard. Additionally, the IL-15 portion can be derived from human sources, animal sources (including insects), fungal sources (including yeast), and plant sources.

[0152] The IL-15 fraction can be obtained, for example, according to the procedure described by Grabstein et al. [See Grabstein et al. (1994) Science. 264 :965-968]. The IL-15 portion can also be prepared using recombinant methods, such as those described in European Patent No. 0 772 624 B2 to Immunex Corporation. Alternatively, the IL-15 portion can be commercially available from, for example, GenScript USA Inc. (Piscataway, NJ) and Peprotech (Rockyhill, NJ).

[0153] The IL-15 portion can be expressed in bacteria [e.g., E. coli, see, e.g., Fischer et al. (1995) Biotechnol. Appl. Biotechnol. 21 (3): 295-311], mammals [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 plants [see, e.g., Mor et al. (2001) Biotechnol. Bioeng. 75 (3): 259-266] expression system. Expression can be via exogenous expression (when the host cell naturally contains the desired genetic code) or via endogenous expression.

[0154] Although recombinant-based methods for preparing proteins can vary, recombinant methods typically involve constructing a nucleic acid encoding the desired polypeptide or fragment, cloning the nucleic acid into an expression vector, transforming a host cell (e.g., a plant, bacteria, yeast, a transgenic animal cell, or a mammalian cell, such as a Chinese hamster ovary cell or a baby hamster kidney cell), and expressing the nucleic acid to produce the desired polypeptide or fragment. Methods for producing and expressing recombinant polypeptides in vitro and in prokaryotic and eukaryotic host cells are known to those of ordinary skill in the art.

[0155] To facilitate the identification and purification of recombinant polypeptides, a nucleic acid sequence encoding an epitope tag or other affinity binding sequence can be inserted or added in frame with the coding sequence, thereby producing a fusion protein consisting of the desired polypeptide and a polypeptide suitable for binding. The fusion protein can be identified and purified in the following manner: first, a mixture containing the fusion protein is passed through an affinity column carrying a binding portion (e.g., an antibody) for the epitope tag or other binding sequence in the fusion protein, thereby binding the fusion protein within the column. Thereafter, the fusion protein can be recovered by washing the column with an appropriate solution (e.g., an acid) to release the bound fusion protein. The recombinant polypeptide can also be purified in the following manner: lysing the host cells, isolating the polypeptide, for example, by ion exchange chromatography, affinity binding methods, hydrophobic interaction methods, and thereafter identifying by MALDI or Western blotting, and collecting the polypeptide. These and other methods for identifying and purifying recombinant polypeptides are known to those of ordinary skill in the art. However, in one or more embodiments, the IL-15 portion is not in the form of a fusion protein.

[0156] Depending on the system used to express the protein having IL-15 activity, the IL-15 portion can be non-glycosylated or glycosylated and either can be used. That is, the IL-15 portion can be non-glycosylated or the IL-15 portion can be glycosylated. In one or more embodiments, the IL-15 portion is non-glycosylated.

[0157] The IL-15 portion can be advantageously modified to include and / or replace one or more amino acid residues, such as, for example, lysine, cysteine, and / or arginine, to facilitate attachment of the polymer to atoms within the amino acid side chains. Examples of substitutions of the IL-15 portion are described in U.S. Patent No. 6,177,079. In addition, the IL-15 portion can be modified to include non-naturally occurring amino acid residues. Techniques for adding amino acid residues and non-naturally occurring amino acid residues are well known to those of ordinary 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 (Michael R. Barnes and Ian C Gray, eds.), 2003 John Wiley & Sons, Ltd, Chapter 14, Amino Acid Properties and Consequences of Substitutions, Betts, MJ, and Russell, RB.

[0158] In addition, the IL-15 portion can be advantageously modified to include attachment to a functional group (other than by adding an amino acid residue comprising a functional group). For example, the IL-15 portion can be modified to include a thiol group. In addition, the IL-15 portion can be modified to include an N-terminal alpha carbon. In addition, the IL-15 portion can be modified to include one or more carbohydrate moieties. In addition, the IL-15 portion can be modified to include an aldehyde group. In addition, the IL-15 portion can be modified to include a ketone group. In some embodiments of the present invention, it is preferred that the IL-15 portion is not modified to include one or more of the following: a thiol group, an N-terminal alpha carbon, a sugar, an aldehyde group, and a ketone group.

[0159] [Journal of Biological Chemistry] 272 (4):2312-2318, and Wong et al. (2013) OncoImmunology 2(11), e26442: 1-3. Preferred IL-15 portions include those having an amino acid sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 3, and sequences substantially homologous thereto (wherein even if SEQ ID NOs 2 and 3, and sequences substantially homologous thereto, do not meet the in vitro activity criteria for IL-15 portions provided herein, it should be understood that for the purposes of the present invention, these sequences are also understood to be "IL-15 portions"). Preferred IL-15 portions have 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.

[0160] In some cases, the IL-15 portion will be in a "monomeric" form, in which a single expression of the corresponding peptide is organized into a discrete unit. In other cases, the IL-15 portion will be in a "dimeric" form (e.g., a dimer of recombinant IL-15), in which two monomeric forms of the protein are associated with each other.

[0161] Additionally, a precursor form of IL-15 can be used as the IL- 15 portion. An exemplary precursor form of IL-15 has the sequence SEQ ID NO:3.

[0162] Truncated forms, hybrid variants, and peptide mimetics 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 sequences that maintain at least some degree of IL-15 activity can also serve as IL-15 moieties.

[0163] For any given peptide, protein portion or conjugate, it is possible to determine whether the peptide, protein portion or conjugate has IL-15 activity. Different methods for determining in vitro IL-15 activity are described in the art. An exemplary method is based on pSTAT assay. In brief, if IL-15 dependent CTLL-2 cells are exposed to a test article with IL-15 activity, the start of the signal cascade results including STAT5 phosphorylation at tyrosine residue 694 (Tyr694) can be quantitatively measured. Assay protocols and kits are known and include, for example, MSD phospho (Tyr694) / total STATa, b whole cell lysate kit (Meso Scal Diagnostics, LLC, Gaithersburg, MD, USA). For example, using this method, a pSTAT5 EC of no more than about 300 ng / mL (more preferably no more than about 150 ng / mL) is exhibited for at least 5 minutes or at 10 minutes. 50 The proposed IL-15 portion with a value of is considered an "IL-15 portion" in connection with the present disclosure. Preferably, however, the IL-15 portion used is more potent (e.g., having a pSTAT5 EC of less than 150 ng / mL for at least one of 5 minutes or 10 minutes). 50 values, such as less than about 1 ng / mL, and even more preferably less than 0.5 ng / mL for at least 5 minutes or at 10 minutes).

[0164] Other methods known in the art can also be used to assess IL-15 function, including electrometric, spectrophotometric, chromatographic, and radiometric methods. For another type of such assay, see, for example, Ring et al. (2012) Nat. Immunol. [Natural Immunology] 13(12): 1187-1195.

[0165] Assays used in conjunction with measuring the activity of IL-15 moieties can also be used to measure the activity of the long-acting IL-15R agonists described herein. See, e.g., the supporting examples provided herein.

[0166] As long as the compound exhibits IL-15 agonism in vivo after being administered to a subject, the amount of time that lasts is longer than the situation of administering IL-15, the agonist is considered to be a long-acting IL-15R agonist according to the present disclosure. Conventional methods, such as those involving radiolabeled compounds, administering the compound in vivo, and determining its clearance rate, can be used to assess whether a compound proposed as a long-acting IL-15R agonist is "long-acting" (i.e., whether the clearance rate is longer than that of IL-15 administered in the same in vivo system). For the purposes of this article, the long-acting properties of long-acting IL-15R agonists can be, and typically are, determined using flow cytometry to measure STAT5 phosphorylation in lymphocytes at different time points after administering the agonist to be evaluated in mice. For reference, the signal is lost for about 24 hours in the case of IL-15, but the duration is greater than the time period of the long-acting IL-15 agonist.

[0167] As previously discussed, preferred long-acting IL-15R agonists will typically comprise a single linear PEG (polyethylene glycol) moiety stably covalently linked to an IL-15 amino group via an amide linkage. Interposed between the PEG moiety and the stable amide linkage to the IL-15 amino group is a linear unsubstituted alkylene group (~CH2~) having from 2 to 5 carbon atoms. m (ie, where m = 2, 3, 4, or 5).

[0168] For example, in some embodiments, the unsubstituted alkylene group is (~CH2~)2; or, in some other embodiments, the unsubstituted alkylene group is (~CH2~)3; in still other embodiments, the unsubstituted alkylene group is (~CH2~)4; in still other embodiments, the unsubstituted alkylene group is the unsubstituted alkylene group is (~CH2~)5.

[0169] For example, in some embodiments, the long-acting IL-15 receptor agonist has the following structure:

[0170]

[0171] wherein IL-15 is the interleukin-15 moiety, n is an integer from about 150 to about 3,000; m is an integer from 2-5 (e.g., 2, 3, 4, or 5) and n' is 1. In Formula I (and in similar formulas provided herein), the ~NH~ in the structure represents the amino group of the IL-15 moiety. Formula (I) can also be depicted as follows, wherein the brackets are moved to reflect the terminal PEG methoxy group, And the two formulas can be used interchangeably. Illustrative exemplary compounds include the following compounds encompassed by formula (I):

[0172]

[0173] as well as

[0174]

[0175] 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).

[0176] In some other embodiments, with respect 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 still other embodiments, n is an integer from about 400 to about 1300. In still other embodiments, n is an integer from about 450 to about 1200.

[0177] PEG having a molecular weight corresponding to any of the aforementioned ranges for n values ​​is generally preferred.

[0178] In one or more 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 (wherein n is about 227), or about 15,000 Daltons (wherein n is about 340), or about 20,000 Daltons (wherein n is about 454), or about 25,000 Daltons (wherein n is about 568), or about 30,000 Daltons (wherein n is about 681), or about 40,000 Daltons (wherein n is about 909), or about 50,000 Daltons (wherein n is about 1136), or even about 60,000 Daltons (wherein n is about 1364).

[0179] In addition to the foregoing weight average molecular weights, other exemplary weight average molecular weights for the polyethylene glycol portion of the compound include 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 45,000 Daltons, about 55,000 Daltons, about 65,000 Daltons, about 70,000 Daltons, and about 75,000 Daltons.

[0180] In some preferred embodiments, the polyethylene glycol polymer portion of the compound has a weight average molecular weight of about 40,000 Daltons.

[0181] While the PEG moiety is preferably terminated with a methoxy group as shown above in formula (I), the PEG moiety may be terminated at its terminus with any lower C 1-6 The alkoxy groups may terminate in hydroxyl groups or other suitable terminal capping groups.

[0182] In some embodiments, the long-acting IL-15 receptor agonist composition comprises no more than about 20 mole % (mol %) of a long-acting IL-15 receptor agonist encompassed by the following formula (IL-15-containing molecules in the composition), when considered together:

[0183]

[0184] wherein the values ​​of n and m are as provided above for Formula (I).That is, with respect to the long-acting IL-15 receptor agonist component of such compositions, the composition comprises no more than about 20 mole % of the long-acting IL-15 receptor agonist having Formula (II).

[0185] In some further embodiments, the long-acting IL-15 receptor agonist composition comprises no more than about 15 mole % (mol %) of a long-acting IL-15 receptor agonist encompassed by the following formula (IL-15-containing molecules in the composition), when considered together:

[0186]

[0187] Wherein the values ​​of n and m are as provided above for formula (I). That is, with respect to the long-acting IL-15 receptor agonist component of such compositions, the composition contains no more than about 15 mol% of a long-acting IL-15 receptor agonist having formula (II). In some embodiments, the long-acting IL-15 receptor agonist composition comprises no more than about 0.1 mol%-20 mol% of a compound having formula (II). In embodiments, these compositions comprise no more than about 0.1 mol%-15 mol%, 0.1 mol%-10 mol%, 0.1 mol%-5 mol%, 0.1 mol%-1 mol%, 1 mol%-20 mol%, 1 mol%-15 mol%, 1 mol%-10 mol%, 1 mol%-5 mol%, 5 mol%-20 mol%, 5 mol%-15 mol%, 5 mol%-10 mol%, 10 mol%-20 mol%, 10 mol%-15 mol%, or 15 mol%-20 mol% of a compound having formula (II).

[0188] In certain specific embodiments related to the foregoing, with respect to Formula (Ia), the long-acting IL-15 receptor agonist composition comprises, when considered together, no more than about 15 mole % (mol %) of a long-acting IL-15 receptor agonist encompassed by the following formula (IL-15-containing molecules in the composition):

[0189]

[0190] wherein the values ​​of n and m are as provided above for Formula (Ia).

[0191] In some other preferred embodiments, with respect to Formula (Ib), the long-acting IL-15 receptor agonist composition comprises no more than about 15 mole % (mol %) of a long-acting IL-15 receptor agonist encompassed by the following formula (IL-15-containing molecules in the composition), when considered together:

[0192]

[0193] wherein the values ​​of n and m are as provided above for Formula (Ib).

[0194] In some other embodiments, with respect to Formula (Ic), the long-acting IL-15 receptor agonist composition comprises no more than about 15 mole % (mol %) of a long-acting IL-15 receptor agonist encompassed by the following formula (IL-15-containing molecules in the composition), when considered together:

[0195]

[0196] wherein the values ​​of n and m are as provided above for Formula (Ic).

[0197] In some other embodiments, with respect to Formula (Id), the long-acting IL-15 receptor agonist composition comprises no more than about 15 mole % (mol %) of a long-acting IL-15 receptor agonist encompassed by the following formula (IL-15-containing molecules in the composition), when considered together:

[0198]

[0199] wherein the values ​​of n and m are as provided above for Formula (Id).

[0200] In some embodiments, the long-acting IL-15 receptor agonist comprises no more than about 0.1 mol%-20 mol% of a compound of formula (II), including compounds of formula (IIa), (IIb), (IIc), and (IId). In some further embodiments, the compositions comprise no more than about 0.1 mol%-15 mol%, 0.1 mol%-10 mol%, 0.1 mol%-5 mol%, 0.1 mol%-1 mol%, 1 mol%-20 mol%, 1 mol%-15 mol%, 1 mol%-10 mol%, 1 mol%-5 mol%, 5 mol%-20 mol%, 5 mol%-15 mol%, 5 mol%-10 mol%, 10 mol%-20 mol%, 10 mol%-15 mol%, or 15 mol%-20 mol% of a compound of formula (II), including compounds of formula (IIa), (IIb), (IIc), and (IId). In some embodiments, the compositions comprise no more than about 0.1 mol%, 1 mol%, 5 mol%, 10 mol%, 15 mol%, or 20 mol% of a compound of formula (II), including compounds of formula (IIa), (IIb), (IIc), and (IId). It will be appreciated that the compositions may be purified by methods known in the art for compounds of formula (I) such that no, trace amounts, or substantially no compound of formula (II) is present in the composition.

[0201] For example, in some embodiments, the long-acting IL-15 receptor agonist composition comprises no more than about 12 mol%, or no more than about 10 mol%, of a long-acting IL-15 receptor agonist encompassed by formula (II), including compounds having formula (IIa), (IIb), (IIc), and (IId), when considered together.

[0202] In some additional embodiments of the foregoing, the composition comprises no more than about 7 mol% of a long-acting IL-15 receptor agonist having n' equal to 2, 3, or greater than 3 (i.e., a higher order PEG polymer). In still other embodiments, the composition comprises no more than about 5 mol% of a long-acting IL-15 receptor agonist having n' equal to 2, 3, or greater than 3 (i.e., 2 or greater).

[0203] In some other embodiments, the composition comprises a long-acting IL-15 receptor agonist according to formula (I),

[0204] wherein n and m are as described above, and n' represents the average number of polyethylene glycol moieties covalently attached to the amino groups of IL-15 (for the composition), and n' for the composition ranges from 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 "monoPEGylated," with it being understood that there is some variability with respect to the degree of PEGylation as described above. In some preferred embodiments of the formulae described herein, "m" is equal to 3.

[0205] Compositions of long-acting IL-15R agonists can comprise a single species, wherein n' is equal to about 1 and the PEG moieties of substantially all IL-15 conjugates in the composition are attached at the same position; or alternatively, can comprise a mixture of monoPEGylated conjugate species, wherein attachment of the linear polyethylene glycol moiety occurs at different sites on the interleukin-15 moiety (i.e., wherein the specific attachment site is not the same for all monoPEGylated IL-15 species contained in the composition). Thus, such compositions are substantially homogeneous with respect to the number of PEG moieties attached to the IL-15 (e.g., a monomer), but are heterogeneous with respect to the position on the IL-15 molecule to which the amino group is attached.

[0206] While alternative PEG architectures and linkage chemistries can be used to achieve long-acting IL-15R agonists, compounds such as those described above are preferred in one or more embodiments, as will become apparent upon consideration of the supporting examples. However, additional long-acting IL-15R agonists having structures as provided herein are also contemplated.

[0207] 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 encompassed by Formula (I) (including Formulas (Ia-Id)) (IL-15-containing molecules in the composition). 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 having Formula (I).

[0208] As described above, the long-acting IL-15R agonist can be in the form of a pharmaceutically acceptable salt. Typically, such salts are formed by reacting with a pharmaceutically acceptable acid or acid equivalent. The term "pharmaceutically acceptable salt" in this regard will generally refer to relatively non-toxic inorganic and organic acid addition salts. These salts can be prepared in situ during the administration of a vehicle or dosage form manufacturing process, or by reacting a long-acting interleukin-15 receptor agonist as described herein with a suitable organic or inorganic acid and separating the salts thus formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, oxalate, methanesulfonate, glucoheptonate, lactobionate, and laurylsulfonate, etc. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts," J. Pharm. Sci. 66: 1-19.) Thus, salts as described can be derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like, or prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, itaconic acid, and the like.

[0209] In some embodiments, the long-acting IL-15 receptor agonist composition comprises at least about 1 mol%-5 mol% of free IL-15 protein (the IL-15-containing molecules in the composition), when considered together. In some other embodiments, the long-acting IL-15 agonist composition comprises no more than about 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, or 5 mol% of free (i.e., unconjugated) IL-15.

[0210] 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) with a PEG reagent functionalized with a succinimidyl group (or other activated ester group). Using this method, the succinimidyl-activated PEG can be attached to the amino group on the IL-15 moiety in aqueous medium at a pH of about 7.0 to 9.0, although using different reaction conditions (e.g., a lower pH such as 6 to 7 or 7 to 8, or a different temperature and / or less than 15° C.) can result in the PEG moiety being attached to a different position on the IL-15 moiety.

[0211] Long-acting IL-15R agonists can be prepared as described in Example 1. For example, long-acting IL-15R agonists can generally be prepared by reacting interleukin-15 (e.g., purified IL-15, such as recombinant IL-15) with an activated PEG reagent (e.g., activated ester, methoxy PEG-succinimidyl butyrate, mPEG-SBA). Other suitable activated PEG reagents include methoxy PEG-succinimidyl propionate, methoxy PEG-succinimidyl pentanoate, and methoxy PEG-succinimidyl hexanoate. Although a succinimidyl activating group is typically used, any suitable active ester or activating group can be used, wherein such a reactive group is suitable for forming a desired stable amide linkage. Generally, interleukin-15 is dissolved in a suitable buffer, such as, for example, phosphate buffered saline (PBS). The PEG reagent can be added typically in a solution in a suitable buffer, in a molar ratio equal to IL-15 (relative to the molar amount of interleukin-15), or in an amount of up to about 15 times molar excess, for example 2 times molar excess, or 5 times molar excess, or 7 times molar excess, or 10 times molar excess, or even 12 times molar excess or more molar excess (based on the molar amount of IL-15). In certain embodiments, the PEG reagent is added in an amount of about 5 to 10 times molar excess. The PEG reagent can be added in solid form or as a solution in a suitable solvent (e.g., an aqueous acid solution, such as dilute hydrochloric acid).

[0212] In some other embodiments of the methods, interleukin-15 is initially (i.e., prior to mixing with the methoxy PEG-succinimidyl alkanoate reagent) present in the solution at a concentration of about 0.5 mg / mL to about 10 mg / mL. Additional illustrative concentration ranges in solution include, for example, about 0.5-5 mg / mL, about 0.5-4 mg / mL, about 0.5-3 mg / mL, about 0.5-2 mg / mL, about 0.5-1.5 mg / mL, about 0.5-1 mg / mL, about 1-10 mg / mL, about 1-5 mg / mL, about 1-4 mg / mL, about 1-3 mg / mL, about 1-2 mg / mL, about 1-1.5 mg / mL, about 1.5-10 ... g / mL, about 1.5-5 mg / mL, about 1.5-4 mg / mL, about 1.5-3 mg / mL, about 1.5-2 mg / mL, about 2-10 mg / mL, about 2-5 mg / mL, about 2-4 mg / mL, about 2-3 mg / mL, about 3-10 mg / mL, about 3-5 mg / mL, about 3-4 mg / mL, about 4-10 mg / mL, about 4-5 mg / mL, or about 5-10 mg / mL interleukin- 15. In some specific but non-limiting embodiments, the concentration of interleukin-15 in the solution is about 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, or 10 mg / mL.

[0213] It should be understood that any suitable buffer can be used or added to the reaction mixture. Some exemplary buffers include sodium phosphate (NaPi), sodium acetate (NaAc), borate, N-bicine (bicine), citrate, and bis-TRIS buffer.

[0214] In some embodiments, the pH of the IL-15 solution is adjusted to about pH 8 prior to the addition of the PEG reagent.

[0215] After adding the PEG reagent, the reaction mixture can then be adjusted to a suitable pH, such as from about 7.0-8.5 or to about 8.0, if necessary. In certain embodiments, the reaction mixture is adjusted to a pH of about 7.0-8.0 or to about 7.4-8.5. In certain specific embodiments, the reaction mixture is adjusted to a pH of about 8.0. It should be understood that the pH can be adjusted as needed before and after adding the PEG reagent to achieve a desired pH.

[0216] Interleukin-15, like many proteins, undergoes deamidation, especially at higher pH, while lower pH levels can lead to a number of potential disadvantages, such as, for example, a lower degree of conjugation at the ipsilon (ε) amine, and / or increased and / or undesirable positional isomers, and protein aggregation. Deamidation introduces negative charges into proteins, which can lead to changes in the activity, structure, function, stability of the protein and / or can alter the protein's susceptibility to degradation. Thus, one of the challenges addressed by the present agonists and related methods is to provide long-acting interleukin-15 receptor agonists that maintain sufficient activity (i.e., to be therapeutically useful) while balancing, among other considerations, at least (i) a desired degree of conjugation, (ii) a low amount of deamidation of the interleukin-15 moiety before and after conjugation with the subject PEG reagents (which deamidation can, for example, lead to reduced interleukin-15 activity), and (iii) protein aggregation (e.g., before and after conjugation).

[0217] Based on the competing and conflicting challenges associated with the reaction parameters used to prepare the long-acting interleukin-15 receptor agonists as described herein, the applicants have discovered that by adjusting the pH of the interleukin-15 solution, and / or the IL-15-PEG reagent reaction mixture (before or after reaction with the PEG reagent), optimal (lower levels) of deamidation can be achieved while also promoting conjugation of the PEG moiety to the interleukin-15 moiety (e.g., at the epsilon amine and N-terminus) to provide the long-acting IL-15R agonists as described herein. While not being bound by theory, based on a series of reactions in which various reaction parameters were varied, it appears that a pH range of from about 7.0 to about 8.5, or from about 7.5 to about 8.2, or from about 7.8 to about 8.2, or at about 8.0 effectively provides lower levels of deamidation in the product while also promoting conjugation of the PEG moiety to form the products as described herein, which also maintain the desired therapeutic profile.

[0218] For example, the methods described herein are effective to produce PEGylated interleukin-15 that is less than about 35% deamidated, or in some embodiments, less than about 30% deamidated, or less than about 25% deamidated, or less than about 20% deamidated. In some embodiments, the deamidation level of the product is in the range of from about 20%-35%, or in the range of about 20%-25%, or in the range of about 25%-35%, or in the range of about 25%-30%. Alternatively, in some embodiments, it is contemplated that the degree of deamidation is less than that of PEGylated interleukin-15 stated above. As shown in Experiment 2 of Example 1, adjusting the pH in the range of about 7.0-8.5 resulted in a deamidation level of 21.29% (Composition 1) or 33.26% (Composition 2).

[0219] Typically reactant is mixed up to and including about 5 to 10 hours. In certain embodiments, reactant is mixed up to and including about 2 to 5 hours. In certain embodiments, reactant is mixed up to and including about 2 hours. In some exemplary embodiments, reactant is mixed for about 30 minutes to about 3.0 hours, or from about 30 minutes to 2.5 hours, or from about 30 minutes to 2 hours, or from about 30 minutes to 1.5 hours, or from about 45 minutes to about 3.0 hours, or from about 45 minutes to about 2.5 hours, or from about 45 minutes to about 2.0 hours, or from about 45 minutes to about 1.5 hours, or from about 45 minutes to about 1.0 hour. Mixing is typically carried out under mild conditions (for example, from about 20 ℃ to about 65 ℃, or from about 20 ℃ to about 40 ℃), or at ambient temperature or room temperature (for example, about 22 ℃). Lower temperatures can be used to facilitate lower PEGylation degrees. Can, for example, be quenched by adding amino acids (such as glycine).

[0220] In an embodiment, the pH of the composition can be further adjusted to mitigate deamidation. In some embodiments, the composition is adjusted to a pH of about 6.5-7.5 or about 6.5-7.0. In some embodiments, the composition is adjusted to a pH of about 6.5, 6.8, 7.0, or 7.5.

[0221] The PEGylated rIL-15 reaction product can then be purified generally by any suitable method (e.g., ion exchange chromatography) to obtain the desired product. For example, anion exchange chromatography can be employed. The chromatography product pool can then be concentrated and diafiltered into a suitable formulation buffer (e.g., sodium acetate buffer containing sucrose) using, for example, tangential flow filtration (TFF). Analysis can be performed by any suitable method, such as, for example, SDS-PAGE, reversed-phase HPLC, or any other suitable analytical method.

[0222] 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-15R agonist such as that encompassed by Formula (I). For example, considering the exemplary IL-15 amino acid sequence provided herein, it is apparent that there are seven lysine residues, each with an ε-amino acid available for conjugation. In addition, the N-terminal amine of methionine can also serve as a point of attachment to the PEG moiety. It should be understood that the polyethylene glycol moiety can 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 located at Lys 10 and Lys 11 (Using the numbering as shown in, for example, SEQ ID NO: 2 or using the Lys 11 and Lys 12) in one or more of the following. In some embodiments, the polyethylene glycol moiety is attached at the N-terminal amine. It should be understood that any of the lysine sites may be suitable as an attachment site for the PEG moiety (e.g., Lys 37 or Lys 42 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 primarily located at the N-terminus (that is, within the collection of positional isomers, the isomer having the PEG moiety attached at the N-terminus is present in the highest amount when compared to the other positional isomers).

[0223] If desired, the product pool can 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 ion exchange chromatography using an ion exchange column, e.g., Sepharose 5000 available from Amersham Biosciences. TM Ion Exchange Column. Any method can be used to separate PEG-IL-15 positional isomers (ie, positional isoforms) having the same molecular weight.

[0224] Gel filtration columns suitable for performing this type of separation include Superdex ® available from GE Healthcare (Buckinghamshire, UK). TM and Sephadex TM Column. The choice of a particular column will depend on the desired range of fractionation. Elution is typically performed using a suitable buffer such as phosphate, acetate, etc. The collected fractions can be analyzed by a variety of different methods, such as (i) absorbance at 280 nm for protein content, (ii) dye-based protein analysis using bovine serum albumin (BSA) as a standard, (iii) iodine test for PEG content (Sims et al. (1980) Anal. Biochem. 107 :60-63), (iv) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) followed by staining with barium iodide, and (v) high performance liquid chromatography (HPLC).

[0225] It has been found that the long-acting IL-15R agonists of the present invention have certain significant and advantageous features. While it is believed that the features described below are generally applicable to compounds as provided herein and encompassed by formula (I), one or more of the following features may be particularly exhibited by compounds according to formula (Ib) and, by extension, according to formula (IIb). Long-acting IL-15R agonists may have one or more of the following features. For example, in some embodiments, the long-acting IL-15 receptor agonist exhibits an EC50 value (ng / mL, CTLL-2pSTAT5) of no more than about 7-fold reduction 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 EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 6-fold decrease in EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 5.5-fold decrease in EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 5-fold decrease in EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 4.5-fold decrease in EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 4-fold decrease in EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 3.5-fold decrease in EC50 value (ng / mL, CTLL-2 pSTAT5), or even no more than about a 3-fold decrease in EC50 value (ng / mL, CTLL-2 pSTAT5) when compared to IL-15. Exemplary long-acting IL-15R agonists according to the foregoing characteristics are described herein and in the accompanying Examples.

[0226] As described in Example 10, the in vitro activities of the illustrative conjugates (1, 3, and 5) all induced IL-15 signaling in human PBMCs, with Conjugate 1 being the most potent in inducing this signaling. Further experiments were performed to investigate the in vitro activity of Conjugate 1 on human CD8 T cells, NK cells, and CD4 T cells (Examples 16, 22, and 26-27). Figures 10A-10B As shown in , at least conjugate 1 induced similar or increased signaling compared to IL-15 in CD56bright and CD56light cells. Although conjugate 1 was less potent than IL-15 in engaging CD8 and CD56bright NK cells (Example 22), it is important to note that conjugate 1 achieved the same maximal response as regular IL-15 (see Figures 38A-38BAs described in Example 16 for the mouse model, a single injection of Conjugate 1 at two different doses induced sustained pSTAT signaling in CD8 and NK cells. As described in the mouse model of Example 26, a single injection of Conjugate 1 resulted in a 5% increase in pSTAT compared to IL-15. In the mouse model, NK cells were most sensitive to a single dose of the conjugate, followed by CD8 T cells, with CD4 T cells being the least sensitive of the cells tested.

[0227] Conjugate 1 also induced signaling in NK cells, CD8 T cells, and CD4 T cells in a non-human primate model (the cynomolgus monkey model in Example 27). As in the murine model, NK cells were most sensitive to induction by Conjugate 1.

[0228] In some further embodiments, the long-acting IL-15 receptor agonist exhibits receptor α binding (K D That is, in some related embodiments, the long-acting IL-15 receptor agonist exhibits receptor α binding (K D , pM), or exhibit no more than about 45% reduction in receptor α binding (K D , pM), or exhibit no more than about 40% reduction in receptor α binding (K D , pM), or even exhibit no more than about 35% reduction in receptor α binding (K D , pM) is not more than about 30% reduction.

[0229] Preferably, the long-acting IL-15 receptor agonist exhibits no more than about a 7-fold decrease in EC50 value (ng / mL, CTLL-2pSTAT5) compared to unmodified IL-15 and exhibits receptor α binding (K D , pM) by no more than about 50%, including the above EC50 values ​​or K D Any specific combination of one or more values ​​that decreases.

[0230] Optionally, the long-acting IL-15 receptor agonist is included in a composition comprising one or more pharmaceutically acceptable excipients. Exemplary excipients include, but are not limited to, those selected from the group consisting of carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, amino acids, and combinations thereof.

[0231] Carbohydrates, such as sugars, derivatized sugars (such as sugar alcohols, aldonic acids, esterified sugars and / or sugar polymers), can 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 sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, inositol, cyclodextrin, etc.

[0232] Excipients may also include inorganic salts or buffers, such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium dihydrogen phosphate, sodium hydrogen phosphate, and combinations thereof.

[0233] The composition may also include an antimicrobial agent for preventing or curbing the growth of microorganisms. Non-limiting examples of antimicrobial agents suitable for one or more embodiments of the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimerosal, and combinations thereof.

[0234] Antioxidants can also be present in the composition. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of other components of the conjugate or formulation. Antioxidants suitable for use in one or more embodiments of the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[0235] 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 esters; steroids such as cholesterol; and IL-15 chelators such as EDTA, zinc, and other such suitable cations.

[0236] Acid or alkali can be present in the composition as excipient.The limiting examples of operable acid include those 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.The example of applicable alkali includes but is not limited to the alkali 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.

[0237] One or more amino acids can be present in the compositions described herein as excipients. In this regard, exemplary amino acids include arginine, lysine, and glycine. Additional suitable pharmaceutically acceptable excipients include, for example, those described in Handbook of Pharmaceutical Excipients, 7th edition, Rowe, RC, ed., Pharmaceutical Press, 2012.

[0238] The amount of long-acting IL-15R agonist included in the composition will vary depending on a number of factors, but it will optimally be a therapeutically effective dose when the composition is stored in a unit dose container (e.g., a vial). Alternatively, the pharmaceutical formulation 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-15R agonist to determine the amount that produces the clinically desired endpoint as 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 by routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining stability and other parameters, and then determining the range that yields optimal performance without significant adverse effects.

[0239] Long-acting IL-15R agonists are suitable for administration to patients suffering from a condition that responds to treatment with interleukin-15. The method comprises administering a therapeutically effective amount of a long-acting IL-15R agonist to the patient, preferably provided as part of a pharmaceutical composition, typically parenterally. As previously described, long-acting IL-15R agonists can be administered parenterally (e.g., intramuscularly, subcutaneously, intravenously, or intraperitoneally). Suitable formulation types for parenteral administration include injection-ready solutions, dry powders that are combined with solvents before use, injection-ready suspensions, dry insoluble compositions that are combined with vehicles before use, and emulsions and liquid concentrates that are diluted before administration, etc. In some specific embodiments, the long-acting IL-15 receptor agonist is provided in a formulation suitable for intravenous administration and is administered intravenously. In some other embodiments, the long-acting IL-15 receptor agonist is provided in a formulation suitable for subcutaneous administration and is administered subcutaneously.

[0240] Methods of administering a long-acting IL-15 receptor agonist (e.g., provided as part of a pharmaceutical composition) can optionally be performed to localize the agonist to a specific area. For example, liquid, gel, and solid formulations containing the agonist can also be surgically implanted into a diseased area (such as within a tumor, near a tumor, in an inflamed area, or near an inflamed area). Conveniently, organs and tissues can also be imaged to ensure optimal exposure to the conjugate at the desired location.

[0241] The method of administration can be used to treat any condition that can be remedied or prevented by administering a long-acting IL-15R agonist, such as, for example, cancer. For example, a long-acting agonist can be used alone or in combination with another drug therapy to treat a patient suffering from a condition that is responsive to IL-15 therapy, such as cancer.

[0242] As used herein with respect to treating a patient suffering from cancer, the terms "treatment," "treat," and "treating" are intended to encompass the full spectrum of interventions with the cancer afflicting the subject, such as administering a combination to alleviate, slow, terminate, or reverse one or more symptoms of the cancer or to delay the progression of the cancer, if not actually eliminate it. Treatment can include, for example, reducing the severity of symptoms, the number of symptoms, or the frequency of recurrences, e.g., inhibiting tumor growth, halting tumor growth, or causing regression of an existing tumor.

[0243] " complete response " refers to the absence of clinically detectable disease, wherein any previously abnormal X-ray radiographic study, bone marrow and cerebrospinal fluid (CSF) or abnormal monoclonal protein measurements are normalized. " partial response " refers to the absence of new lesions, and all measurable tumor loads (that is, the number of malignant cells present in the subject or the volume of the tumor mass measured or the quantity of abnormal monoclonal proteins) are reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. The term "treatment" encompasses both complete and partial responses.

[0244] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.

[0245] As used herein, "tumor" and "solid tumor" refer to all lesions and neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

[0246] Exemplary conditions are cancers such as, for example, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, tumor), leiomyosarcoma, rhabdomyosarcoma, colon cancer, 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, liver cancer, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder 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, and leukemia.

[0247] In one specific method, a long-acting IL-15R agonist is used to treat a hematological malignancy, such as a leukemia or lymphoma. In yet another method, a long-acting IL-15R agonist is used to treat a solid cancer.

[0248] In some embodiments, the long-acting IL-15R agonist or composition is effective in stimulating NK activation and / or proliferation when administered to a subject at a therapeutically effective dose.

[0249] In the exemplary mouse model described in Example 11, Conjugate 1 effectively induced the proliferation and sustained increase in the number of NK cells, as evidenced by an increase in cell number (cells / μ, e.g. Figures 12B-12C ) and an increase in Ki67% (e.g. Figure 11A ) as shown. Ki67% was used as a marker for proliferating cells. Figures 12A-12D As seen in the NK cells of all maturity levels (terminal effector cells, precursor NK cells, high effector cells and early NK cells), an increase in cell number was found. As shown in Example 17, the increase in NK cell number continued for at least 96 hours at all dose levels, with the medium and high dose levels continuing for at least 144 hours. Compared to the vehicle, administration of conjugate 1 in the mouse model induced an increase in Ki67% that lasted for at least 120 hours at all dose levels. % Ki67 at least the medium dose range (e.g., 0.1 mg / kg and 0.3 mg / kg) induced an increase in Ki67% that lasted for at least 144 hours.

[0250] The effects of Conjugate 1 can be induced and sustained with a single dose. In the exemplary murine model described in Example 11, administration of a single dose of Conjugate 1 induced and sustained an increase in cell number. A single dose induced and sustained Ki67% levels in murine CD49b cells comparable to repeated (e.g., Q7dx3) dosing at the same level (see Figure 28A ).

[0251] In some other embodiments, the long-acting IL-15R agonist or composition is effective in increasing NK cell activation when administered at a therapeutically effective dose, as demonstrated by increased expression of NK cell cytotoxic proteases. In an exemplary non-human primate model as described in Example 28, a single dose of Conjugate 1 induced and enhanced expression of the NK cell lytic enzymes granzyme B and perforin. Figures 48A-48C As seen in Figure 2, all dose levels increased granzyme B expression and the middle / higher doses increased expression (MFI) by at least three-fold compared to pre-dose levels. Figures 49A-49C As seen in Figure 3, all dose levels increased perforin expression and the middle / higher doses doubled expression (MFI) compared to pre-dose levels. Thus, Conjugate 1 effectively increased NK cell cytotoxicity.

[0252] In yet other embodiments, the long-acting IL-15R agonist or composition is effective in supporting CD8 T cell survival and memory formation when administered to a subject at a therapeutically effective dose.

[0253] In an exemplary mouse model, a single dose of Conjugate 1 at all dose levels increased cell proliferation as evidenced by an increase in the % Ki-67 positivity in CD8 cells ( Figure 30D ) and increases in central and effector memory CD8 subsets ( Figures 30E-30F As shown in Example 17, administration of Conjugate 1 induced a significant increase in total CD8 T cells in the blood in a murine model (see Figure 30A The lowest dose increased CD8 Tcm and CD8 Tem (see Figures 30B-30C In an exemplary murine model, a single intravenous injection of Conjugate 1 maintained increased cell numbers for at least 240 hours compared to vehicle administration (see, e.g., Figure 30A ). Notably, when conjugate 1 was administered at some dose levels, the number of CD8 and CD8 memory T cells did not return to baseline 240 hours after injection. Thus, conjugate 1 maintained the CD8+ memory T cell population for an extended period of time. At all dose levels, a single dose of conjugate 1 also increased Ki-67 positivity in all CD8 and CD8 subsets, indicating increased proliferation of these cells. Repeated administration of conjugate 1 resulted in further increases in CD8, CD8 Tcm, and CD8 Tem populations (see Figures 31A-31C Repeated dosing also resulted in a long-term increase in cell proliferation for at least 240 hours for each of CD8, CD8 Tcm, and CD8 Tem in mice.

[0254] Conjugate 1 also induced proliferation and sustained increases in the number of NK cells and CD8 T cells in a non-human primate model (the cynomolgus monkey model in Example 27) compared to vehicle administration. Each dose level of Conjugate 1 induced and sustained increases in the number of NK cells for at least 14 days (see Figure 44A At each dose level, Conjugate 1 induced and sustained increases in CD8 T cell numbers for at least 10 days.

[0255] In yet one or more other embodiments, the IL-15R agonist is administered intravenously. In even other embodiments, the IL-15R agonist is administered subcutaneously.

[0256] In yet other embodiments, upon administration, the IL-15R agonist is effective to induce sustained signaling in lymphocytes, resulting in proliferation of CD8 T cells and preferential expansion of the CD8 central memory population.

[0257] The actual dosage to be administered will vary according to the age, weight and general condition of the subject, as well as the severity of the condition being treated, the judgment of a health professional, and the conjugate being administered. A therapeutically effective amount is known to those of ordinary skill in the art and / or described in the relevant reference texts and literature. In general, a therapeutically effective amount will be in the range of from about 0.001 mg to 100 mg, preferably in a dosage from 0.01 mg / day to 75 mg / day and more preferably in a dosage from 0.10 mg / day to 50 mg / day. A given dose can be administered regularly until, for example, a clinician determines that an appropriate endpoint (e.g., cure, regression, partial regression, etc.) is achieved.

[0258] In some embodiments, the therapeutically effective dose is in the range of from about 0.25-25 mcg / kg. In other embodiments, the therapeutically effective dose is in the range of from about 0.25 mcg / kg to about 0.1 mg / kg per day, from about 0.01 mg / kg to about 0.1 mg / kg per day, or from about 0.03 mg / kg to about 0.1 mg / kg per day. In other embodiments, the therapeutically effective dose is in the range of from about 1-10 mcg / kg, from about 0.03 mg / kg to about 0.1 mg / kg. In some specific but non-limiting embodiments, the therapeutically effective dose is about 0.25mcg / kg, 0.3mcg / kg, 0.5mcg / kg, 1mcg / kg, 2mcg / kg, 3mcg / kg, 5mcg / kg, 6mcg / kg, 7mcg / kg, 10mcg / kg, 15mcg / kg, 20mcg / kg, 25mcg / kg, 0.01mg / kg, 0.03mg / kg, 0.05mg / kg, or 0.1mg / kg. With reference to the doses mentioned in the examples herein, one of ordinary skill in the art can convert animal (e.g., mouse) doses into corresponding doses in humans using conversion methods known in the art (e.g., Nair et al., J. Basic and Clin. Pharmacy [Basic and Clinical Pharmacy Journal] (2016) 7:27-31).

[0259] According to the judgment of the clinician, the needs of the patient, etc., any given conjugate of a unit dose can be administered in a variety of dosage regimens (again, preferably provided as part of a pharmaceutical formulation). Specific dosage regimens will be known to those of ordinary skill in the art or can be determined experimentally using conventional methods. Exemplary dosage regimens include, but are not limited to, daily administration, three times a week, twice a week, weekly administration, monthly administration twice (e.g., q / 14 days), monthly administration (e.g., q / 30 or 31 days or q / 21 days), and any combination thereof. Once the desired clinical endpoint has been achieved, the administration of the composition is suspended or reduced. In some embodiments, a single unit dose of any given conjugate can be administered to provide a sustained effect.

[0260] It should be understood that although the present invention has been described in conjunction with the preferred specific embodiments of the present invention, the foregoing description and the following examples are intended to illustrate rather than limit the scope of the present invention. Other aspects, advantages and modifications within the scope of this disclosure will be apparent to those skilled in the art to which the present invention belongs.

[0261] All articles, books, patents, and other publications cited herein are incorporated by reference in their entirety. In the event of a discrepancy between the teachings of this specification and the art incorporated by reference, the teachings and definitions in this specification control (particularly with respect to terms used in the claims appended hereto). For example, if this application and a publication incorporated by reference define the same term differently, the definition of that term will be retained within the teachings of the document in which the definition appears.

[0262] Examples

[0263] It should be understood that the foregoing description and the examples that follow are intended to illustrate rather than limit the scope of the invention provided herein. Other aspects, advantages, and modifications will be apparent to those skilled in the art to which this disclosure pertains.

[0264] In the following examples, efforts have been made to ensure accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, temperatures are in ° C. and pressures are at or near atmospheric pressure at sea level. Each of the following examples is considered to be instructive for one of ordinary skill in the art to perform one or more embodiments described herein.

[0265] Materials and Methods

[0266] In the following examples, recombinant IL-15 ("rIL-15") SEQ ID NO: 1 (e.g., Figure 1), but any suitable IL-15 portion may be similarly employed. SEQ ID NO: 1, a recombinant human IL-15 from E. coli, is a non-glycosylated single polypeptide chain containing 115 amino acids with a molecular weight of 12.9 kDa.

[0267] The reactive polymer reagent, linear mPEG-succinimidylbutyrate (40 kDa) ("mPEG-SBA") has the following structure,

[0268]

[0269] wherein n corresponds to the number of monomer subunits that provides a polymer having a weight average molecular weight of about 40 kilodaltons, i.e., wherein n is about 909. Additional mPEG-succinimidylbutyrate reagents suitable for use include those having a weight average molecular weight of, for example, about 10 kD, 15 kD, 20 kD, 25 kD, 30 kD, 40 kD, 50 kD, or 60 kD. This activated polymer reagent is effective in forming a stable amide linkage between the IL-15 portion and the polyethylene glycol portion upon reaction with an amino group (e.g., a lysine or the N-terminus) of IL-15.

[0270] The reactive fluorenyl-PEG reagent PEG2-CAC-FMOC-20kD-NHS has the following structure:

[0271]

[0272] wherein mPEG is methoxy(polyethylene glycol) and the weight average molecular weight of the polymer reagent is about 20 kilodaltons (i.e., wherein the weight average molecular weight of each mPEG moiety is about 10 kilodaltons). Additional PEG2-CAC-FMOC reagents having different molecular weights are correspondingly designated, for example, PEG2-CAC-FMOC-10kD-NHS, PEG2-CAC-FMOC-15kD-NHS, PEG2-CAC-FMOC-30kD-NHS, PEG2-CAC-FMOC-40kD-NHS, wherein such reagents have the structures shown above and differ only in the molecular weight of the "mPEG" attached to the FMOC core.

[0273] SDS-PAGE analysis

[0274] Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using an Invitrogen gel electrophoresis system (XCell SureLock Mini-Cell). The samples were mixed with sample buffer. The prepared samples were then loaded into NuPAGE Novex precast gels and run for approximately 30 minutes.

[0275] RP-HPLC analysis

[0276] Reverse phase chromatography (RP-HPLC) analysis was performed on an Agilent 1200 HPLC system (Agilent). Samples were analyzed at 60°C using a Poroshell 300SB-C3 column (2.1 x 75 mm, Agilent). The mobile phases used were 0.1% TFA / H2O (A) and 0.1% TFA / CH3CN (B). The column flow rate was 0.5 ml / min. The eluted protein and PEG-protein conjugate were detected using UV at 280 nm.

[0277] Bioassay

[0278] STAT5 phosphorylation-based potency assay in CTLL-2 cells (mouse T cells)

[0279] In the phospho-STAT5 assay, following receptor binding, downstream cell signaling can then activate signal transducer and activator of transcription 5 (STAT5) through phosphorylation to promote gene expression and thereby induce cell proliferation. Phospho-STAT5 activation was measured in CTLL-2 cells (murine T lymphocyte line) in response to sample and reference treatments for approximately 10 minutes using a phospho-STAT5 / total STAT5 multiplex assay (Meso Scale Discovery, Maryland).

[0280] The day before the assay, CTLL-2 cells were isolated into fresh growth medium [RPMI 1640 supplemented with 10% FBS, 10% T-cell culture supplement (Cat. No. 354115, Corning, Inc., Tewksbury, MA), 2 mM L-glutamate, and 1 mM sodium pyruvate]. On the day of the assay, cells were pre-incubated in assay medium (RPMI 1640 supplemented with 1% FBS, 2 mM L-glutamate, and 1 mM sodium pyruvate) for at least 4 hours and then seeded at 50,000 cells / well in assay medium in 96-well plates. Dilutions of the test article were prepared in the appropriate buffer just before the assay. Stimulation of CTLL-2 cells was initiated by transferring 25X test article solution to triplicate wells containing CTLL-2 cells. The plates were incubated at 37° C., 5% CO 2 for 10 minutes, and the reaction was stopped by cell lysis. Phospho-STAT5 and total STAT5 protein levels in cell lysates were measured using the MSD Phospho (Tyr694) / Total STATa,b Whole Cell Lysate Kit (Meso Scale Discovery, MD). Recombinant human IL-15 obtained from PeproTech demonstrated IL-15 activity by inducing STAT5 phosphorylation in CTLL-2 cells after a 10-minute treatment, with an average EC 50 was 0.27 ng / mL, which served as a control.

[0281] Human PBMC-pStat5 assay

[0282] The efficacy of IL-15 or long-acting IL-15R agonists on various human lymphocyte subsets was determined by a phospho-STAT5 (Y694) dose-response assay. Frozen human PBMCs from various donors were supplied by AllCells. 1 x 10 6 Cells / 100ul were cultured in complete RPMI medium for 2 hours and then incubated with IL-15 or conjugates at the indicated concentrations (10,000ng / ml-0.001ng / ml serial dilutions) at 37°C for 20 minutes. The cells were then fixed (using BD Cytofix) and permeabilized (using 100% pre-cooled methanol) and stained with antibodies against CD3, CD4, CD8, CD4-Treg (CD4+CD25+Foxp3+), CD56, and phosphorylated STAT5 (Y694) before analysis by flow cytometry. EC was calculated using a concentration-response relationship. 50 value.

[0283] Receptor affinity of long-acting rIL-15 receptor agonists for IL-15Rα

[0284] The affinity of IL-15 and exemplary long-acting rIL-15 receptor agonists is measured using surface plasmon resonance (" SPR ") using a BIAcore™ SPR system. Briefly, a 1:1 NHS:EDC mixture was used to activate the surface of a Biacore CM5 sensor chip to produce active NHS esters. Goat anti-human Fc antibodies were injected into 10 mM sodium acetate (pH 4) for five minutes to be covalently attached to the surface. Approximately 8000 RU of antibody were present to bind to the surface. Any remaining NHS esters were then quenched with ethanolamine.

[0285] At the beginning of each injection cycle, IL-15-Ra-Fc was captured on the sensor chip channel by a five-minute injection step in PBSP. Typically, 150-200 RU of receptor were bound to the surface.

[0286] The long-acting rIL-15 receptor agonist test article was diluted to 10 μM in PBS (containing 0.05% Tween 20 and 0.1 mg / ml BSA). A series of 3-fold dilutions were made and injected onto the sensor chip coated with IL-15Rα. The affinity was determined by measuring k a and k d The rate is measured, and k d With k a The ratio between them is used to calculate K d value.

[0287] Example 1

[0288] Preparation of long-acting IL-15 receptor agonist,

[0289]

[0290] Mono(methoxy PEG-N-butyramide) 40kD Interleukin-15

[0291] Experiment 1: 2.7 ml of IL-15 solution (1.23 mg / ml in PBS buffer, pH 7.4) was transferred to a small reaction bottle. 300 μl of 0.6 M borate buffer at pH 8 was added to adjust the pH to pH 8. mPEG-SBA (40 kDa) stored at -20 ° C. under argon was warmed to ambient temperature. A ten-fold excess (relative to the molar amount of IL-15) of mPEG-SBA-40K was dissolved in 2 mM HCl to form a 10% PEG reagent solution. The 10% PEG reagent solution was quickly added to the IL-15 solution and mixed thoroughly. After adding mPEG-SBA-40K, the pH of the reaction mixture was determined using conventional techniques and adjusted to pH 8. In order to couple mPEG-SBA-40K to IL-15 (i.e., via formation of a stable amide bond), the reaction solution was placed on a slow laboratory rotator at room temperature for 1.5 hours to facilitate conjugation. The reaction was quickly quenched by adding glycine solution.

[0292] Figure 2 The chromatogram following RP-HPLC analysis of the conjugation reaction mixture is shown. The reaction produced 40% monoconjugate (i.e., having a single PEG moiety attached to IL-15), 24% diconjugate (having two PEG moieties attached to IL-15), and 6% triconjugate (having three PEG moieties attached to IL-15) species. Approximately 30% unreacted IL-15 remained in the reaction mixture, but the reaction conditions were not optimized.

[0293] The desired monoconjugate was isolated by anion exchange chromatography using a Q Sepharose high performance column and sodium phosphate buffer as the elution phase. The purified mono-mPEG-SBA40K-IL-15 conjugate (also referred to herein as mono-mPEG-butyramide-40K-IL-15 or mono-(methoxy PEG-N-butyramide) 40kD Interleukin-15, or single mPEG 40K -C4-amide-IL-15) was characterized by HPLC and SDS-PAGE. For the remaining examples, the purified mono-mPEG-SBA40K-IL-15 was designated as Conjugate 1.

[0294] Figure 3 The FPLC purification profile of the anion exchange chromatography column is provided. Figure 4 Shown is an SDS gel of the purified mono-mPEG-SBA-40K-IL-15 conjugate. As indicated by the gel, the purified conjugate has a high level of purity and is free of unreacted IL-15. Figure 5RP-HPLC analysis of purified mono-mPEG-SBA-40K-IL-15 is shown. As can be seen from the HPLC results, the purified mono-mPEG-SBA-40K-IL-15 composition contains less than about 10% (molar amount) of di- or higher-order conjugates.

[0295] Using this same method, conjugates such as mono-mPEG-SBA-10K-IL-15, mono-mPEG-SBA-15K-IL-15, mono-mPEG-SBA-20K-IL-15, mono-mPEG-SBA-25K-IL-15, mono-mPEG-SBA-30K-IL-15, mono-mPEG-SBA-40K-IL-15, mono-mPEG-SBA-50K-IL-15, and mono-mPEG-SBA-60K-IL-15 were prepared using mPEG-SBA with different weight average molecular weights.

[0296] Experiment 2: A solution of approximately 2 mg / ml IL-15 in a buffer (50 mM sodium phosphate, 100 mM sodium chloride, 10% sucrose, pH 7.4) was transferred to each of two different reaction vessels (referred to herein as Composition 1 and Composition 2). To adjust the pH to 8.0, borate buffer (0.4 M or 0.6 M) at pH 8 was added. A tenfold excess (relative to the molar amount of IL-15) of mPEG-SBA-40K (mPEG-SBA, 40 kDa) diluted in 2 mM HCl was added to each IL-15 solution and mixed thoroughly. After the addition of mPEG-SBA-40K, the pH of the reaction mixture was set to pH 8 or adjusted as necessary by adding additional borate buffer. The final IL-15 concentration in the reaction was targeted at 1 g / L, with additional diluent used as necessary (buffer containing 50 mM sodium phosphate, 100 mM sodium chloride, 10% sucrose, pH 7.4 for Composition 1 and water for Composition 2). To couple mPEG-SBA-40K to IL-15 (i.e., via formation of a significantly stable amide linkage), the reaction solution was mixed at room temperature for 45 or 60 minutes for Composition 1 or Composition 2, respectively, to facilitate conjugation. The reaction was quenched by adding a 71-fold excess of glycine relative to the molar amount of PEG initially added to the reaction at pH 8.0 for 30 minutes. For Composition 1, the pH was adjusted by titration to pH 7.0 using 0.2 M phosphoric acid.

[0297] The resulting composition was characterized by reverse phase HPLC (RP-HPLC), SDS-PAGE, and ion exchange HPLC (IEX-HPLC).The results of the RP-HPLC analysis are provided in Table 1A below.

[0298] Table 1A.

[0299]

[0300] RRT is relative retention time.

[0301] The results of the SEC-HPLC analysis are provided in Table 1B below.

[0302] Table 1B.

[0303]

[0304] HMW = high molecular weight mPEG-IL-15 conjugate with 3 or more PEGs covalently linked to interleukin-15.

[0305] The results of the IEX-HPLC analysis are provided in Table 1B below.

[0306] Table 1C.

[0307]

[0308] The prepared composition contained primarily mPEG-SBA-40K monoPEGylated species with less than 10% PEG dimers (i.e., having 2 PEG moieties attached to IL-15) and even lower amounts of higher PEG species (i.e., having 3 or more PEG moieties attached to IL-15).

[0309] Additionally, these compositions have a relatively low degree of deamidation (identified as "acidic region" in Table 1C). Composition 1 is 21.29% deamidated and Composition 2 is 33.26% deamidated.

[0310] Example 2

[0311] Preparation of long-acting IL-15 receptor agonist,

[0312]

[0313] Under nitrogen purge, mPEG2-CAC-fmoc-20K-NHS stored at -80 ° C. under nitrogen was warmed to ambient temperature. A stock solution of mPEG2-CAC-fmoc-20K-NHS (200 mg / mL) was prepared in 2 mM HCl and added to rIL-15 at a molar ratio of mPEG2-CAC-fmoc-20K-NHS to rIL-15 ranging from 5:1 to 100:1. The final concentration of rIL-15 in the mixture was 0.5 mg / mL (0.031 mM). Sodium bicarbonate buffer (1 M, pH 8.0) was added to the mixture to a final concentration of 100 mM, and conjugation was allowed to proceed for thirty minutes to provide [mPEG2-CAC-fmoc-20K-NHS]-[rIL-15] conjugate (wherein the informal name of the conjugate reflects the polymer reagent used to prepare the conjugate, it being understood that for one or more of the resulting products, the reactive moiety in the polymer reagent has been replaced by the linkage to IL-15 (in this example). After thirty minutes, quenching was achieved by adding 1 M glycine (pH 6.0) to the reaction mixture to a final concentration of 100 mM. The pH of the quenched reaction mixture was then adjusted to 4.0 using glacial acetic acid prior to column chromatography purification and characterization.

[0314] The reaction mixture was analyzed by RP-HPLC analysis. Based on SDS-PAGE, the reaction mixture contained approximately 10%-20% monoconjugate, approximately 50%-70% diconjugate, and approximately 20%-30% triconjugate - that is, the reaction mixture contained primarily diconjugated species. The conjugate mixture was separated by anion exchange chromatography using a Q Sepharose high performance column and sodium phosphate buffer as the elution phase to provide purified [mPEG2-CAC-FMOC-20kD-NHS]-IL-15 with an average degree of PEGylation of approximately 2 (degree of PEGylation ranged from approximately 1.7 to 2.5), such that n' in the above structure for this purified composition is approximately 2.

[0315] For the remaining examples, the purified [mPEG2-CAC-FMOC-20kD-NHS]-IL-15 was referred to as Conjugate 2.

[0316] Example 3

[0317] Preparation of long-acting IL-15 receptor agonist,

[0318] [Mono-PEG2-RU-ButryALD-40K]-IL15

[0319]

[0320] The branched mPEG-butyraldehyde PEG reagent is mono-PEG2-RU-ButryALD-40K, The subject long-acting IL-15R agonist is prepared, wherein the weight average molecular weight of the PEG reagent used to prepare the agonist is about 40,000 Daltons.

[0321] 2.7 ml of IL-15 (1.23 mg / ml in PBS buffer, pH 7.4) was transferred to a small reaction bottle and 0.3 ml of 1 M sodium acetate buffer (pH 5) was added to adjust the pH to pH 6. mPEG2-ru-ButyrALD (40 kDa) stored at -20 ° C. under nitrogen was warmed to ambient temperature. A fifteen-fold excess (relative to the amount of IL-15) of mPEG2-ru-ButyrALD was dissolved in MilliQ H2O to form a 10% reagent solution. The 10% reagent solution was quickly added to the IL-15 solution and thoroughly mixed, and placed on a RotoMixer for 15 min. 1 / 100 volume of 1 M NaCNBH3 / H2O was then added to the reaction mixture. To couple mPEG2-ru-ButyrALD to IL-15 via a secondary amine linkage, the reaction solution was placed on a RotoMixer at 4°C for 17 hours and then quenched with a glycine solution. Because the PEGylation reaction is carried out at an acidic pH, attaching the PEG derivative to IL-15 is more selective for the N-terminus. Anion exchange chromatography using a Q Sepharose high-performance column and sodium phosphate buffer was also developed to purify the conjugate. The purified monoPEG2-ru-ButyrALD-40K-IL-15 conjugate was characterized by HPLC and SDS-PAGE.

[0322] For the remaining examples and in the accompanying disclosure, purified [monoPEG2-RU-ButryALD-40K]-IL-15 (i.e., having a single PEG moiety with the structure shown above covalently attached to IL-15 via an amine linkage) is referred to as Conjugate 3.

[0323] Using this same method, conjugates were prepared using PEG2-RU-ButryALD- with varying weight average molecular weights. For example, mono-PEG2-RU-ButryALD-20kD]-IL-15 (referred to herein as Conjugate 4) was prepared as described above using a 20 kD polymer reagent.

[0324] Example 4

[0325] Preparation of long-acting IL-15 receptor agonist,

[0326] Single mPEG-ButyrALD-40K-IL-15

[0327]

[0328] The PEG reagent-having the structure Linear mPEG-butyraldehyde (40 kDa) ("mPEG-ButyrALD") is used to prepare the subject long-acting IL-15R agonists, wherein the weight average molecular weight of the PEG reagent used to prepare the agonist is approximately 40,000 Daltons.

[0329] 2.7 ml of IL-15 (1.23 mg / ml in PBS buffer, pH 7.4) was transferred to a small reaction bottle and 0.3 ml of 1 M sodium acetate buffer (pH 5) was added to adjust the pH to pH 6. mPEG-ButyrALD (40 kDa) stored at -20 ° C under nitrogen was warmed to ambient temperature. A ten-fold excess (relative to the amount of IL-15) of mPEG-ButyrALD was dissolved in MilliQ H2O to form a 10% reagent solution. The 10% reagent solution was quickly added to the IL-15 solution and mixed thoroughly, and placed on a RotoMixer for 15 min. 1 / 100 volume of 1 M NaCNBH3 / H2O was then added to the reaction mixture. In order to couple mPEG-ButyrALD to IL-15 via secondary amine linkage, the reaction solution was placed on a RotoMixer for 17 hours at 4 ° C and subsequently quenched with a glycine solution. Because the PEGylation reaction is carried out at acidic pH, attachment of the PEG derivative to IL-15 is more selective for the N-terminus. Anion exchange chromatography using a Q Sepharose high-performance column and sodium phosphate buffer was also developed to purify the conjugate. The purified mono-mPEG-ButyrALD-40K-IL-15 conjugate was characterized by HPLC and SDS-PAGE.

[0330] For the remaining examples, mono-mPEG-ButryALD-40k-IL-15 is referred to as Conjugate 5.

[0331] Example 5

[0332] Evaluation of the receptor bias of long-acting IL-15 receptor agonists for IL-15Rα

[0333] The affinity of an exemplary long-acting IL-15 receptor agonist (test article) for the IL-15α receptor was measured and compared to IL- 15. Affinity was measured by BIAcore using IL-15Rα:Fc captured by immobilized anti-Fc.

[0334] The test article was diluted to 10 μM in PBS (containing 0.05% Tween 20 and 0.1 mg / ml BSA). A series of 3-fold dilutions were made and injected onto a sensor chip coated with IL-15Rα. Affinity was determined by measuring k a and k d The rate is measured, and k d With k a The ratio between them is used to calculate K d value.

[0335] Preferred conjugates are generally those that retain as much affinity for IL-15Rα as possible after PEGylation compared to unmodified IL- 15. Conversely, preferred conjugates are generally those in which the affinity for IL-15Rα is minimally reduced relative to the affinity for unmodified IL-15.

[0336] For example, in some embodiments, preferred conjugates exhibit no more than about a 7-fold decrease in EC50 value (ng / mL, CTLL-2 pSTAT5) and receptor α binding (K D For example, conjugate 1 has about a two-fold reduction in potency when compared to IL-15 and retains about 80% of the alpha receptor affinity of IL-15.

[0337] Table 2A.

[0338] Test items <![CDATA[k a (M -1 sec -1 )]]> <![CDATA[K d (sec -1 )]]> <![CDATA[K D (pM)]]> IL-15 <![CDATA[5.78x 10 5 ]]> <![CDATA[1.49x 10 -4 ]]> 258 Conjugate 1 <![CDATA[4.92x 10 5 ]]> <![CDATA[1.03x 10 -4 ]]> 209

[0339] As indicated in the table above, Conjugate 1 retains its high affinity for the IL-15α receptor (ie, when compared to IL-15), a characteristic that is particularly preferred for long-acting IL-15 receptor agonists. The affinity constants (K) for the additional conjugates are provided below. D )(in pM).

[0340] Table 2B. Additional Characteristics of Exemplary Long-Acting IL-15 Receptor Agonists

[0341]

[0342] Example 6

[0343] In vivo studies: single-dose PK studies in mice

[0344] C57BL / 6 mice (n=3 / group) were administered a single intravenous dose of IL-15 (control) (at a dose of 0.3 mg / kg) or conjugate 2 (at a dose of 0.3 mg / kg). Following administration, blood samples were collected at various time points after administration (24 hours, 48 ​​hours, 78 hours, 96 hours). The samples were pooled and evaluated by flow cytometry for pharmacodynamic analysis of drug effects on lymphocyte populations, expressed as fold change relative to vehicle control (results are described in the subsequent examples below). In addition to changes in cell number, functional markers and activity markers were quantified. Finally, at each time point, the plasma concentration of the drug was determined. See Figure 6 .

[0345] like Figure 6 As shown in , after administration, Conjugate 2 maintains measurable concentrations in plasma (solid squares) for an extended period of time (e.g., for greater than 1 week), T 1 / 2 In contrast, a rapid decline in plasma levels was observed for non-long-acting IL-15 (filled circles) over a period of approximately 20-30 hours.

[0346] Example 7

[0347] In vivo studies: Single-dose PK studies in rats

[0348] Rats (n=3 / group) were administered a single intravenous dose of Conjugate 2 at doses of 0.3, 0.15, and 0.075 mg / kg, or a single subcutaneous dose of Conjugate 2 at 0.15 mg / kg. Following administration, blood was collected on days 1-7 after administration (with multiple samples collected within the first 24 hours after administration). At each time point, the plasma concentration of the drug was determined. Figure 7 .

[0349] like Figure 7 As shown in Figure 6 Similar to the results shown for mice in , administration of Conjugate 2 resulted in sustained and dose-proportional drug exposure.

[0350] Example 8

[0351] In vivo IL-15 signaling studies in mice

[0352] Mice were dosed as described above in Example 6 to assess in vivo signaling, as assessed by the degree of STAT5 phosphorylation. STAT5 phosphorylation in various lymphocytes (CD4, CD8, and NK cells) was assessed by staining whole blood for leukocyte surface markers and pSTAT5, followed by flow cytometry. The results for IL-15 and Conjugate 2 are shown in Table 1. Figure 8A and8B middle.

[0353] STAT5 phosphorylation is an early and transient event in IL-15 / IL-2 receptor signaling. Figure 8A As seen in Figure 2, the in vivo signaling activity of IL-15 is extremely short-lived, while exemplary conjugate 2 induces persistent STAT5 phosphorylation, which is most pronounced in NK cells (inverted solid triangles ▼) and CD8 cells (normal solid triangles ▲), with measurable STAT5 phosphorylation activity noted in NK and CD8 cells after more than 72 hours. STAT5 phosphorylation activity of CD4 cells is also shown (closed squares ■).

[0354] Example 9

[0355] In vivo IL-15 signaling studies in non-human primates

[0356] In this study, cynomolgus monkeys (cynomolgus monkeys) (one female and one male) were each administered a single dose of conjugate 2 (0.5 mg / kg) intravenously. Serial blood samples were taken from each animal before treatment (day -6 and day -1) and at various time intervals after treatment to assess STAT5 phosphorylation in various lymphocyte types (CD4, CD8, and NK cells) by flow cytometry. The results are presented in Figure 9A (CD4), 9B (CD8), and 9C (NK).

[0357] like Figures 9A-9C As shown in , the results are similar to those observed in mice (Example 7), but in non-human primates, STAT5 phosphorylation was also observed in CD4 cells ( Figure 9A ). STAT5 phosphorylation in each of the three cell types increased significantly after administration, reaching a maximum level between about 3 and 4 days after administration of the illustrative long-acting IL-15 agonist and returning to levels close to day -1 (before administration) by about days 5-10. Similar to Example 7, these results indicate the continued presence of active IL-15 species.

[0358] Example 10

[0359] The NK cell line of human peripheral blood mononuclear cells (PBMC) is expressed as follows:

[0360] In vitro IL-15 activity in subsets

[0361] The in vitro activity of exemplary long-acting IL-15 receptor agonists (e.g., Conjugates 1, 3, and 5) was evaluated by studying signaling in NK cell subsets of human PBMCs, as shown in Figure 10A(CD56 bright cells) and 10B (CD56 dim cells). STAT5 phosphorylation was assessed as previously described for the evaluation of IL-15 signaling activity of long-acting IL-15 receptor agonists.

[0362] Table 3.

[0363]

[0364] The results are shown in Figure 10A (CD56 bright) and 10B (CD56 dim).

[0365] As can be seen, each of the illustrative conjugates induced IL-15 signaling in human PBMCs, with Conjugate 1 being the most potent. Of the conjugates tested (full data not shown), Conjugate 1 exhibited the highest potency / activity on human PBMCs. The data demonstrate that even while maintaining the same degree of PEGylation (i.e., number of PEG moieties) and the same size of PEG moieties for each IL-15 protein, different PEG architectures and linkers can induce very different effects on the bioactivity of the resulting conjugates.

[0366] A second study was conducted to investigate / compare the pStat5 responses of human PBMCs (CD3, CD4, CD8, CD56 (bright and dim) and CD4-Tregs (CD25+Foxp3+)) obtained from two donors to IL-15, Conjugate 1, and Conjugate 5. An 11-point dose response was examined using 10-fold dilutions with a dose range of 0.001-10,000 ng / ml and 20 minutes of stimulation. Each test article was diluted in IL-15 buffer + 0.1% BSA. The results are provided in the table below.

[0367] Table 4. pSTAT5% and pSTAT5 MFI in CD3 and CD4 cells

[0368]

[0369] Based on the data in Table 3 above, IL-15 appears to be approximately 4-6 times more potent than Conjugate 1 for both CD3 and CD4 induction; the potency of Conjugate 1 and Conjugate 5 appears to be similar for both CD3 and CD4 induction.

[0370] Table 5. pSTAT5% and pSTAT5 MFI in CD4-Treg and CD8 cells

[0371]

[0372] Based on the data in Table 5 above, IL-15 appears to be approximately 3-5 times more potent than Conjugate 1 for Treg and CD8 induction; for CD4 and CD8 induction, the potencies of Conjugates 1 and 5 appear essentially similar.

[0373] Table 6. pSTAT5% and pSTAT5 MFI in CD56 bright and CD56 dim cells

[0374]

[0375] Based on the data in Table 6, IL-15 appears to be approximately 10-fold more potent than Conjugate 1 for CD56 induction. However, Conjugate 1 appears to be more potent than Conjugate 5 in inducing both CD56 Bright and CD56 Dim.

[0376] Based on the aforementioned data, IL-15, Conjugate 1 and Conjugate 5 showed similar pSTAT5 induction for all cell populations, with the maximal responses appearing to be higher for CD56 bright and Treg cells.

[0377] Table 7. Summary table

[0378]

[0379] Based on the foregoing data, IL-15 was approximately 3-4 times more potent than Conjugate 1 and approximately 5-8 times more potent than Conjugate 5 in inducing CD3, CD4, CD8, and Treg cells, while IL-15 was approximately 12 times more potent than Conjugate 1 and approximately 40-60 times more potent than Conjugate 5 in inducing CD56bright and CD56dim cells - indicating certain unforeseen and particularly advantageous features of Conjugate 1.

[0380] Example 11

[0381] In vivo studies: Single-dose PD studies in mice - cell proliferation and activation

[0382] Balb / c mice (n=3 / group) were administered a single intravenous dose of vehicle (50 mM sodium phosphate, 100 mM sodium chloride, 10% sucrose, pH 7.4) or conjugate 1 at a dose of 0.03 mg / kg (Figure 11, low dose), 0.3 mg / kg (Figure 11, medium dose) or 1 mg / kg (Figure 11, high dose). Following administration, blood samples were collected at various time points (24 hours, 48 ​​hours, 78 hours, 96 hours, 120 hours) after administration. Pharmacodynamic analysis of drug effects on lymphocyte populations was performed on samples from each mouse by flow cytometry. In addition to changes in cell number, functional markers and activity markers were also examined.

[0383] Additional administrations of Conjugate 1 were performed at doses of 0.01 mg / kg, 0.1 mg / kg, and 1.5 mg / kg.

[0384] Results are available on Figure 11A and 11B These results illustrate the proliferation of NK cells in mice administered 0.03 mg / kg (Figure 11, low dose), 0.3 mg / kg (Figure 11, medium dose), and 1 mg / kg (Figure 11, high dose) of each of Conjugate 1.

[0385] NK cells and their proliferation are defined using CD45+CD3-CD49b+ and CD45+CD3-CD49b+Ki67+ marker combinations. After administration, blood samples were collected on a Fortessa flow cytometer running FACS DIVA software. Flowjo software was used for analysis and Prism was used to plot the absolute NK cell count and Ki67 positive rate % in the NK cells.

[0386] Figure 11A is a curve of Ki67 expression (in percentage) over time; Figure 11B NK cell numbers over time are provided.These curves illustrate the ability of Conjugate 1 to induce sustained NK cell proliferation in mice.

[0387] The effects of an exemplary long-acting IL-15 receptor agonist on NK cells at all maturation levels were explored. Peripheral NK cell pools can be delineated by CD27 expression, where CD27 低 / - NK cells and CD27 高 NK cells have greater cytotoxicity and produce more cytokines than NK cells (Hayakawa Y et al., J Immunol. 2006; 176: 1517-1524). The mature peripheral NK cell population has been further subdivided into four maturation stages, which are defined by the sequential upregulation of CD11b expression followed by downregulation of CD27, with the most immature NK cells being CD27. - CD11b - And the most mature NK cells are CD27 - CD11b + (Chiossone L. et al., Blood, 2009; 113: 5488-5496).

[0388] In mice, four different maturation states of NK cells are defined by the expression of CD27 and CD11b. Once triple-positive cells for NK markers (CD49b+), naturally activating NK receptors (NKp46+), and IL-15 / IL-2RB (CD122+) are identified, immature (CD11b-CD27-), early (CD11b-CD27+), high-effector (CD11b+CD27+), and terminal-effector (CD11b+CD27-) NK cells are quantified by flow cytometry.

[0389] In the mice after the third dosage of the single dose of 0.01,0.03,0.1,0.3,1.0 and 1.5mg / kg accepting conjugate 1 or q7dx3 scheme, the NK cells in various mature states are quantitatively tested. Using flow cytometry, interested NK colony is identified by CD49b, NKp46 and CD122 positive rate. Then CD11b and CD27 are used to further differentiate the NK colony into immature (CD11b-CD27-), early stage NK (CD11b-CD27+), high effect (CD11b+CD27+) and terminal effect (CD11b+CD27-) subpopulation. Peripheral blood is run on Fortessa flow cytometer and BD FACSDIVA software is used, during sample collection, counting beads are used to measure the absolute value of each colony. Flow cytometry analysis is carried out using Flowjo software and in Prism, data are mapped.

[0390] The results are shown in Figures 12A-12D Other results are shown in Figures 22A-22D The results of q7dx3 are shown in Figures 28A-28D As can be seen from the curve, conjugate 1 effectively increases the number of NK cells at all maturity levels (terminal effector cells, precursor NK cells, high effector cells and early NK cells). A dose-dependent increase in NK cells in all mature subpopulations was observed, and the effect lasted for at least 120 hours.

[0391] Flow cytometric analysis of the anti-NKG2D signal was used to demonstrate the surface expression of NKG2D, which was expressed as the mean fluorescence intensity (MFI) in NK cells. Similarly, flow cytometric detection of the anti-granzyme B signal was used to demonstrate the level of intracellular granzyme B, which was also expressed as the MFI in NK cells. After detecting NKG2D and granzyme B signals using a Fortessa flow cytometer and FACS Diva software, Flowjo software was used for analysis. MFI values ​​were plotted using Prism. The results are shown in Figure 13A and 13BThese results further demonstrate the ability of Conjugate 1 to increase NK cell activation, as demonstrated by the ability of Conjugate 1 to achieve sustained increases in NK cell expression of both NKG2D and granzyme B (a pro-apoptotic serine protease) compared to vehicle, with increases being most pronounced at the mid- and high-dose levels. Dose-dependent increases in both NK activation markers were observed after a single dose of Conjugate 1.

[0392] In mice, CD8 T cells were defined as CD45+CD3+CD4-CD8+. Blood and spleen from mice were immunophenotyped using a Fortessa flow cytometer and analyzed using FlowJo software. Absolute CD8 cell counts were plotted in Prism as shown in Figure 2. Figure 14 (blood) and Figure 24 (Spleen) shown. Figure 14 and Figure 24 The results show that after a single intravenous administration of Conjugate 1 in mice at each dose, the conjugate 1 can induce CD8 T cell proliferation and sustained increases in number. The effects were most pronounced at the medium (0.1 mg / kg, 0.3 mg / kg) and high (1.0 mg / kg, 1.5 mg / kg) doses.

[0393] In mice, CD8 effector memory (Tem) and CD8 central memory (Tcm) T cells were identified as CD45+CD3+CD4-CD8+CD44+CD62- and CD45+CD3+CD4-CD8+CD44+CD62L+. The proliferation of these memory populations was performed using Ki-67 positivity. Following a single dose of conjugate 1 or IL-15, blood and spleen were immunophenotyped using a Fortessa flow cytometer, DIVA acquisition software, and Flowjo analysis software. Graphs were drawn in Prism. For blood, conjugate 1 induced a dose-dependent increase in both effector and central memory CD8 T cells, while a single dose of IL-15 did not. Figure 15A and 15B For the spleen, conjugate 1 induced a dose-dependent increase in both Ki67 and granzyme B, whereas a single dose of IL-15 did not. Figure 25 and 26 As shown in . Both effector and central memory populations proliferated in response to administration of Conjugate 1, an exemplary long-acting IL-15 agonist.

[0394] Example 12

[0395] In vivo studies: single-dose PD studies in non-human primates

[0396] In this study, cynomolgus monkeys (one female and one male) were intravenously administered 500 μg / kg of conjugate 2. Serial blood samples were taken from each animal before treatment (day -6 and day -1) and at various time intervals after a single dose of treatment to assess lymphocyte numbers (NK cells, CD8 T cells, etc.) and activation by flow cytometry.

[0397] NK cell counts were determined to assess the ability of exemplary conjugate 2 to induce sustained NK cell proliferation in non-human primates; the results are shown in Figure 16A and 16B NK cells and their proliferation in the blood of cynomolgus monkeys were identified by flow cytometry. NK cells (CD45+CD3-CD16+) and their proliferation status (CD45+CD3-CD16+Ki67+) were collected and analyzed using BD FACS DIVA software. The absolute values ​​of NK cells and proliferating NK cells were used to calculate the Ki67 positivity rate within the NK population. The values ​​before and after treatment were plotted using Prism.

[0398] As shown therein, single-dose administration of Conjugate 2 effectively induced sustained NK cell proliferation in non-human primates.

[0399] Also like Figure 17 As shown in , CD8 T cell counts were measured for each animal from pre-dose to 14 days after administration. Specifically, CD8 T cells were defined as CD45+CD3+CD4-CD8+. Blood from monkeys was also immunophenotyped as previously described. In monkeys, CD8 T cells increased in a sustained manner, with this effect lasting for at least 10 days. This curve further illustrates the ability of the exemplary long-acting IL-15 receptor agonist conjugate 2 to induce CD8 T cell proliferation and a sustained increase in number after administration.

[0400] In monkeys, CD8 T EM Cells were defined as CD45+CD3+CD4-CD8+CD45Ra-CD197- and CD8 T cells were CM Defined as CD45+CD3+CD4-CD8+CD45Ra-CD197+. Immunophenotyping was performed by flow cytometry, with sample collection performed on DIVA software and data analysis performed on Flowjo software. Prism was used to plot the curves. The CD8 T effector memory cells (T) of each animal were measured from pre-dose to 14 days after administration. EM cells) and CD8 T central memory cells (T CM ) count, such as Figure 18A and 18BAs shown in . Conjugate 2 induces a significant and sustained increase in CD8 effector and central memory T cell populations in cynomolgus monkeys. The figure shows that both CD8 effector and central memory T cell populations proliferate in response to exemplary conjugate 2.

[0401] Example 13

[0402] Evaluation of antitumor activity in a CT26-induced subcutaneous lung metastasis tumor model in BALB / C mice

[0403] On day 0, 1 × 10 5 Six- to eight-week-old female Balb / c mice were inoculated with CT-26 cells. On day 1, 24 hours after administration of the CT-26 cells, the mice were divided into ten groups. Each group consisted of 6-9 animals (for Conjugate 2) or 9-12 animals (for Conjugate 1). (Two separate studies were conducted for the administration of Conjugate 1 and Conjugate 2, but the study protocols were essentially identical in both studies.) Each group received one intervention as follows: vehicle, phosphate-buffered saline (Group A); initial IL-15 alone (Group B); conjugate 2 at a dose of 0.03 mg / kg (Group C); conjugate 2 at a dose of 0.1 mg / kg (Group D); conjugate 2 at a dose of 0.3 mg / kg (Group E); conjugate 2 at a dose of 1.0 mg / kg (Group F); conjugate 2 at a dose of 3.0 mg / kg (Group G); for conjugate 1: vehicle, phosphate-buffered saline (Group H); conjugate 1 at a dose of 0.03 mg / kg (Group I) and conjugate 1 at a dose of 0.3 mg / kg (Group J). Animals were dosed on days 1, 5, and 10.

[0404] Thirteen days after the administration of CT-26 tumor cells, mice were anesthetized, and blood and spleen cells were collected for further analysis of immunophenotyping markers, while lungs were fixed in Bouins solution containing picric acid and formaldehyde for 24-48 h.

[0405] The number of lung tumor nodules per lung was counted at autopsy, and the mean lung nodules per group were determined. Statistical significance was also obtained between the vehicle group and the intervention group using an unpaired Student's t-test.

[0406] The lung metastasis results for the treatment groups corresponding to conjugate 2 and conjugate 1, respectively, are shown in Figure 19 and 20 While both illustrative long-acting IL-15 receptor agonists were effective in promoting a reduction in lung metastases, Conjugate 2 provided a 65% reduction in metastases compared to vehicle, whereas Conjugate 1 provided an 85% reduction in metastases.

[0407] On day 13, blood and spleen cells were analyzed for changes in immunophenotyping markers using flow cytometry and marker antibodies conjugated to various fluorescent dyes. Conjugate 2 administered at 0.3, 1, and 3 mg / kg induced a dose-dependent increase of 1.5, 2.5, and 3.3-fold in CD8 T cells in the blood, respectively, relative to vehicle. Similar observations were obtained in the spleen, with increases of 1.3, 1.7, and 2.2-fold at 0.3, 1, and 3 mg / kg dose levels relative to vehicle. Ki-67 immunophenotyping revealed a significant dose-dependent increase in CD8 T cell proliferation: at the same low, medium, and high dose levels, there were 1.7, 4.6, and 5.3-fold changes in the blood and 2.5, 5.7, and 6.9-fold changes in the spleen compared to vehicle. In addition, conjugate 2 treatment increased the pro-survival Bcl-2+ MFI of CD8 in both blood and spleen by up to 1.5-fold.

[0408] Table 8.

[0409]

[0410] Example 14

[0411] In vitro and in vivo cytotoxicity of NK cells following treatment with conjugate 1

[0412] The NK cell-mediated cytotoxicity against target tumor cells was evaluated in vitro using a flow cytometry-based assay. NK cells were isolated from the spleen of Balb / c mice using negative selection magnetic cell separation (mouse NK cell enrichment kit, Stemcell Technologies Co., Ltd. (Stemcell Technologies)) and used as effector cells. For in vitro studies, before being used for cytotoxicity assays, isolated NK cells were stimulated overnight with conjugate 1 at a concentration of 3000, 1000, 300, 30, 3 or 0 (unstimulated) ng / mL in a humidified incubator at 37°C, 5% CO2. For in vivo studies, mice were administered 0.3 mg / kg conjugate 1, and spleen NK cells were separated 24, 48 and 72 h after administration and used directly in cytotoxicity assays.

[0413] YAC-1 T cells labeled with PKH26 were used as target cells. To monitor NK cell cytotoxicity, NK and YAC-1 cells were co-cultured at various effector: target ratios (50:1, 25:1, and 12.5:1) at 37°C, 5% CO2 for 4 hours, and then stained with 7-AAD for 10 minutes to mark dead cells. Cells were immediately analyzed by flow cytometry. Lysed target cells were identified as PKH26 + 7-AAD + .

[0414] In vitro results: Cytotoxicity was assessed by flow cytometry after 4 hours of co-culture. Figure 21 middle.

[0415] In vivo results: Cytotoxicity was assessed after 24, 48, and 72 hours following treatment with 0.3 mg / kg. Figure 27 middle.

[0416] The data demonstrate a dose-dependent increase in NK cell cytotoxicity following treatment with Conjugate 1 both in vitro and in vivo.

[0417] Example 15

[0418] Induction of granzyme B by conjugate 1

[0419] NK cell granzyme B expression over time was measured after treatment with conjugate 1 at doses of 0.01 mg / kg, 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, and 1.5 mg / kg. Whole blood was collected from 24 to 240 hours after administration for immunophenotyping. After erythrocyte lysis, white blood cells were labeled with viability dyes and markers specific for CD45, CD3, and CD49b to identify live NK cells. The cells were then fixed and permeabilized simultaneously for intracellular granzyme B staining. The stained blood was run on a Fortessa flow cytometer, collected by DIVA software, and analyzed using Flowjo software. Data were expressed as the percentage of NK cells that were positive for granzyme B expression.

[0420] The results are shown in Figure 23 The data indicate that treatment with Conjugate 1 increases granzyme B expression by NK cells.

[0421] Example 16

[0422] In vivo studies: single dose of IL-15 and conjugates 1PK and JAK / STAT in mice

[0423] Signal transduction research

[0424] For PK analysis, conjugate 1 was administered with a single intravenous dose of 0.3 mg / kg in balb / c mice (n=3). After administration, mice were humanely killed and plasma was collected 24, 48, 72, 96, 120, and 144 hours after treatment. According to independent studies, a single intravenous dose of IL-15 (0.5 mg / kg) was administered to mice. Samples from these mice were collected at designated time points within 6 hours of treatment. [PK methods previously described herein] For pharmacodynamics, balb / c mice (n= / group) were injected intravenously with 0.03 or 0.3 mg / kg of conjugate 1 or vehicle (50 mM sodium phosphate, 100 mM sodium chloride, 10% sucrose, pH 7.4) and blood was collected 15 min, 1, 24, 48, 72, 96, and 120 hours before and after administration. Samples were analyzed separately by flow cytometry and expressed as pSTAT5 positive rate percentages in CD8 and NK cells.

[0425] Figure 29A

[00145]

[00146] Figures 2 and 3 are plasma concentrations of the test article (IL-15 or Conjugate 1) over a 144 hour time course following administration of a single intravenous dose of the test article at 0.5 and 0.3 mg / kg, respectively, in balb / c mice.

[0426] Results: Conjugate 1 exhibited a half-life of approximately 12 hours, whereas IL-15 was rapidly cleared from plasma with a half-life of less than 1 hour.

[0427] Figure 29B is a graph of the percentage of pSTAT5 positivity in CD8 T cells in mice after a single injection of Conjugate 1 at 0.03 and 0.3 mg / kg.

[0428] Results: Conjugate 1 induced sustained pSTAT5 signaling in CD8 T cells at both dose levels. A 120-hour time course is shown, including pre-dose.

[0429] Figure 29C is a graph showing the percentage of pSTAT5 positivity in murine NK cells following a single injection of Conjugate 1 at 0.03 and 0.3 mg / kg.

[0430] Results: Conjugate 1 at both dose levels induced robust and sustained pSTAT5 signaling in NK cells.

[0431] Example 17

[0432] In vivo single-dose and Q7DX3 pharmacodynamic studies in mice - cell number and proliferation

[0433] Balb / c mice (n=3 / group) were administered a single dose, or once weekly dose, three times of conjugate 1 (at 0.01, 0.03, 0.1, 0.3, 1 or 1.5 mg / kg) or vehicle. Mice were sacrificed and blood was collected at various time points (24, 48, 72, 96, 120, 144, 240 hours) after administration. Flow cytometry analysis was performed on samples from each mouse to examine the pharmacodynamic effects and functional markers of interest (cell counts of CD8 T cells, CD8 memory T cells, and NK cells, and the percentage of Ki-67 positivity within each population) within the lymphocyte population. The results are shown in Figures 30A-30F , 31A-31C, and 32A-32C.

[0434] Figures 30A-30C As described in Example 17, after a single administration of conjugate 1 at 0.01, 0.03, 0.1, 0.3, 1 or 1.5 mg / kg, the curves of the total CD8, CD8 central memory (Tcm) and CD8 effector memory (Tem) cell numbers, respectively. As described in Example 17, conjugate 1 at a dose level equal to or greater than 0.03 induced a significant increase in total CD8 T cells in the blood. The lowest dose of 0.01 mg / kg increased CD8 Tcm and CD8 Tem. At 0.3 mg / kg, conjugate 1 increased CD8, CD8 Tcm and CD8 Tem by 6.4X, 37.9X and 14.5X. It is noteworthy that when conjugate 1 was administered at 0.3-1.5 mg / kg, the number of CD8 and CD8 memory T cells did not return to baseline 240 hours after injection, which demonstrated the sustained PD effect of conjugate 1.

[0435] Figure 30D 、 30E 30F and 30F are plots of the percentage of Ki-67 positivity within the total CD8, CD8 Tcm, and CD8 Tem populations, respectively, in mice as described in Example 17. A single dose of Conjugate 1 increased Ki-67 positivity in all CD8 and CD8 subset populations at all dose levels.

[0436] Figure 31A 、 31B 31C and 31C are plots of CD8 and CD8 memory subset T cell numbers following single (dashed line) or Q7dx3 (solid line) administration of Conjugate 1 at 0.03 and 0.3 mg / kg, as described in Example 17. Repeated dosing further increased these populations, with CD8, CD8 Tcm, and CD8 Tem increasing by 35.3X, 183X, and 73.8X, respectively. At the end of the time course (240 hours after the first or last dose at 0.3 mg / kg), cell numbers in mice had not returned to baseline.

[0437] Figure 32A and 32B Figure 1 is a graph of NK cell numbers and Ki-67 positive percentages following single doses of Conjugate 1 from 0.01 to 1.5 mg / kg in mice as described in Example 17. At all dose levels, NK cell numbers increased significantly above the dose control and returned to baseline by 240 hours post-dose. All dose levels induced a robust increase in the percentage of Ki-67 positive NK cells.

[0438] Figure 32C Figure 1 is a graph of murine NK cell numbers following single (solid line) or Q7dx3 (dashed line) administration of Conjugate 1 at 0.03 and 0.3 mg / kg, as described in Example 17. Repeated administration of Conjugate 1 at 0.3 mg / kg induced slightly fewer, but still significant, NK cell numbers compared to a single dose. Similar NK cell numbers were achieved with single versus repeated administration at 0.03 mg / kg.

[0439] Results: Conjugate 1 at dose levels equal to or greater than 0.03 induced a significant increase in total CD8 T cells in the blood. The lowest dose of 0.01 mg / kg increased CD8 Tcm (central memory) and CD8 Tem (effector memory). At 0.3 mg / kg, conjugate 1 increased CD8, CD8 Tcm, and CD8 Tem by 6.4X, 37.9X, and 14.5X, respectively. Notably, when conjugate 1 was administered at 0.3-1.5 mg / kg, the number of CD8 and CD8 memory T cells did not return to baseline 240 hours after injection, demonstrating the beneficial sustained PD effect of conjugate 1.

[0440] Example 18

[0441] Measurement of in vitro NK cytotoxicity and blood NK cell granzyme B analysis in mice treated with conjugate 1

[0442] Balb / c mice (n=2 / group) were treated with conjugate 1 (0.006, 0.03 or 0.3 mg / kg), IL-15 (1 mg / kg) or vehicle control. Spleens were isolated 24, 72 and 96 hours after treatment to achieve NK cell isolation. NK cells were isolated by a magnet-based negative selection method and incubated at 12.5:1, 25:1 and 50:1 NK (effector) to YAC-1 (target cell) ratios (E:T) at 37°C, 5% CO2 for 4 hours. YAC-1 target cells were pre-labeled and then stained with 7AAD after NK cell incubation. Detection of cleaved (7AAD+) target cells (PKH26+) was performed by flow cytometry. Blood from these mice was also collected at the same time points and subjected to flow cytometric measurement of granzyme B expression by NK cells. The results are shown in Figure 33A and 33B middle.

[0443] Figure 33A In vitro NK cytotoxicity assays are described that measure changes in NK-mediated target cell lysis following test article treatment in mice. A time course of the percentage of specific lysis of YAC-1 cells by splenic NK cells isolated from balb / c mice treated with 0.006, 0.03, or 0.3 mg / kg conjugate 1 or 1 mg / kg IL-15 is shown over the indicated hours. Splenic NK cells from mice dosed with vehicle served as controls.

[0444] Results: Conjugate 1 administered at 0.3 mg / kg induced a superior increase in NK cytotoxicity in both magnitude and duration compared to NK cells from mice that received a single injection of IL-15 at 1 mg / kg.

[0445] Figure 33B yes Figure 33A Figure 2 is a graph showing the percentage of granzyme B positive NK cells in blood from the same mice used for the NK in vitro cytotoxicity assay.

[0446] Results: Conjugate 1 administered at 0.03 and 0.3 mg / kg induced a significant increase in NK granzyme B expression, with a robust and sustained increase seen at 0.3 mg / kg.

[0447] Example 19

[0448] Conjugate 1 Single Agent Efficacy in the CT-26 Lung Metastasis Model

[0449] Balb / c mice received 1 x 10 5The mice were injected with CT-26 colorectal cancer cells through the tail vein. The next day, mice (n=9 / group) were treated twice weekly with Conjugate 1 (0.03 or 0.3 mg / kg) or vehicle control. Five days after the second injection, mice were humanely sacrificed and lung nodules were counted. The results are presented in Figure 34A and 34B middle.

[0450] Figure 34A and 34B Depicted are the percent inhibition of lung nodules in balb / c mice receiving intravenous CT-26 tumor cell injections followed by treatment with Conjugate 1 at 0.03 or 0.3 mg / kg administered twice one week apart.

[0451] Results: Injection of conjugate 1 at 0.03 and 0.3 mg / kg inhibited lung nodule formation by 40% and 80%, respectively. The same mice dosed at 0.3 mg / kg were followed up for 32 days after tumor cell injection to assess survival. Treatment with conjugate 1 significantly increased survival compared to tumor-injected mice receiving vehicle control.

[0452] Example 20

[0453] Evaluation of NK cell dependence of Conjugate 2 efficacy in the CT-26 lung metastasis model

[0454] CT-26 mice (n=7-11 / group) were injected with anti-asialo GM1 to deplete NK cells or two different controls (IgG or PBS), followed by injection of 1×10 5 CT-26 tumor cells. Mice were then treated with Conjugate 2 or vehicle control at 0.3 mg / kg on days 1, 5, and 10 after tumor cell injection. Three days after the last day of treatment, the mice were sacrificed and lung nodules were counted. The results are shown in Figure 35 middle.

[0455] Figure 35 Is a graph showing the percent inhibition of lung nodules in CT-26 injected mice treated with Conjugate 2 that received antibody-mediated NK cell depletion (olive green), IgG control (blue), or PBS (orange). Data are expressed as percent inhibition of lung nodules relative to CT-26 injected mice that were not subjected to NK cell depletion and treated with vehicle control (black). When mice lack NK cells, Conjugate 2 efficacy in this tumor model is abolished.

[0456] Example 21

[0457] In vivo pharmacodynamics of a single dose of conjugate 1 in non-human primates

[0458] In this study, cynomolgus monkeys (cynomolgus monkeys) (one female and one male) were each administered a single dose of conjugate 1 (0.1 mg / kg) intravenously. Serial blood samples were taken from each animal over a 14-day period and subjected to flow cytometric analysis of various lymphocytes (CD8 T cells, percentage of Ki-67 positivity within total CD8, CD8 central memory T cells (Tcm) and CD8 effector memory T cells (TEM), NK cells, and percentage of Ki-67 positivity of NK cells). The results are shown in Figures 36A-36D and 37A-37B.

[0459] Figure 36A and 36B The graphs illustrate the two-week time course of CD8 cell number and percentage of Ki-67 positivity as a measure of proliferation in one male (dashed line) and one female (solid line) cynos following intravenous administration of 0.1 mg / kg of Conjugate 1. As can be seen, Conjugate 1 induces a significant increase in CD8 T cells in the cynos, with a 7-10X increase in cell number after a single dose.

[0460] Figure 36C and 36D Demonstrated is an increase in the number of cyno CD8 Tcm and CD8 Tem cells following a single injection of conjugate 1. CD8 Tcm and Tem numbers increased 27-30X and 21-33X, respectively.

[0461] Figure 37A and 37B is a graph of the number of NK cells and the percentage of Ki-67 positivity in cynos following a single 0.1 mg / kg dose of Conjugate 1. NK cells increased 9-10X following treatment with Conjugate 1.

[0462] Example 22

[0463] Comparison of in vitro activity of IL-15 and conjugate 1 in CD8 and CD56 NK cells in human PBMCs

[0464] In vitro activity of Conjugate 1 was assessed by studying NK and CD8 JAK / STAT signaling following treatment of human PBMCs with IL-15 or Conjugate 1 at a dose range of 0.001-10000 ng / ml. STAT5 phosphorylation was assessed as previously described.

[0465] Table 9.

[0466]

[0467] The results are shown in Figure 38A and 38BMiddle; These graphs are EC50 curves of IL-15 (●) versus Conjugate 1 (■) treatment of human PBMCs and subsequent measurement of the percentage of pSTAT5 positivity in CD8 and CD56 bright NK cells.

[0468] Results: Conjugate 1 was 5.5 and 15X less potent than IL- 15 at engaging CD8 and CD56 bright NK cells, respectively. Importantly, however, conjugate 1 achieved the same maximal response as regular IL-15.

[0469] Example 23

[0470] In vivo studies: single-dose PK studies in mice

[0471] Balb / c mice (n=3 / group) were administered a single intravenous dose of IL-15 (500 ug / kg) or Conjugate 1 (at 10, 30, 100, 300, and 1000 μg / kg). Blood samples were collected at designated time points after administration (Conjugate 1: 24, 48, 72, 96, 120, 144, 240 hours; IL-15 control: 0.03, 0.08, 0.25, 0.5, 1, 2, 4, 6, 8 hours) and the plasma concentration of the drug was determined. Figure 39 .

[0472] like Figure 39 As shown in , Conjugate 1 exhibited prolonged pharmacokinetics, i.e., measurable concentrations in plasma, with a half-life of approximately 14 hours, compared to plasma levels observed for non-long-acting IL-15, which was rapidly cleared.

[0473] Example 24

[0474] In vivo studies: Single-dose PK studies in rats

[0475] Sprague Dawley rats (n=3) were administered a single intravenous dose of Conjugate 1 at 10, 75, and 150 μg / kg. Plasma concentrations of the drug were determined at the indicated time points after injection (0.03, 0.08, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 72, 96, 120, 144 hours). Figure 40 .

[0476] like Figure 40 As shown in Figure 40 Compared to the plasma levels observed for the rapidly cleared, non-long-acting IL-15 shown in , Conjugate 1 exhibited sustained pharmacokinetics, i.e., measurable concentrations in plasma with a half-life of approximately 18 hours.

[0477] Example 25

[0478] In vivo studies: single-dose PK studies in non-human primates

[0479] A single intravenous dose of Conjugate 1 was administered to cynomolgus monkeys (n=2, 1 male and 1 female) at 10, 50, and 100 μg / kg. IL-15 was administered as a control at a single intravenous dose of 50 μg / kg. Plasma concentrations of the drug were determined at designated time points after injection (0.03, 0.25, 1, 4, 12, 24, 48, 72, 96, 120, 144, 168 hours). Figure 41 .

[0480] like Figure 41 As shown in , in contrast to the non-long-acting IL-15 which is rapidly cleared from plasma, Conjugate 1 exhibits sustained pharmacokinetics, ie, measurable concentrations in plasma with a half-life of approximately 30 hours for a 100 μg / kg dose.

[0481] Conjugate 1 achieved prolonged and sustained plasma exposure in multiple species (mice, rats, and cynomolgus monkeys) after a single dose (see Figures 39-41 ).

[0482] Example 26

[0483] In vivo studies: Single-dose PD studies in mice - cell number, proliferation, and engagement of JAK / STAT signaling

[0484] Balb / c mice (n=3 / group) were administered a single intravenous dose of vehicle (as described in Example 11) or Conjugate 1 (at a dose of 0.3 mg / kg or 0.03 mg / kg). Following administration, blood samples were collected at time points after administration (24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, 144 hours, 240 hours). The samples were immunophenotyped to obtain the number of CD4 T cells at the indicated time points (see Figure 42A ) and Ki-67% (see Figure 42B ).

[0485] CD4 T cells and their proliferation were defined by CD45+CD3+CD4+CD8- and CD45+CD3+CD4+CD8-Ki-67+ markers, respectively. Figure 42A is a curve of the number of CD4 T cells over time and Figure 42Bis a plot of CD4 T cell proliferation as measured by Ki-67 positivity over time. Compared to CD8 and NK cells, CD4 T cells are the least sensitive population to treatment with Conjugate 1, with relatively small increases in number and Ki-67 expression % observed 72-144 hours after dosing (see, e.g., Example 11). In mice, NK cells are more sensitive to stimulation of the proliferative response induced by a single dose of Conjugate 1 than either CD4 T cells or CD8 T cells.

[0486] STAT5 phosphorylation in CD4 T cells was determined by using the CD3+CD4+CD8-pSTAT5+ marker combination. Figure 43 Figure 2 is a graph of the percentage of pSTAT5 phosphorylation-positive CD4 T cells over time (0.25, 1, 6, 24, 48, 72, 96, and 120 hours after administration) at a dose of 0.03 mg / kg (blue, solid squares) or 0.3 mg / kg (orange, solid circles). Vehicle (black) and pre-dose (open circles) levels over time are also shown.

[0487] Results: Compared with CD8 and NK cells, CD4 T cells were the least sensitive population to treatment with Conjugate 1, with relatively small increases in pSTAT5 expression observed from 0.25 to 72 hours after administration. In mice, NK cells were more sensitive to stimulation with a single dose of Conjugate 1 in terms of proliferative responses compared with either CD8 or CD4 T cells.

[0488] Example 27

[0489] In vivo studies: Minimum effective dose studies in non-human primates (NHPs)

[0490] Cynomolgus monkeys (n=3-4 males) were administered a single intravenous injection of Conjugate 1 (at 0.003, 0.01, 0.1 mg / kg) or vehicle control. Blood samples were collected before and at designated time points after dosing (-5, -2, 1, 2, 3, 4, 5, 6, 7, 10, 14, 17 days) and analyzed by flow cytometry to examine the pharmacodynamic effects within lymphocyte populations. The cell counts of NK, CD8 T, and CD4 T cells were examined and the results are shown in Table 1. Figures 44A-44C The proliferation of NK cells, CD8 T cells, and CD4 T cells (Ki-67%) and JAK / STAT signaling (pSTAT5%) were examined, and the results are shown in Figures 45A-45C and Figures 46A-46C In. Check CD8 subsets (T 初始 、T em 、T cm and Tscm ) proliferation (Ki-67%), the results are shown in Figures 47A-47D middle.

[0491] In NHPs, after a single dose of conjugate 1, the number of NK (CD45+CD3-CD16+) cells increased significantly and in a dose-dependent manner. At the 0.1 and 0.01 mg / kg dose levels, the maximum cell number was observed five days after administration and lasted for up to 14 days. The lowest dose level that produced a significant increase in NK cells was 0.01 mg / kg. Confirming the observations on the number of NK cells, conjugate 1 also drove a dose-dependent and robust induction of Ki-67 expression, which reached a maximum approximately 3 to 4 days after treatment and could last for up to about 14 days. A significant increase in Ki-67% could be detected after the 0.001 mg / kg dose level. Conjugate 1 also robustly engaged the JAK / STAT signaling pathway in NK cells, with a dose-dependent increase in pSTAT5%, which could be detected at dose levels as low as 0.001 mg / kg.

[0492] Figure 44A 、 45A 46A and 46B are curves of NK cell number, Ki-67% and pSTAT5% over time after conjugate 1 treatment, respectively.

[0493] In NHPs, conjugate 1 induced a significant increase in total CD8 T cells (defined as CD45+CD3+CD4-CD8+), with maximum cell numbers reached approximately day 5 after treatment. This effect persisted for more than seven days, returning to baseline between 10 and 14 days after dosing. The effect of conjugate 1 on total CD8 cell numbers could be detected at 0.003 mg / kg. Supporting these findings, conjugate 1 induced a substantial Ki-67 positivity % in CD8 T cells, which could be detected at a low dose of 0.01 mg / kg. Conjugate 1 was also robust in engaging the JAK / STAT signaling pathway in CD8 T cells, with pSTAT5 increasing in a dose-dependent manner at 0.1 and 0.01 mg / kg dose levels.

[0494] Figure 44B 、 45B 46A and 46B are curves of CD8 cell number, Ki-67% and pSTAT5% over time after conjugate 1 treatment, respectively.

[0495] Compared to NK and CD8 T cells, Conjugate 1 had relatively little effect on total CD4 T cells (defined as CD45+CD3+CD4+CD8-) in NHPs. Conjugate 1, administered at the highest dose level of 0.1 mg / kg, induced a small increase in CD4 T cell number, Ki-67%, and pSTAT5%.

[0496] Figure 44C 、 45C and 46C are curves of CD4 T cell number, Ki-67% and pSTAT5 over time after Conjugate 1 treatment.

[0497] In NHPs, NK cells showed the greatest sensitivity to Conjugate 1 dose response compared to CD8 T cells or CD4 T cells in vivo.

[0498] In cynomolgus monkeys, CD8 naive and memory subsets were defined by CD45Ra, CD197, and CD95. Examination of the proliferation (Ki -67%) of CD8 T naive (CD45+CD3+CD4-CD8+CD45Ra+CD197+), CD8 Tscm (CD45+CD3+CD4-CD8+CD45Ra+CD197+CD95+), CD8 Tem (CD45+CD3+CD4-CD8+CD45Ra-CD197-), and CD8 Tcm (CD45+CD3+CD4-CD8+CD45Ra-CD197+) revealed that the CD8 memory subset had increased sensitivity to conjugate 1 compared to CD8 naive T cells. Conjugate 1 induced robust Ki-67 expression in a dose-dependent manner within the CD8 Tem, Tcm, and Tscm populations, with detectable increases in proliferation marker positivity beginning as early as day 2, reaching a maximum on day 5, and returning to baseline between days 10 and 14. Ki-67 expression and kinetics within the CD8 population support the findings of Examples 27 and Figures 47A-47D The sustained increase in the number of CD8 T cells was shown in Figure 3.

[0499] Figures 47A-47D CD8 T cells after conjugate 1 treatment 初始 、T scm 、T cm and T em Figure 3 Ki-67% of the population over time. As seen in the figure, in NHPs, the memory CD8 T cell population showed increased sensitivity to a single dose of Conjugate 1 compared to naive CD8 T cells in vivo.

[0500] Example 28

[0501] Induction of granzyme B or perforin by conjugate 1

[0502] After a single dose of conjugate 1, the expression of NK cell lytic enzymes, granzyme B and perforin was examined in cynomolgus monkeys. The expression of granzyme B and perforin was quantified by mean fluorescence intensity (MFI) in NK cells at 0.001 mg / kg, 0.01 mg / kg or 0.1 mg / kg dosage levels. At 0.01 and 0.1 mg / kg, conjugate 1 increased the MFI of granzyme B by approximately 3 times (peak value, relative to before administration). At 0.01 and 0.1 mg / kg, conjugate 1 also increased the MFI of perforin by approximately 2 times (peak value, relative to before administration). In summary, conjugate 1 not only induces the robust expansion of NK cells, but also enhances their function.

[0503] Figures 48A-48C is the curve of granzyme B at the following two times and Figures 49A-49C is a plot of perforin MFI at two times: before dosing (baseline) and at the time of peak levels after Conjugate 1 treatment (0.0001-0.1 mg / kg) in NHPs.

[0504] Conjugate 1 increased the protein levels of cytotoxic enzymes, such as constitutively expressed granzyme B and perforin, in NHP NK cells.

[0505] Sequence Listing

[0506] SEQ ID NO: 1 (rhIL-15)

[0507]

[0508] SEQ ID NO:2

[0509]

[0510] SEQ ID NO:3

[0511]

Claims

1. A composition comprising: A long-acting interleukin-15 receptor agonist compound or a pharmaceutically acceptable salt thereof, wherein the agonist compound has the following structure: Formula (I) Wherein IL-15 is the interleukin-15 part; The value of n corresponds to a weight-average molecular weight of polyethylene glycol of approximately 40,000 daltons; m is 3; n' is 1; and -NH- in the structure represents the amino group of the IL-15 moiety, and contains no more than about 15 mole % of a compound having the formula: Formula (II) wherein the variables n and m in formula (II) are as defined for formula (I), wherein the average number of polyethylene glycol moieties covalently attached to the amino groups of IL-15 in the composition is in the range of 1.0 to about 1.

3.

2. The composition of claim 1, wherein the IL-15 portion has an amino acid sequence selected from any one of SEQ ID NOs: 1-3.

3. The composition of claim 1, which is effective to stimulate natural killer cell activation and / or proliferation when administered to a subject at a therapeutically effective dose.

4. The composition of claim 1, which is effective in supporting CD8 T cell survival and / or memory formation when administered to a subject at a therapeutically effective dose.

5. The composition of claim 1 comprising no more than about 10 mole % of a long-acting IL-15 receptor agonist encompassed by formula (II) when considered together.

6. The composition of claim 1 comprising, when considered together, no more than about 7 mole % of a long-acting IL-15 receptor agonist encompassed by formula (II).

7. The composition of claim 1 comprising no more than about 5 mole % of a long-acting IL-15 receptor agonist encompassed by formula (II) when considered together.

8. The composition of claim 1, wherein the average number of polyethylene glycol moieties covalently attached to the amino groups of IL-15 in the composition is selected from the group consisting of 1.0, 1.1, 1.2, and about 1.

3.

9. The composition of claim 1, which exhibits a reduced EC50 value for CTLL-2 pSTAT5 in ng / mL when compared to the EC50 value of unmodified IL-15 for CTLL-2 pSTAT5 in ng / mL.

10. The composition of claim 1, which exhibits an EC50 value in ng / mL for CTLL-2 pSTAT5 that is no less than about 6.5-fold reduced when compared to the EC50 value in ng / mL of unmodified IL-15 for CTLL-2 pSTAT5.

11. The composition of claim 1 having a receptor alpha binding value K of 0.05 in pM for unmodified IL-15. D When compared to the receptor α binding value K in pM, D The reduction does not exceed approximately 50%.

12. The composition of claim 11, having a receptor alpha binding value K in pM for unmodified IL-15. D When compared to the receptor α binding value K in pM, D The reduction does not exceed approximately 45%.

13. The composition of claim 1, which exhibits an EC50 value for CTLL-2 pSTAT5 in ng / mL that is no less than about 7-fold less than the EC50 value for CTLL-2 pSTAT5 in ng / mL of unmodified IL-15, and a receptor alpha binding value K of 1% in pM that is less than the EC50 value for CTLL-2 pSTAT5 in ng / mL of unmodified IL-15. D When compared to the receptor α binding value K in pM, D The reduction does not exceed approximately 50%.

14. The composition of claim 1, wherein the compound contained in the composition is less than about 35% deamidated.

15. The composition of claim 1 comprising a mixture of positional isomers of the compound according to formula (I).

16. The composition of claim 15, wherein for the mixture of positional isomers of formula (I), the amino group is predominantly located at the N-terminus of the IL-15 moiety.

17. The composition of claim 1, wherein the IL-15 portion according to formula (I) is not glycosylated.

18. The composition of claim 1 formulated for parenteral administration.

19. The composition of claim 1, further comprising a pharmaceutically acceptable excipient.

20. The composition of claim 19, wherein the pharmaceutically acceptable excipient is a buffer.

21. The composition of claim 20, wherein the buffer is potassium phosphate.

22. The composition of claim 19, wherein the pharmaceutically acceptable excipient is a carbohydrate.

23. The composition of claim 22, wherein the carbohydrate is trehalose.

24. The composition of claim 19, wherein the pharmaceutically acceptable excipient is a surfactant.

25. The composition of claim 24, wherein the surfactant is polysorbate 20.

26. The composition of claim 1, wherein the composition is contained in an aqueous medium at a pH of about 6.5 to 7.

0.

27. The composition of claim 26, wherein the composition is contained in an aqueous medium at a pH of about 6.

8.

28. The composition of claim 19, which is effective to stimulate natural killer cell activation and / or proliferation when administered to a mammalian subject in a therapeutically effective dose.

29. The composition of claim 19, which is effective in supporting CD8 T cell survival and memory formation when administered to a mammalian subject at a therapeutically effective dose.

30. The composition of claim 19, formulated for intravenous administration.

31. The composition of claim 19, comprising a long-acting IL-15 receptor agonist at a dose of 0.25-25 micrograms / kg.

32. The composition of claim 31, wherein the dosage of the long-acting interleukin-15 receptor agonist is 1-10 micrograms / kg.

33. Use of the composition of any one of claims 1 to 32 in the preparation of a medicament for treating cancer, wherein the cancer is selected from the group consisting of head and neck cancer, colon cancer, adenocarcinoma, neuroblastoma, glioma, lymphoma and myeloma.

34. The use of claim 33, wherein the cancer is lymphoma.

35. The use of claim 33, wherein the cancer is myeloma.

36. A method for preparing a water-soluble polyethylene glycol polymer conjugate of IL-15, wherein the water-soluble polyethylene glycol polymer conjugate of IL-15 has the following structure: Formula (I) IL-15 is the interleukin-15 part; The value of n corresponds to a weight-average molecular weight of polyethylene glycol of approximately 40,000 daltons; m is 3; n' is 1; and -NH- in the structure represents the amino group of the IL-15 moiety, The method comprises contacting the IL-15 moiety with a linear unsubstituted polyethylene glycol polymer reagent functionalized with a succinimidyl group or other activated ester group in an aqueous medium at a pH of 7.0 to 9.0 under conjugation conditions.

37. The method of claim 36, wherein the polyethylene glycol polymer reagent is a methoxy PEG-succinimidyl alkanoate reagent.

38. The method of claim 37, wherein the polyethylene glycol polymer reagent is selected from the group consisting of: , wherein n is an integer from about 150 to about 3,000.

39. The method of claim 37, wherein the methoxy PEG-succinimidyl alkanoate reagent is methoxy PEG succinimidyl butyrate.

40. The method of claim 37, wherein the methoxy PEG-succinimidyl alkanoate reagent is added in an equimolar amount to the interleukin-15 moiety.

41. The method of claim 37, wherein the methoxy PEG-succinimidyl alkanoate reagent is added in a molar excess relative to the interleukin-15 moiety.

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