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 to U.S. Provisional Patent Application No. 62/536,966, filed July 25, 2017; and to U.S. Provisional Patent Application No. 62/582,186, filed November 6, 2017; and to U.S. Provisional Patent Application No. 62/648,240, filed March 26, 2018, the disclosures of which are incorporated herein by reference in their entireties..

FIELD

[0002] The instant disclosure is directed to (among other things) a long acting interleukin-15 ("IL-15") receptor agonist, related compositions and methods of preparation and use, for example, in the treatment of conditions responsive to therapy effective to provide, for example, sustained immune activation and anti-tumor activity.

BACKGROUND

[0003] Interleukin-15 ("IL-15") is a pleiotropic cytokine that was first reported by

Grabstein et al. (Grabstein et al. (1994) Science 264:965-968). Secreted as a 162-amino acid precursor, human IL-15 contains a 29-amino acid leader sequence and a 19-amino acid pro sequence; the mature protein is therefore 114 amino acids in length. Belonging to the four a-helix bundle family of cytokines, IL-15 binds to a heterotrimeric receptor, wherein a unique a subunit (IL-15Ra) confers receptor specificity to IL-15, and the β and γ subunits of this receptor share commonality with one or more other cytokine receptors. Giri et al. (1995) EMBO J.

14:3654-3663.

[0004] As a cytokine, IL-15 has effects on both the innate immune system and the adaptive immune system (DiSabitino et al. (2011) Cytokine Growth Factor Rev. 22: 19-33). With respect to the innate immune system (which defends the host from foreign invaders generically), IL-15 causes the development of and maintains the survival of natural killer cells ("NK cells") and natural killer-T cells ("NK-T cells"), in addition to having other properties. Consistent with their role in the innate immune system, K cells do not specifically attack the invading pathogen, rather, these cells destroy compromised host cells (such as tumor cells or virus-infected cells). NK-T cells generate immunomodulatory cytokines, particularly interferon-γ, which result in a general activation of the immune response.

[0005] With respect to the adaptive immune system (which defends the host from a specific foreign invader following an initial encounter with that particular pathogen), IL-15 is necessary for the maintenance of the immunomodulatory cytokine-generating helper T cells. Importantly, IL-15 also supports the long-term maintenance of "antigen-experienced" memory T cells, which have the ability to rapidly reproduce, thereby generating a faster and stronger immune response upon re-exposure to the particular foreign pathogen invading the host.

[0006] Finally, notwithstanding its specific roles within both the innate and adaptive immune systems, IL-15 has significant and broad effects across both categories of immune systems. In particular, IL-15 inhibits or reduces apoptosis (or cell death) of a number of cells types (including dendritic cells, neutrophils, eosinophils, mast cells, CD4+ T cells, and B cells) associated within both categories of immune systems.

[0007] Because it stimulates the proliferation and maintenance of many cells within the immune system that can fight against cells that appear to the host as foreign (or "non-self), IL-15 has been proposed for use in the treatments of individuals suffering from cancer (Steel et al. (2012) Trends Pharmacol. Sci. 33(1):35-41). For example, an IL-15-based agonist has been proposed to treat myelomas (Wong et al. (2013) Oncolmmunology 2(11), e26442: 1-3). In addition, IL-15 pharmacotherapy has been proposed for treating individuals suffering from viral infections, such as HIV infection.

[0008] Despite its potential for use in the treatment of individuals suffering from a number of diseases, IL-15-based therapies face a number of challenges. For example, IL-15 is rapidly cleared from plasma and is relatively unstable under physiological conditions. Moreover, in vivo signaling activity of IL-15 is similarly short-lived, and the molecule unfavorably requires daily dosing or multi-day continuous infusion for optimal activity. Certain approaches attempt to overcome these limitations by complexing IL-15 with the IL-15 receptor alpha subunit. Such an approach, however, may abrogate the desirable signaling that occurs uniquely through the IL-15 receptor alpha, expressed on multiple cell types. A non-releasable PEGylation with a

succinimidyl carbonate-terminated polymer of relatively small molecular weight (5kDa) has

been reported, but this resulted in significant alteration of IL-15's biological activity. Pettit et al. (1997) J: Biol Chem. 272(41:2312-2318.

[0009] Notwithstanding the foregoing approaches, however, there remains a need for new IL-15 receptor agonists having improved characteristics and profiles, such as, for example, potent immune stimulatory effects, low systemic toxicity, stability and/or improved

pharmacokinetics. Thus, among other things, the instant disclosure provides a long-acting IL-15 receptor agonist having a number of advantageous features to be described in greater detail below, as well as compositions and kits comprising such agonist, as well as related methods of preparation and use, as described herein, which are believed to be new and completely unsuggested by the art.

SUMMARY

[0010] In a first aspect, provided herein is a long acting IL-15 receptor agonist including pharmaceutically acceptable salt forms thereof. The long acting IL-15 receptor (IL-15 R) agonist comprises at least a single linear PEG (polyethylene glycol) moiety stably covalently attached to an IL-15 amino group via an amide linkage. Intervening between the linear PEG strand and the stable amide linkage to an IL-15 amino group may be a linear unsubstituted alkylene group (~CH2~)m having from 2 to 5 carbon atoms (i.e., m=2, 3, 4, or 5).

[0011] For example, in some embodiments, the unsubstituted alkylene group is (~CH2~)2; or, in some additional embodiments, the unsubstituted alkylene group is (~CH2~)3; in yet some further embodiments, the unsubstituted alkylene group is (~CH2~)4; in yet some further embodiments, the unsubstituted alkylene group is (~CH2~)5.

[0012] For example, in some embodiments, the long acting IL-15 receptor agonist has the following structure:

Formula (I)

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) may also be depicted as [CFLO-(CH2CH20)n(CH2)mC(0)-NH-]n'-IL15, and the two formulae may be used interchangeably. In

Formula I (and in similar formulae provided herein) the -NH- in the structure represents an amino group of the IL-15 moiety.

[0013] In some further 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.

[0014] In yet one or more additional embodiments, m is 2 or 3, such that the linear alkylene group separating the PEG moiety from the stable amide linkage to IL-15 is either ~(CH2)2~ or (CH2)3~. In some preferred embodiments, m is 3.

[0015] In one or more embodiments, n is an integer having a value that corresponds to a polyethylene glycol polymer having a weight average molecular weight selected from the group consisting of 10,000 daltons (e.g., n is -227), 15,000 daltons (e.g., n is -340), 20,000 daltons (e.g., n is -454), 25,000 daltons (e.g., n is -568), 30,000 daltons (e.g., n is -681), 40,000 daltons (e.g., n is -909), 50,000 daltons (e.g., n is -1136) and 60,000 daltons (e.g., n is -1364).

[0016] In one or more illustrative embodiments, provided is a composition comprising a long acting IL-15 receptor agonist in accordance with Formula (I), including without limitation each and every one of its related embodiments as provided herein.

[0017] In some embodiments, the long acting IL-15 receptor agonist composition comprises no more than about 15 mole percent of long-acting IL-15 receptor agonists, when considered collectively, encompassed by the formula:

O

THE 15

CH3-(OCH2CH2)nO-(CH2)mC

2,3 or greater than 3

Formula (II),

where the values of n and m are as provided for Formula (I) above. That is to say, in terms of the long-acting IL-15 receptor agonist component of such compositions, no more than about 15 mol percent of long-acting IL-15 receptor agonists comprised in the composition are of Formula (II).

[0018] For example, in some embodiments, the long acting IL-15 receptor agonist composition comprises no more than about 10 mole percent of long-acting IL-15 receptor agonists, that when considered collectively, are encompassed by Formula (II).

[0019] In some additional embodiments of the foregoing, the composition comprises no more than about 7 mole percent of long acting IL-15 receptor agonists having n' equal to 2, 3, or greater than 3 (i.e., higher PEGmers, also referred to as "high-mers"). In yet some other embodiments, the composition comprises no more than about 5 mole percent, 6 mole percent, 9 mole percent or 10 mole percent of long acting IL-15 receptor agonists having n' equal to 2, 3 or greater than 3 (i.e., of 2 or greater).

[0020] In some further embodiments, the composition comprises a long-acting IL-15 receptor agonist according to Formula (I),

5

[0021]

where n and m are as described above, and n' represents the average number of polyethylene glycol moieties covalently attached to IL-15 amino groups (for the composition), and n' for the composition is in a range 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.

[0022] In yet another aspect, provided herein is a method of preparing a long acting interleukin-15 receptor agonist such as described in Formula (I), e.g., Formulae (la), (lb), (Ic), (Id), and in Formula (II), e.g., (Ila), (lib), (lie), and (lid). In the method, interleukin-15 (generally dissolved in a buffer such as phosphate buffered saline or any other suitable buffer, at a pH of around 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 a methoxyPEG-succinimidyl alkanoate (where n is an integer from about 150 to about 3,000) according to the following structure:

for a period of time sufficient to form,

O

CH 3 0 (CH 2 CH 2 0) n (CH 2 ) m C 1 IL-15

n , where n' is 1. Exemplary methoxyPEG-succinimidyl alkanoate reagents for reacting with interleukin-15 include the following:

[0023] In some preferred embodiments, the methoxyPEG-succinimidyl alkanoate

CH30(CH2CH20)n(CH2)

is
t mPEG-succinimidyl butanoate.

[0024] In some embodiments, the methoxyPEG-succinimidyl alkanoate reagent has a weight average molecular weight selected from the group consisting of about 10,000 daltons (e.g., n is -227), about 15,000 daltons (e.g., n is -340), about 20,000 daltons (e.g., n is -454), about 25,000 daltons (e.g., n is -568), about 30,000 daltons (e.g., n is -681), about 40,000 daltons (e.g., n is -909), about 50,000 daltons (e.g., n is -1136) and about 60,000 daltons (e.g., n is -1364).

[0025] In one or more embodiments of the method, the methoxyPEG-succinimidyl alkanoate reagent is added in an equimolar amount (i.e., equimolar ratio) to interleukin-15.

[0026] In one or more alternative embodiments, the methoxyPEG-succinimidyl alkanoate reagent is added in a molar excess of interleukin-15. In some particular embodiments, the methoxyPEG-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 ten-fold molar excess, or even a 12-fold molar excess or more. In some embodiments, the methoxyPEG-succinimidyl alkanoate reagent is added at a 5 to 10-fold molar excess.

[0027] In some embodiments, the methoxyPEG-succinimidyl alkanoate reagent is added as a solid.

[0028] In some other embodiments, the methoxyPEG-succinimidyl alkanoate reagent is dissolved in a suitable solvent. In a particular embodiment, the methoxyPEG-succinimidyl alkanoate reagent is dissolved in an aqueous acid, such as, for example, dilute hydrochloric acid, although any suitable acid may be employed.

[0029] In some further embodiments of the method, the interleukin-15 is initially present in solution, i.e., prior to mixing with the methoxyPEG-succinimidyl alkanoate reagent, 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.

[0030] In some further embodiments, the pH of the interleukin-15 solution is adjusted to about 8.0 prior to addition of the methoxyPEG-succinimidyl alkanoate reagent.

[0031] In some further embodiments, the pH of the reaction mixture is adjusted to about

8.0 following addition of the methoxyPEG-succinimidyl alkanoate reagent.

[0032] In some further embodiments, the resulting reaction mixture is stirred (or mixed) for a period of time sufficient to allow reaction between the reactants. In some embodiments, the reactants are mixed for up and including from about 15 minutes to about 10 hours. In some further embodiments, the reactants are mixed from about 30 minutes to about 5 hours, or from about 30 minutes to about 2 hours.

[0033] In some embodiments, the reaction is carried out under ambient conditions, e.g., at room temperature, i.e., absent the addition of heat. Illustrative temperatures 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 further embodiments, the reaction is carried out at a temperature from about 20 0 C to about 25° C.

[0034] In some embodiments of the method, the reaction is quenched by addition of an amino acid. In some related embodiments, the reaction is quenched by addition of glycine.

[0035] In some further embodiments of the method, conjugation products, i.e., methoxyPEG-alkanoate-interleukin-15 conjugates, are separated from the reaction mixture.

[0036] In some additional embodiments, the reaction mixture comprising methoxyPEG-alkanoate-interleukin-1 conjugates is purified.

[0037] In some particular embodiments, the reaction results in formation of

where n' is 1.

[0038] In some further embodiments, the reaction is effective to form a composition comprising no more than about 15 mole percent (mol%) of long-acting IL-15 receptor agonists (of the IL-15 containing molecules in the composition), when considered collectively, encompassed by the formula:

0

I L-1 5

CH3-(OCH2CH2)NO-(CH2)MC— NH- 2,3 or greater than 3

Formula (II),

where the values of n and m are as provided for Formula (I) above.

[0039] In yet some additional embodiments, the reaction is effective to product

PEGylated interleukin-15 that is less than 20-35% or less than about 25% deami dated.

[0040] In some embodiments, the long-acting IL-15 receptor agonist exhibits no more than about a 7-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5) when compared to unmodified (i.e., 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 reduction in EC50 value (ng/mL, CTLL-2 pSTAT5), or no more than about a 6-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5), or no more than about a 5.5-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5), or no more than about a 5-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5), or no more than about a 4.5-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5), or no more than about a 4-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5), or no more than about a 3.5-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5), or even no more than about a 3-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5) when compared to IL-15. [0041] In some further embodiments, the long-acting IL- 1 receptor agonist exhibits no more than about a 50% reduction in receptor alpha binding (KD, pM) when compared to unconjugated IL-15, e.g., when measured using a technique suitable for determining receptor alpha binding, such as, for example surface plasmon resonance (SPR). In some related embodiments, the long-acting IL-15 receptor agonist exhibits no more than about a 45% reduction in receptor alpha binding (KD, pM), or exhibits no more than about a 40% reduction in

receptor alpha binding (KD, pM), or exhibits no more than about a 35% reduction in receptor alpha binding (KD, pM), or even exhibits no more than about a 30% reduction in receptor alpha binding (KD, pM) when compared to unconjugated IL-15.

[0042] In yet some further embodiments, the long-acting IL-15 receptor agonist exhibits no more than about a 7-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5) when compared to unmodified IL-15 and no more than about a 50% reduction in receptor alpha binding (KD, pM) when compared to IL-15, including any one or more particular combinations of reductions in EC50 values or KD values described above.

[0043] In yet one or more embodiments, provided is a composition comprising a long acting IL-15 R agonist as described herein, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

[0044] In yet some further embodiments, the long acting IL-15 R agonist or composition is effective when administered at a therapeutically effective dose to a subject to stimulate NK activation and/or proliferation.

[0045] In yet one or more further embodiments, the long acting IL-15 R agonist or composition is effective when administered at a therapeutically effective dose to a subject to support CD8 T-cell survival and/or memory formation.

[0046] In another aspect, provided herein is a method of treating a condition that is responsive to treatment with IL-1 by administering to a subject having the condition a therapeutically effective dose of a long-acting IL-15 R agonist, or a composition comprising such agonist, as provided herein.

[0047] In yet a further aspect, provided is a method for treating a cancer by administering to a subject having cancer a therapeutically effective dose of a long-acting IL-15 R agonist or composition as provided herein.

[0048] Additional aspects and embodiments are set forth in the following description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] FIG. 1 provides the amino acid sequence of an exemplary recombinant human

IL-15 from E. coli (SEQ ID NO: 1), a single, non-glycosylated polypeptide chain containing 115 amino acids, with a molecular weight of 12.9 kDa.

[0050] FIG. 2 is a chromatogram illustrating the RP-HPLC analysis of an exemplary conjugation reaction mixture as described in Example 1.

[0051] FIG. 3 is an FPLC purification profile from an anion-exchange chromatography column as described in Example 1.

[0052] FIG. 4 is an SDS-PAGE of an exemplary purified long-acting IL-15 receptor agonist, mono-mPEG-butanamide-IL-15, as described in Example 1. Lane 1 provides molecular weight markers as indicated; Lane 2 is the unconjugated parent molecule, IL-15, and Lane 3 is mono-mPEG-butanamide-IL- 15.

[0053] FIG. 5 is a RP-HPLC analysis of purified mono-mPEG-butanamide-IL-15, as described in Example 1.

[0054] FIG. 6. is a plot of plasma concentration of test article (IL-15, solid circle, or mPEG2-CAC-FMOC-20K-NHS-IL-15, also referred to as N-(2-methoxyPEG-ethyl)-7-(4-((2-methoxyPEG-ethyl)amino)-4-oxobutyl)-9-ethyl-9 H-fluorene-4-carboxamide carbamate-IL-15, or Conjugate 2, solid square) over time following administration of a single intravenous dose of test article in mice as described in Example 6.

[0055] FIG. 7 is a plot of mean plasma concentration of mPEG2-CAC-FMOC-20K- NHS-IL-15, also referred to as N-(2-methoxyPEG-ethyl)-7-(4-((2-methoxyPEG-ethyl)amino)-4-oxobutyl)-9-ethyl-9 H-fluorene-4-carboxamide carbamate-IL-15, or Conjugate 2, over time following administration of a single intravenous dose of Conjugate 2 in rats at dosage amounts of 0.3 (■), 0.15 (A) and 0.075 mg/kg (·), or a single subcutaneous dose of Conjugate 2 at 0.15 mg/kg (♦) as described in Example 7.

[0056] FIGS. 8A and 8B illustrate the degree of STAT5 phosphorylation in various lymphocytes, i.e., CD4 (■), CD8 (A), and NK cells (T) following administration of a single i.v. dose of either IL-15 (FIG. 8A) or Conjugate 2 (0.3 mg/kg, FIG. 8B) as described in Example 8.

[0057] FIGS. 9A, 9B and 9C are plots demonstrating the degree of STAT5

phosphorylation in various lymphocytes, i.e., CD4, CD8, and NK cells, respectively, following administration of a single i.v. dose of Conjugate 2 (0.5 mg/kg) in cynomolgus monkeys, as described in Example 9.

[0058] FIGS. 10A and 10B are plots demonstrating in-vitro activity of exemplary long acting IL-15 receptor agonists (Conjugates 1, 3 and 5) as measured by signaling in NK subsets of human PBMCs, CD56bright (FIG. 10A) and CD56dim (FIG. 10B) NK cells, respectively, as described in detail in Example 10.

[0059] FIGS. 11A and 11B are plots illustrating NK cell proliferation in mice following i.v. administration of mono-mPEG-SBA40K-IL-15 (also referred to herein as Conjugate 1) at doses of 0.03 mg/kg (low dose, open squares), 0.3 mg/kg (medium dose, solid circles, solid line), or 1 mg/kg (high dose, diamonds) when compared to vehicle as described in Example 11. FIG. 11 A illustrates Ki67 expression (expressed as a percentage) over time, while FIG. 1 IB illustrates NK cell counts (cells/ul) versus time post administration for each of the sample groups.

[0060] FIGS. 12A-D are plots illustrating increasing numbers of NK cells of all maturation levels in mice following i.v. administration of Conjugate 1 at doses of 0.03 mg/kg (low dose, open squares), 0.3 mg/kg (medium dose, solid circles, solid line), or 1.0 mg/kg (high dose, diamonds) compared to vehicle (solid circles, dashed line) as described in Example 11. Subsets of NK cells were defined by CD1 lb and CD27 expression. FIG. 12A demonstrates increases in numbers of terminal effector cells, expressed in cells/μΕ from 24 hours to 120 hours post administration; FIG. 12B demonstrates increases in numbers of pre-NK cells, expressed in cells/ μΕ from 24 hours to 120 hours post administration; FIG. 12C demonstrates increases in numbers of high effector cells, expressed in cells^L from 24 hours to 120 hours post administration and FIG. 12D demonstrates increases in numbers of early NK cells, expressed in cells/ μΕ from 24 hours to 120 hours post administration.

[0061] FIGS. 13A and 13B are plots illustrating levels of expression of NKG2D (FIG.

13A) and Granzyme B (FIG. 13B) by NK cells in mice following i.v. administration of

Conjugate 1 at doses of 0.03 mg/kg (low dose, open squares), 0.3 mg/kg (medium dose, solid circles, solid line), or 1 mg/kg (high dose, diamonds) compared to vehicle (solid circles, dashed line) as described in Example 11.

[0062] FIG. 14 is a plot illustrating numbers of CD8 T cells, expressed in cells/μΐ, both pre-dose and from 24 hours to 120 hours post administration following i.v. administration of Conjugate 1 in mice at doses of 0.03 mg/kg (low dose), 0.3 mg/kg (medium dose), or 1 mg/kg (high dose) as described in Example 11.

[0063] FIGS. 15A and 15B are plots illustrating illustrates levels of Ki67 expression

(expressed as a percentage) over time for T effector memory cells and T central memory cells, respectively, versus time post administration following i.v. administration of Conjugate 1 in mice at doses of 0.3 mg/kg or 1.0 mg/kg in comparison to vehicle and IL-15 as described in Example 11.

[0064] FIGS. 16A and 16B are plots illustrating NK cell proliferation in cynomolgus monkeys following i.v. administration of Conjugate 2 at a dose of 500 μg/kg as described in Example 12. FIG. 16A illustrates Ki67 expression (expressed as a percentage) in NK cells from pre-dose to 15 days post-administration, while FIG. 16B illustrates NK cell counts from pre-dose to 15 days post-administration.

[0065] FIG. 17 is a plot illustrating CD8 T-cell counts in cynomolgus monkeys (shown for each animal) following i.v. administration of Conjugate 2 at a dose of 500 μg/kg as described in Example 12 from pre-dose to 14 days post-administration.

[0066] FIGS. 18 A and 18B are plots illustrating numbers of CD8 T effector memory cells (TEM cells) and CD8 T central memory cells (TCM), respectively, in cynomolgus monkeys versus time post administration (from pre-dose to 14 days post-administration) following i.v. administration of Conjugate 2 at a dose of 500 μg/kg as described in Example 12.

[0067] FIG. 19 illustrates total lesions per mouse in the lungs of female Balb/c mice inoculated with mouse CT-26 colon cancer cells, followed by treatment with one of the following test articles: vehicle, phosphate buffered saline (Group A); native 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) as described in detail in Example 13.

[0068] FIG. 20 illustrates total lesions per mouse in the lungs of female Balb/c mice inoculated with mouse CT-26 colon cancer cells, followed by 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, as described in detail in Example 13.

[0069] FIG. 21 illustrates the percent specific lysis as a function of E:T (effectortarget) ratio at 1000 ng/mL (squares), 3000 ng/mL (circles), 300 ng/mL (triangles), 30 ng/mL (upper Xs), 3 ng/mL (diamonds), and unstimulated (lower Xs), as described in Example 14. This data illustrates dose-dependent increased cytotoxicity of NK cells in vitro following culturing with Conjugate 1.

[0070] FIGS. 22A-D are plots illustrating increasing numbers of NK cells of all maturation levels in mice following i.v. 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. FIG. 22A demonstrates increases in numbers of terminal effector cells, expressed in cells^L from 24 hours to 120 hours post administration; FIG. 22B demonstrates increases in numbers of pre-NK cells, expressed in cells^L from 24 hours to 120 hours post administration; FIG. 22C

demonstrates increases in numbers of high effector cells, expressed in cells^L from 24 hours to 120 hours post administration and FIG. 22D demonstrates increases in numbers of early NK cells, expressed in cells^L from 24 hours to 120 hours post administration.

[0071] FIG. 23 is a plot illustrating Granzyme B expression as a function of 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 indicates treatment with

Conjugate 1 increases NK-cell Granzyme B expression.

[0072] FIG. 24 is a plot illustrating numbers of CD8 T cells isolated from mouse spleen, expressed in cells/μΐ, both pre-dose and from 24 hours to 96 hours post administration following

1. v. administration of Conjugate 1 in mice at doses of 0.03 mg/kg (filled circles, dashed line) and 0.3 mg/kg (filled circles, solid line), with vehicle IL-15 buffer (open circles, dashed line) as described in Example 11.

[0073] FIG. 25 is a plot illustrating levels of Ki67 expression (expressed as a percentage) for CD49b cells versus time post administration following i.v. administration of Conjugate 1 in mice at doses of 0.03 mg/kg or 0.3 mg/kg in comparison to vehicle IL-15 buffer, as described in Example 11.

[0074] FIG. 26 is a plot illustrating levels of Granzyme B expression (expressed as a percentage) over time for CD49b cells versus time post administration following i.v.

administration of Conjugate 1 in mice at doses of 0.03 mg/kg or 0.3 mg/kg in comparison to vehicle IL-15 buffer, as described in Example 11.

[0075] FIG. 27 is a plot illustrating in vivo cytotoxicity results after treatment with

Conjugate 1 at 0.3 mg/kg, as described in Example 14. Cytotoxicity was assessed at 24 hours, 48 hours, and 72 hours following treatment.

[0076] FIGS. 28A-D are plots illustrating increasing numbers of NK cells of all maturation levels in mice following i.v. administration of Conjugate 1 at doses of 0.03 mg/kg and 0.3 mg/kg on a single dose or following the third dose in a q7dx3 schedule, as described in Example 11. FIG. 28 A demonstrates increases in percentage of Ki67 CD49b cells, from 24 hours to 240 hours post administration; FIG. 22B demonstrates increases in percentage of Granzyme B CD49b cells, from 24 hours to 240 hours post administration; FIG. 28C

demonstrates increases in number of CD49b cells, expressed in cells/ μΕ from 24 hours to 240 hours post administration; and FIG. 28D demonstrates increases in percentage of granB+ MFI CD49b cells, from 24 hours to 240 hours post administration.

[0077] FIGS. 29A-C are plots related to the study described in Example 16. FIG. 29A is a plot of plasma concentration of test article (IL-15 or Conjugate 1) during a 144 hour time course following administration of a single intravenous dose of test articles in balb/c mice at 0.5 and 0.3 mg/kg, respectively. Conjugate 1 exhibits a half-life of approximately 12 hours, whereas IL-15 is quickly cleared from the plasma with a half-life of less than 1 hour. FIG. 29B is a graph of pSTAT5 percent positivity within CD8 T cells in mice after a single injection of Conjugate 1 at 0.03 and 0.3 mg/kg. Conjugate 1 at both dose levels induces sustained pSTAT5 signaling in CD8 T cells. A 120 hour time course, including pre-dose, are shown. FIG. 29C is a graph of pSTAT5 percent positivity within murine NK cells after a single injection of Conjugate 1 at 0.03 and 0.3 mg/kg. Conjugate 1 at both dose levels induces robust and sustained pSTAT5 signaling in NK cells.

[0078] FIGS. 30A-C are plots of total CD8, CD8 central memory (Tcm) and CD8 effector memory (Tem) cell numbers, respectively, 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. Conjugate 1 at dose levels equal to or greater than 0.03 induce a significant increase in total CD8 T cells in the blood as described in Example 17. 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, respectively. Notably, CD8 and CD8 memory T cell numbers did not return to baseline at 240 hours post injection when Conjugate 1 was dosed at 0.3-1.5 mg/kg, demonstrating the sustained PD effects of Conjugate 1.

[0079] FIGs. 30D, 30E and 30F are plots of Ki-67 percent positivity within total CD8,

CD8 Tcm and CD8 Tem populations, respectively, in mice, as described in Example 17. A

single dose of Conjugate 1, at all dose levels, increases Ki-67 positivity in all CD8 and CD8 subpopulations.

[0080] FIGS. 31A, 31B and 31C are plots of CD8 and CD8 memory subpopulation T cell numbers after Conjugate 1 was dosed singly (dotted lines) or Q7dx3 (solid lines) at 0.03 and 0.3 mg/kg as described in Example 17. Repeated dosing increased these populations further with CD8, CD8 Tcm, CD 8 Tern increases of 35.3X, 183X and 73.8X, respectively. At the end of the time course (240 hours after first or last dose at 0.3 mg/kg), cell numbers had not returned to baseline in mice.

[0081] FIGS. 32A and 32B are plots of NK cell numbers and Ki-67 percent positivity after a single dose of Conjugate 1 from 0.01 to 1.5 mg/kg in mice as described in Example 17. NK cell numbers increase significantly above vehicle control at all dose levels and return back to baseline by 240 hours post dose. All dose levels induce a robust increase of Ki-67 percent positivity in NK cells.

[0082] FIG. 32C is a plot of murine NK cell numbers after single (solid lines) or Q7dx3

(dashed lines) dosing of Conjugate 1 at 0.03 and 0.3 mg/kg as described in Example 17.

Repeated dosing of Conjugate 1 at 0.3 mg/kg induced slightly less, although still significant, numbers of NK cells compared to a single dose. Similar NK numbers were achieved with single versus repeat dosing at 0.03 mg/kg.

[0083] FIG. 33A illustrates an in vitro NK cytotoxicity assay that measures changes in

NK-mediated target cell lysis after test article treatment in mice as described in Example 18. A time course at the indicated hours of percent 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 lmg/kg of EL-15 is shown. Splenic NK cells from mice dosed with vehicle served as a control. Conjugate 1 dosed at 0.3 mg/kg induced an elevation of NK cytotoxicity superior in magnitude and duration compared to NK cells from mice receiving a single injection of IL-15 at 1 mg/kg.

[0084] FIG. 33B is a graph of Granzyme B percent positivity in blood NK cells from the same mice devoted to the NK in vitro cytotoxicity assay in FIG. 33A. See Example 18.

Conjugate 1 dosed at 0.03 and 0.3 mg/kg induced significant increases in NK Granzyme B expression, with a robust and sustained elevation seen at 0.3 mg/kg.

[0085] FIGS. 34A and 34B illustrate the percent lung nodule inhibition in balb/c mice receiving intravenous CT-26 tumor cell injection followed by Conjugate 1 treatment dosed

twice, one week apart, at 0.03 or 0.3 mg/kg as described in Example 19. Conjugate 1 injection 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 for 32 days post tumor cell injection to assess survival. Treatment with Conjugate 1 increased survival significantly compared to tumor-injected mice receiving vehicle control.

[0086] FIG. 35 is a graph of percent lung nodule inhibition in CT-26-injected mice treated with Conjugate 2, that received antibody-mediated depletion of NK cells (olive green), IgG control (blue), or PBS (Orange) as described in Example 20. Data is represented as percent lung nodule inhibition relative to CT-26-injected mice that were not NK cell depleted and treated with a vehicle control (black). Conjugate 2 efficacy in this tumor model was abolished when mice lacked NK cells.

[0087] FIGS. 36A and 36B are plots illustrating a two week time course for CD8 cell numbers and Ki-67 percent positivity as a measure of proliferation of one male (dotted line) and one female (solid line) cyno following intravenous administration of Conjugate 1 at a dose of 0.1 mg/kg as described in Example 21. Conjugate 1 induces significant CD8 T cell increase in cyno with a 7-1 OX increase in cell numbers after a single dose.

[0088] FIGS. 36C and 36D illustrate the increases in cyno CD8 Tern and CD8 Tern cell numbers after a single injection of Conjugate 1 as described in Example 21. CD8 Tcm and Tern numbers increased 27-30X and 21-33X, respectively.

[0089] FIGS. 37A and 37B are graphs of NK cell numbers and Ki-67 percent positivity in cyno after a single dose of Conjugate 1 at 0.1 mg/kg. See Example 21. NK cells increased 9-10X after treatment with Conjugate 1.

[0090] FIGS. 38A and 38B are EC50 curves for IL-15 (red, solid circle) versus

Conjugate 1 (green, solid square) treatment of human PBMCs and subsequent measurement of pSTAT5 percent positivity in CD8 and CD56 bright NK cells as described in Example 22.

Conjugate 1 is 5.5 and 15X less potent than IL-15 in engaging CD8 and CD56 bright NK cells, respectively. However, Conjugate 1 achieves the same maximum response as conventional IL-15.

[0091] FIG. 39 is a plot of plasma concentration IL-15 at 500 μg/kg (green, solid circles) or Conjugate 1 at ^g/kg (pink, solid squares), 30 μg/kg (purple, solid upwards triangles), 100 μg/kg (red, solid downwards-facing triangles), 300 μg/kg (orange, solid diamonds) or 1000

μ§/1¾ (dark red, open hexagons) over time following administration of a single intravenous dose in mice as described in Example 23.

[0092] FIG. 40 is a plot of plasma concentration of Conjugate 1 at 10 μg/kg (pink, solid squares), 75 μg/kg (purple, solid upwards triangles) or 150 μg/kg (orange, solid downwards-facing triangles) over time following administration of a single intravenous dose in rats as described in Example 24.

[0093] FIG. 41 is a plot of plasma concentration of IL-15 at 50 μg/kg (green, solid circles) or Conjugate 1 at 10 μg/kg (red, solid downwards facing triangles), 50 μg/kg (blue, solid diamonds) or 100 μg/kg (orange, solid squares) over time following administration of a single intravenous dose of test article in cynomolgus monkeys as described in Example 25.

[0094] FIGS. 42A and 42B are plots of CD4 T cell numbers and Ki-67 percent positivity, respectively, after a single dose of Conjugate 1 at a dose of 0.03 mg/kg (blue, solid circles) or 0.3 mg/kg (orange, solid circles) in mice as described in Example 26. The 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 of Ki-67 percent positivity in CD4 T cells.

[0095] FIG. 43 is a graph of pSTAT5 percent positivity within CD4 T cells in mice after a single injection of Conjugate 1 at 0.03 mg/kg (orange, solid circles) or 0.3 mg/kg (blue, solid squares) as described in Example 26. Conjugate 1 at both dose levels induced increased pSTAT5 signaling in CD4 T cells, with the 0.3 mg/kg inducing a greater increase. A 120-hour time course, including vehicle (black) and pre-dose (open circles), are shown.

[0096] FIGS. 44A, 44B and 44C are plots illustrating cell counts versus time post administration for NK cells (FIG. 44A), CD8 T cells (FIG. 44B) and CD4 T cells (FIG. 44C) in cynomolgus monkeys following i.v. administration of a 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) as described in Example 27.

[0097] FIGS. 45A, 45B and 45C are plots illustrating Ki-67 percent positivity after 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. The vehicle (black) levels are also shown. All dose levels returned to baseline by at least 17 days post dose. The 0.1 mg/kg and 0.003 mg/kg dose levels induced a

robust increase of Ki-67 percent positivity in NK cells and CD8 T cells. The 0.1 mg/kg dose level induced an increase of Ki-67 percent positivity in all cell types tested.

[0098] FIGS. 46A, 46B and 46C illustrate the degree of STAT 5 phosphorylation in NK cell (FIG. 46A), CD8 T cell (FIG. 46B) and CD4 T cell (FIG. 46C) lymphocytes following administration of a single i.v. dose of either vehicle (black) or 0.001 mg/kg (purple, solid squares), 0.01 mg/kg (green, diamonds) or 0.1 mg/kg (orange, solid squares) of Conjugate 1 in cynomolgus monkeys as described in Example 27.

[0099] FIGS. 47A-D are plots illustrating Ki-67 percent positivity after 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. The vehicle (black) levels are also shown. FIG. 47A shows results for CD8 Tm e cells, FIG. 47B shows results for CD8 TSCm cells. FIG. 47C shows results for CD8 Tcm cells. FIG. 47D shows results for CD8 Tem cells.

[00100] FIGS. 48A, 48B, and 48C are plots illustrating Granzyme B expression as a function of time following treatment with Conjugate 1 at doses of 0.001 mg/kg (FIG. 48A), 0.01 mg/kg (FIG. 48B) and 0.1 mg/kg (FIG. 48C) as described in Example 28. This data indicates that treatment with Conjugate 1 increases levels of Granzyme B in NK cells in non-human primates (NHPs).

[00101] FIGS. 49A, 49B, and 49C are plots illustrating Perforin expression as a function of time following treatment with Conjugate 1 at doses of 0.001 mg/kg (FIG. 49A), 0.01 mg/kg (FIG. 49B) and 0.1 mg/kg (FIG. 49C) as described in Example 28. This data indicates treatment with Conjugate 1 increases levels of Perforin in NK cells in NHPs.

DETAILED DESCRIPTION

[00102] Before describing one or more aspects or embodiments of the present disclosure in detail, it should be noted that the presented disclosure is not intended to be limited to the particular synthetic techniques, IL-15 moieties, and the like, as such may vary as would be understood by one having ordinary skill in the art to which this disclosure applies.

[00103] In describing and claiming certain features of this disclosure, the following terminology will be used in accordance with the definitions described below unless indicated otherwise.

[00104] 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.

[00105] In describing and claiming one or more embodiments, the following terminology will be used in accordance with the definitions described below.

[00106] A "physiologically cleavable" or "hydrolyzable" or "degradable" bond is a relatively labile bond that reacts with water (i.e., is hydrolyzed) 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 these atoms. Appropriate hydrolytically unstable or weak linkages may include but are not limited to carboxylate ester, phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides, oligonucleotides, thioesters, and carbonates.

[00107] An "enzymatically degradable linkage" means a linkage that is subject to degradation by one or more enzymes.

[00108] A "stable" linkage or bond refers to a chemical bond that is substantially stable in water, that is to say, does not undergo hydrolysis under physiological conditions to any appreciable extent over an extended period of time. Examples of hydrolytically stable linkages generally include but are not limited to the following: carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, amines, and the like. Generally, a stable linkage is one that exhibits a rate of hydrolysis of less than about 1-2% per day under physiological conditions. Hydrolysis rates of representative chemical bonds can be found in most standard chemistry textbooks.

[00109] A covalent "releasable" linkage, for example, in the context of a polyethylene glycol that is covalently attached to an active moiety such as interleukin-15, is one that releases or detaches a polyethylene glycol polymer from the active moiety under physiological conditions, e.g., by any suitable mechanism, at a rate that is clinically useful and includes, for example and without limitation, hydrolyzable bonds and enzymatically degradable linkages.

[00110] "Substantially" or "essentially" means nearly totally or completely, for instance, 95% or greater of a given quantity.

[00111] Similarly, "about" or "approximately" as used herein means within plus or minus 5% of a given quantity.

[00112] Optional" or "optionally" means that the subsequently described circumstance may but need not necessarily occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[00113] "Pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to a component that may be included in a composition as described herein and causes no significant adverse toxicological effects to a subject.

[00114] The phrases "pharmaceutically effective amount" and "pharmacologically effective amount" and "therapeutically effective amount" and "physiologically effective amount" are used interchangeably herein and refer to the amount of a long acting IL-15 R agonist as provided herein that is needed to provide a desired level of the substance in the bloodstream or in a target tissue to produce a desired biological or medicinal response. For example, such a response may be to destroy target cancer cells or to slow or arrest the progression of cancer in a subject. The term also applies to a dose that will induce a particular response in target cells. . The precise amount will depend upon numerous factors, such as for example, the particular condition being treated, the intended patient population, individual patient considerations, the components and physical characteristics of the therapeutic composition to be administered, and the like.

[00115] Reference to a long acting IL-15 R agonist as described herein is meant to encompass pharmaceutically acceptable salt forms thereof.

[00116] The term "patient," or "subject" as used herein refers to a living organism suffering from or prone to a condition that can be prevented or treated by administration of a compound or composition as provided herein. Subjects include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and preferably are human.

[00117] Molecular weight in the context of a water-soluble polymer, such as PEG, can be expressed as either a number average molecular weight or a weight average molecular weight. Unless otherwise indicated, all references to molecular weight herein refer to the weight average molecular weight. Both molecular weight determinations, number average and weight average, can be measured using gel permeation chromatography or other liquid chromatography techniques (e.g. gel filtration chromatography). Most commonly employed are gel permeation chromatography and gel filtration chromatography. Other methods for etermining molecular

weight include end-group analysis or the measurement of colligative properties (e.g., freezing-point depression, boiling-point elevation, or osmotic pressure) to determine number average molecular weight or the use of light scattering techniques, ultracentrifugation, MALDI TOF, or viscometry to determine weight average molecular weight. PEG polymers are typically polydisperse (i.e., the number average molecular weight and the weight average molecular weight of the polymers are not equal), possessing low polydispersity values of preferably less than about 1.2, more preferably less than about 1.15, still more preferably less than about 1.10, yet still more preferably less than about 1.05, and most preferably less than about 1.03.

[00118] The terms "active," "reactive" or "activated" when used in conjunction with a particular functional group, refer to a reactive functional group that reacts readily with an electrophile or a 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., a "non-reactive" or "inert" group).

[00119] As used herein, the term "functional group" or any synonym thereof is meant to encompass protected forms thereof as well as unprotected forms.

[00120] The terms "spacer moiety," "linkage" and "linker" may be used herein to refer to a bond or an atom or a collection of atoms optionally used to link interconnecting 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 hydrolysable, enzymatically degradable, or otherwise releasable linkage. Unless the context clearly dictates otherwise, a spacer moiety optionally exists between any two elements of a compound (e.g., an IL-15 moiety and a water-soluble polymer such as a PEG can be attached directly or indirectly through a spacer moiety).

[00121] "Alkyl" refers to a hydrocarbon chain, typically ranging from about 1 to 15 atoms in length. Such hydrocarbon chains are preferably but not necessarily saturated and may be branched or straight chain, although typically straight chain is preferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 3-methylpentyl, and the like.

[00122] "Lower alkyl" refers to an alkyl group containing from 1 to 6 carbon atoms, and may be straight chain or branched, as exemplified by methyl, ethyl, //-butyl, /'-butyl, and i-butyl.

[00123] "Alkoxy" refers to an -OR group, wherein R is alkyl or substituted alkyl, preferably Ci-6 alkyl (e.g., methoxy, ethoxy, propyloxy, and so forth).

[00124] The term "substituted" as in, for example, "substituted alkyl," refers to a moiety (e.g., an alkyl group) substituted with one or more noninterfering substituents, such as, but not limited to: alkyl, C3-8 cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g., fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl; substituted phenyl; and the like.

"Substituted aryl" is aryl having one or more noninterfering groups as a substituent. For substitutions on a phenyl ring, the substituents may be in any orientation (i.e., ortho, meta, or para).

[00125] "Noninterfering substituents" are those groups that, when present in a molecule, are typically nonreactive with other functional groups contained within the molecule.

[00126] "Aryl" means one or more aromatic rings, each of 5 or 6 core carbon atoms. Aryl includes multiple aryl rings that may be fused, as in naphthyl or unfused, as in biphenyl. Aryl rings may also be fused or unfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclic rings. As used herein, "aryl" includes heteroaryl.

[00127] "Heteroaryl" is an aryl group containing from one to four heteroatoms, preferably sulfur, oxygen, or nitrogen, or a combination thereof. Heteroaryl rings may also be fused with one or more cyclic hydrocarbon, heterocyclic, aryl, or heteroaryl rings.

[00128] "Heterocycle" or "heterocyclic" means one or more rings of 5-12 atoms, preferably 5-7 atoms, with or without unsaturation or aromatic character and having at least one ring atom that is not a carbon. Preferred heteroatoms include sulfur, oxygen, and nitrogen.

[00129] "Substituted heteroaryl" is heteroaryl having one or more noninterfering groups as substituents.

[00130] "Substituted heterocycle" is a heterocycle having one or more side chains formed from noninterfering substituents.

[00131] An "organic radical" as used herein shall include alkyl, substituted alkyl, aryl, and substituted aryl.

[00132] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.

[00133] The term "IL-15 moiety," as used herein, refers to a peptide or protein moiety having human IL-15 activity. In addition, the term "IL-15 moiety" encompasses both the IL-15 moiety prior to conjugation as well as the IL-15 moiety residue following conjugation. As will be explained in further detail below, one of ordinary skill in the art can determine whether any given moiety has IL-15 activity. Proteins comprising an amino acid sequence corresponding to any one of SEQ ID NOs: 1 through 3 is an IL-15 moiety, as well as any protein or polypeptide substantially homologous thereto. As used herein, the term "IL-15 moiety" includes such peptides and proteins modified deliberately, as for example, by site directed mutagenesis or accidentally through mutations. These terms also include analogs having from 1 to 6 additional glycosylation sites, analogs having at least one additional amino acid at the carboxy terminal end of the peptide or protein wherein the additional amino acid(s) includes at least one glycosylation site, and analogs having an amino acid sequence which includes at least one glycosylation site. The term includes naturally, recombinantly and synthetically produced moieties.

[00134] The term "substantially homologous" or "substantially identical" means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences. For purposes herein, a sequence having greater than 95 percent homology (identity), equivalent biological activity (although not necessarily equivalent strength of biological activity), and equivalent expression characteristics to a given sequence is considered to be substantially homologous (identical). For purposes of determining homology, truncation of the mature sequence should be disregarded. Exemplary IL-15 polypeptides for use herein include those sequences that are substantially homologous to SEQ ID NO: 1. SEQ ID NO:2 is nearly identical to SEQ ID NO: l, with the exception that SEQ ID NO:2 has a methionine at the beginning of the sequence that is required for initiating translation in E. coli.

[00135] The term "fragment" means any protein or polypeptide having the amino acid sequence of a portion or fragment of an IL-15 moiety, and having the biological activity, or substantially the biological activity, of IL-15. Fragments include proteins or polypeptides produced by proteolytic degradation of an IL-15 moiety as well as proteins or polypeptides produced by chemical synthesis by methods routine in the art.

[00136] Amino acid residues in peptides are abbreviated as follows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is He or I; Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gin or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; and Glycine is Gly or G.

Overview

[00137] The instant disclosure is directed to providing a long-acting IL-15 receptor agonist. Such an agonist will ideally possess several advantageous and unpredictable features, such as, for example, at least one if not more of the following: (i) the ability to deliver sustained IL-15 activity by providing a measurable pharmacodynamic effect without the need for daily dosing, (ii) retains to a large degree binding to IL-15 receptor a (i.e., in comparison to IL-15), (iii) stimulates NK cell activation and/or proliferation, and/or (iv) supports CD8 T-cell survival and/or memory formation, and (v) provides inhibition of tumor growth. Surprisingly, the Applicants have arrived at a long acting IL-15 R agonist that possesses a unique combination of advantageous properties, to be described in greater detail below.

Long Acting IL-15 R Agonist and Related Compositions

[00138] Generally, a long acting IL-15 receptor agonist or a pharmaceutically acceptable salt form thereof comprises a single linear PEG (polyethylene glycol) moiety stably covalently attached to an IL-1 amino group via an amide linkage. Intervening between the PEG moiety and the stable amide linkage to an IL-15 amino group is a linear unsubstituted alkylene group (~CH2~)m having from 2 to 5 carbon atoms (i.e., m=2, 3, 4, or 5).

[00139] When considering the IL-15 moiety, the term "IL-15 moiety" refers to the IL-15 moiety prior to conjugation as well as to the IL-15 moiety following attachment to a non-peptidic, water-soluble polymer such as a poly(alkylene oxide) (e.g. a poly(ethylene glycol) or PEG). While specific reference is made to PEG hereafter as the non-peptidic, water-soluble polymer below, it will be understood that the disclosure relates generally to a non-peptidic, water-soluble polymer or poly(alkylene glycol). It will be understood, however, that when the original IL-15 moiety is attached to a polyethylene glycol moiety, the IL-15 moiety is slightly altered due to the presence of one or more covalent bonds associated with linkage to the polymer(s).

[00140] The IL-15 moiety can be derived from non-recombinant methods and from recombinant methods, and the disclosure is not limited in this regard. In addition, the IL-15

moiety can be derived from human sources, animal sources (including insects), fungi sources (including yeasts), and plant sources.

[00141] The IL-15 moiety can be obtained according to the procedures described by, for example, Grabstein et al. [See. Grabstein et al. (1994) Science 264:965-9681. The IL-15 moiety can also be prepared using recombinant methods, such as, for example, those described in EP Patent No. 0 772 624 B2 to Immunex Corporation. Alternatively, the IL-15 moiety can be purchased commercially from, for example, GenScript USA Inc. (Piscataway NJ) and Peprotech (Rockyhill, NJ).

[00142] The IL-15 moiety can be expressed in bacterial [e.g., E. coli, see, for example, Fischer et al. (1995) Biotechnol. Appl. Biotechnol. 2J_(3):295-311], mammalian [see, for example, Kronman et al. (1992) Gene 121 :295-304], yeast [e.g., Pichia pastoris, see, for example, Morel et al. (1997) Biochem. J. 328(1): 121-129], and plant [see, for example, Mor et al. (2001) Biotechnol. Bioeng. 75(31:259-266] expression systems. The expression can occur via exogenous expression (when the host cell naturally contains the desired genetic coding) or via endogenous expression.

[00143] Although recombinant-based methods for preparing proteins can differ, recombinant methods typically involve constructing the nucleic acid encoding the desired polypeptide or fragment, cloning the nucleic acid into an expression vector, transforming a host cell (e.g., plant, bacteria, yeast, transgenic animal cell, or mammalian cell such as Chinese hamster ovary cell or 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.

[00144] To facilitate identification and purification of the recombinant polypeptide, nucleic acid sequences that encode for an epitope tag or other affinity binding sequence can be inserted or added in-frame with the coding sequence, thereby producing a fusion protein comprised of the desired polypeptide and a polypeptide suited for binding. Fusion proteins can be identified and purified by first running a mixture containing the fusion protein through an affinity column bearing binding moieties (e.g., antibodies) directed against the epitope tag or other binding sequence in the fusion proteins, thereby binding the fusion protein within the column. Thereafter, the fusion protein can be recovered by washing the column with the

appropriate solution (e.g., acid) to release the bound fusion protein. The recombinant polypeptide can also be purified by lysing the host cells, separating the polypeptide, e.g., by ion-exchange chromatography, affinity binding approaches, hydrophobic interaction approaches, and thereafter identify by MALDI or western blot, and collecting the polypeptide. These and other methods for identifying and purifying recombinant polypeptides are known to those of ordinary skill in the art. In one or more embodiments, however, the IL-15 moiety is not in the form of a fusion protein.

[00145] Depending on the system used to express proteins having IL-15 activity, the IL-15 moiety can be unglycosylated or glycosylated and either may be used. That is, the IL-15 moiety can be unglycosylated or the IL-15 moiety can be glycosylated. In one or more embodiments, the IL-15 moiety is unglycosylated.

[00146] The IL-15 moiety can advantageously be modified to include and/or substitute one or more amino acid residues such as, for example, lysine, cysteine and/or arginine, in order to provide facile attachment of the polymer to an atom within the side chain of the amino acid. An example of substitution of an IL-15 moiety is described in U.S. Patent No. 6, 177,079. In addition, the IL-15 moiety can be modified to include a non-naturally occurring amino acid residue. Techniques for adding amino acid residues and non-naturally occurring amino acid residues are well known to those of ordinary skill in the art. Reference is made to J. March, Advanced Organic Chemistry: Reactions Mechanisms and Structure, 4th Ed. (New York:

Wiley-Interscience, 1992), and Bioinformatics for Geneticists (eds. Michael R. Barnes and Ian C Gray), 2003 John Wiley & Sons, Ltd, Chapter 14, Amino Acid Properties and Consequences of Substitutions, Betts, M.J., and Russell, R. B.

[00147] In addition, the IL-15 moiety can advantageously be modified to include attachment of a functional group (other than through addition of a functional group-containing amino acid residue). For example, the IL-15 moiety can be modified to include a thiol group. In addition, the IL-15 moiety can be modified to include an N-terminal alpha carbon. In addition, the IL-15 moiety can be modified to include one or more carbohydrate moieties. In addition, the IL-15 moiety can be modified to include an aldehyde group. In addition, the IL-15 moiety can be modified to include a ketone group. In some embodiments of the invention, it is preferred that the IL-15 moiety is not modified to include one or more of a thiol group, an N-terminal alpha carbon, carbohydrate, aldehyde group and ketone group.

[00148] Exemplary IL-15 moieties are described herein, in the literature, and in, for example, U.S. Patent Application Publication No. US 2006/0104945, Pettit et al. (1997) J. Biol. Chem. 272(4):2312-2318, and Wong et al. (2013) Oncolmmunology 2(11), e26442: l-3.

Preferred IL-15 moieties include those having an amino acid sequence comprising sequences selected from the group consisting of SEQ ID NOs: 1 through 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 standard of an IL-15 moiety provided herein, it will be understood for purposes of the present invention that these sequences are also understood to be "IL-15 moieties"). A preferred IL-15 moiety has an amino acid sequence corresponding to SEQ ID NO: 1. In some embodiments, the IL-15 moiety is a functional homolog having at least about 85% or at least about 90% identity with any one of SEQ ID NOs: 1-3. In some embodiments, the IL-15 moiety is a functional homolog having at least about 95%, 98% or 99% identity with any one of SEQ ID NOs: 1-3.

[00149] In some instances, the IL-15 moiety will be in a "monomer" form, wherein a single expression of the corresponding peptide is organized into a discrete unit. In other instances, the IL-15 moiety will be in the form of a "dirtier" (e.g., a dimer of recombinant IL-15) wherein two monomer forms of the protein are associated to each other.

[00150] In addition, precursor forms of IL-15 can be used as the IL-15 moiety. An exemplary precursor form of IL-15 has the sequence of SEQ ID NO: 3.

[00151] Truncated versions, hybrid variants, and peptide mimetics of any of the foregoing sequences can also serve as the IL-15 moiety. Biologically active fragments, deletion variants, substitution variants or addition variants of any of the foregoing that maintain at least some degree of IL-15 activity can also serve as an IL-15 moiety.

[00152] For any given peptide, protein moiety or conjugate, it is possible to determine whether that peptide, protein moiety or conjugate has IL-15 activity. Various methods for determining in vitro IL-15 activity are described in the art. An exemplary approach is based on a pSTAT assay. Briefly, if an IL-15 -dependent CTLL-2 cell is exposed to a test article having IL-15 activity, initiation of a signaling cascade results that includes the phosphorylation of STAT5 at tyrosine residue 694 (Tyr694), which can be quantitatively measured. Assay protocols and kits are known and include, for example, the MSD Phospho(Tyr694)/Total STATa,b Whole Cell Lysate Kit (Meso Seal Diagnostics, LLC, Gaithersburg, MD). For example, using this approach,

a proposed IL-15 moiety that exhibits a pSTAT5 EC50 value of no more than about 300 ng/mL (more preferably no more than about 150 ng/mL) at least one of 5 minutes or at 10 minutes is considered an "IL-15 moiety" in connection with the present disclosure. It is preferred, however, that the IL-15 moiety used is more potent (e.g., having a pSTAT5 EC50 value of less than 150 ng/mL at one of least 5 minutes or 10 minutes, such as less than about 1 ng/mL, and even more preferably less than 0.5 ng/mL at least one of 5 minutes or at 10 minutes).

[00153] Other methodologies known in the art can also be used to assess IL-15 function, including electrometry, spectrophotometry, chromatography, and radiometric methodologies. See, for example, Ring et al. (2012) Nat. Immunol. 13(12): 1187-1195 for one such additional type of assay.

[00154] Assays for use in connection with measuring the activity of an IL-15 moiety can also be used to measure the activity of the long acting IL-15 R agonists described herein. See, for example, the supporting examples provided herein.

[00155] A compound is considered to be a long-acting, IL-1 R agonist in accordance with the present disclosure so long as, following administration to a subject, the agonist exhibits IL-15 agonism in vivo for an amount of time that is longer than would be the case for

administration of IL-15. Conventional approaches, such as those involving radiolabeling a compound, administering the compound in vivo, and determining its clearance, can be used to assess whether a compound proposed to be a long-acting, IL-15 R agonist is "long-acting" (i.e., has a clearance that is longer than that of IL-15 administered in the same in vivo system). For the purposes herein, the long acting nature of a long-acting IL-15 R agonist may be, and is typically determined using flow cytometry to measure STAT5 phosphorylation in lymphocytes at various time points after administration of the agonist to be evaluated in mice. As a reference, the signal is lost by around 24 hours with IL-15, but is sustained for a period greater than that for a long-acting IL-15 agonist.

[00156] As previously discussed, a preferred long acting IL-15 R agonist will generally comprise a single linear PEG (polyethylene glycol) moiety stably covalently attached to an IL-15 amino group via an amide linkage. Intervening between the PEG moiety and the stable amide linkage to an IL-15 amino group is a linear unsubstituted alkylene group (~CH2~)m having from 2 to 5 carbon atoms (i.e., where m=2, 3, 4, or 5).

[00157] For example, in some embodiments, the unsubstituted alkylene group is (~CH2~)2; or, in some additional embodiments, the unsubstituted alkylene group is (~CH2~)3; in yet some further embodiments, the unsubstituted alkylene group is (~CH2~)4; in yet some further embodiments, the unsubstituted alkylene group is the unsubstituted alkylene group is (~CH2~ )5.

[00158] For example, in some embodiments, the long acting IL-15 receptor agonist has the following structure:

5

Formula (I)

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. In Formula I (and in similar formulae provided herein) the ~NH~ in the structure represents an amino group of the IL-15 moiety. Formula (I) may also be depicted as follows, where the parentheses are shifted to reflect a terminal PEG

O

CH30 (CH 2 CH 2 0) n (CH 2 ) IL-15

methoxy group, , and the two formulae may be used interchangeably. Illustrative exemplary compounds include the following encompassed by Formula (I):

5

Formula (Ia)

0

CH30(CH2CH20)nCH2CH2CH2C NH- IL-15

Formula (lb)

0

CH30(CH2CH20)nCH2CH2CH2CH2C NH- IL-15

Formula (Ic), and

0

CH The 3 0 (CH The 2 CH The 2 0) N CH The 2 CH The 2 CH The 2 CH The 2 CH The 2 the C NH- IL-15

n' Formula (Id).

[00159] In some preferred embodiments, the long acting IL-15 receptor agonist corresponds to Formula (la) or Formula (lb). In some particularly preferred embodiments, the long acting IL-15 receptor agonist corresponds to Formula (lb).

[00160] In some further embodiments, in reference to the structures and formulae 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 to say, in some embodiments, n is an integer from about 200 to about 2000. In yet some further embodiments, n is an integer from about 400 to about 1300. In yet some further embodiments, n is an integer from about 450 to about 1200.

[00161] PEGs having a molecular weight corresponding to any one of the foregoing ranges of n values are generally preferred.

[00162] In one or more embodiments, n is an integer having a value that corresponds to a polyethylene glycol polymer having a weight average molecular weight selected from the group consisting of about 10,000 daltons (where n is -227), or about 15,000 daltons (where n is -340), or about 20,000 daltons (where n is -454), or about 25,000 daltons (where n is -568), or about 30,000 daltons (where n is -681), or about 40,000 daltons (where n is -909), or about 50,000 daltons (where n is -1136) or even about 60,000 daltons (where n is -1364) or greater.

[00163] Further exemplary weight-average molecular weights for the polyethylene glycol portion of the compound, in addition to the foregoing, 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.

[00164] In some preferred embodiments, the weight-average molecular weight of the polyethylene glycol polymer portion of the compound is about 40,000 daltons.

[00165] While the PEG moiety is preferably end-capped as shown above in Formula (I) with a methoxy group, the PEG moiety may be capped at its terminus with any lower Ci-6 alkoxy group, or may terminate in a hydroxyl group, or other suitable end-capping group.

[00166] In some embodiments, the long acting IL-15 receptor agonist composition comprises no more than about 20 mole percent (mol%) of long-acting IL-15 receptor agonists (of the IL-15 containing molecules in the composition), when considered collectively, encompassed by the formula:

0

C 3 - (DESIRE 2 CH 2 ) n point (C 2 ) nrr ■ NH- IL-15

or greater than 3

Formula (II),

where the values of n and m are as provided for Formula (I) above. That is to say, in terms of the long-acting IL-15 receptor agonist component of such compositions, no more than about 20 mole percent of long-acting IL-15 receptor agonists comprised in the composition are of Formula (II).

[00167] In some additional embodiments, the long acting IL-15 receptor agonist composition comprises no more than about 15 mole percent (mol%) of long-acting IL-15 receptor agonists (of the IL-1 containing molecules in the composition), when considered collectively, encompassed by the formula:

0

CH 3 - (OCH 2 CH 2 ) n O- (CH 2 ) -NH- IL-15

2,3 or greater than 3

Formula (II),

where the values of n and m are as provided for Formula (I) above. That is to say, in terms of the long-acting IL-15 receptor agonist component of such compositions, no more than about 15 mol percent of long-acting IL-15 receptor agonists comprised in the composition are of Formula (II). In some embodiments, the long-acting IL-15 receptor agonist composition comprises no more than about 0.1-20 mol% of compounds of Formula (II). In embodiments, the compositions comprise no more than about 0.1-15, 0.1-10, 0.1-5, 0.1-1, 1-20, 1-15, 1-10, 1-5, 5-20, 5-15, 5-10, 10-20, 10-15, or 15-20 mol% of compounds of Formula (II).

[00168] In some particular embodiments related to the foregoing, the long acting IL-15 receptor agonist composition, in reference to Formula (la), comprises no more than about 15 mole percent (mol%) of long-acting IL-15 receptor agonists (of the IL-15 containing molecules in the composition), when considered collectively, encompassed by the formula:

O

CH 3 0 (CH 2 CH 2 0) n CH 2 CH 2 C NH- IL-15

2,3 or greater than 3

Formula (Ila),

where the values of n and m are as provided for Formula (la) above.

[00169] In some other preferred embodiments, the long acting IL-15 receptor agonist composition, in reference to Formula (lb), comprises no more than about 15 mole percent (mol%) of long-acting IL-15 receptor agonists (of the IL-15 containing molecules in the composition), when considered collectively, encompassed by the formula:

0

CH 3 0 (CH 2 CH 2 0) n CH 2 CH 2 CH 2 C- IL-15

2,3 or greater than 3

Formula (lib),

where the values of n and m are as provided for Formula (lb) above.

[00170] In some other embodiments, the long acting IL-15 receptor agonist composition, in reference to Formula (Ic), comprises no more than about 15 mole percent (mol%) of long-acting IL-15 receptor agonists (of the IL-15 containing molecules in the composition), when considered collectively, encompassed by the formula:

O

CH30(CH2CH20)nCH2CH2CH2CH2C NH- IL-15

2,3 or greater than 3

Formula (lie),

where the values of n and m are as provided for Formula (Ic) above.

[00171] In some other embodiments, the long acting IL-15 receptor agonist composition, in reference to Formula (Id), comprises no more than about 15 mole percent (mol%) of long-acting IL-15 receptor agonists (of the IL-15 containing molecules in the composition), when considered collectively, encompassed by the formula:

O

CH The 3 0 (CH2CH 2 0) N CH2CH 2 CH2CH 2 CH The 2 the C - NH- IL-15

2,3 or greater than 3

Formula (lid),

where the values of n and m are as provided for Formula (Id) above.

[00172] In some embodiments, the long-acting EL-15 receptor agonist composition comprises no more than about 0.1-20 mol% of compounds of Formula (II), including compounds of Formulae (Ila), (lib), (lie), and (lid). In some additional embodiments, the compositions comprise no more than about 0.1-15, 0.1-10, 0.1-5, 0.1-1, 1-20, 1-15, 1-10, 1-5, 5-20, 5-15, 5-10, 10-20, 10-15, or 15-20 mol% of compounds of Formula (II), including compounds of Formulae (Ila), (lib), (lie), and (lid). In some embodiments, the compositions comprise no more than about 0.1, 1, 5, 10, 15, or 20 mol% of compounds of Formula (II), including compounds of Formulae (Ila), (lib), (lie), and (lid). It will be appreciated that the compositions may be purified by methods known in the art for compounds of Formula (I) resulting in no, trace amounts, or substantially no compounds of Formula (II) present in the composition.

[00173] For example, in some embodiments, the long acting IL-15 receptor agonist composition comprises no more than about 12 mole percent, or no more than about 10 mole percent of long-acting IL-15 receptor agonists, that when considered collectively, are

encompassed by Formula (II), including compounds of Formulae (Ila), (lib), (lie), and (lid).

[00174] In some additional embodiments of the foregoing, the composition comprises no more than about 7 mol% of long acting IL-15 receptor agonists having n' equal to 2, 3, or greater than 3 (i.e., higher PEGmers). In yet some other embodiments, the composition comprises no more than about 5 mol% of long acting IL-15 receptor agonists having n' equal to 2, 3 or greater than 3 (i.e., of 2 or greater).

[00175] In some further embodiments, the composition comprises a long-acting IL-15 receptor agonist according to Formula (I),

O

CH3-(OCH2CH2)nO-(CH2)mC— NH-|— IL" 5

, where n and m are as described above, and n' represents the average number of polyethylene glycol moieties covalently attached to IL-15 amino groups (for the composition), and n' for the composition is in a range 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 to say, a preferred long -acting IL-15 receptor agonist according to Formula (I) may be referred to herein as "monoPEGylated", where it is to be understood that some variability exists around the degree of PEGylation as described above. In some preferred embodiments in reference to the formulae described herein, "m" is equal to 3.

[00176] A composition of the long-acting IL-15 R agonist may comprise a single species where n' equals about 1 and the PEG moiety is attached at the same location for substantially all IL-15 conjugates in the composition, or alternatively, may comprise a mixture of

monoPEGylated conjugate species where attachment of the linear polyethylene glycol moiety occurs at different sites on the interleukin-15 moiety, that is to say, where the particular attachment sites are not the same for all of the monoPEGylated IL-15 species comprised in the composition). Thus, such compositions are substantially homogeneous in terms of the number of PEG moieties attached to IL-15 (e.g., 1-mers), but are heterogeneous in terms of the locations of amino group attachment on the IL-15 molecule.

[00177] While additional PEG architectures and linkage chemistries may be employed for arriving at a long acting IL-15 R agonist, compounds such as previously described are preferred in one or more embodiments, as will become apparent when considered in light of the supporting examples. However, additional long acting IL-15 R agonists having structures as provided herein are also contemplated.

[00178] In some embodiments, the long acting IL-15 receptor agonist composition comprises at least about 80 mol% of long acting IL-15 receptor agonists (of the IL-15 containing molecules in the composition), when considered collectively, encompassed by Formula (I), including Formulae (Ia-d). 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 the long acting IL-15 receptor agonists of Formula (I).

[00179] As described above, the long acting IL-15 R agonist may be in the form of a pharmaceutically-acceptable salt. Typically, such salts are formed by reaction with a

pharmaceutically-acceptable acid or an acid equivalent. The term "pharmaceutically-acceptable salt" in this respect, will generally refer to the relatively non-toxic, inorganic and organic acid addition salts. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting a long-acting interleukin-15 receptor agonist as described herein with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, oxylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66: 1-19). Thus, salts as described may be derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; or prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic,

methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.

[00180] In some embodiments, the long acting IL-15 receptor agonist composition comprises no more than about 1-5 mol% of free IL-15 protein (of the IL-15 containing molecules in the composition), when considered collectively. In some further 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.

[00181] To prepare a long acting IL-15 receptor agonist, the IL-15 moiety may, for example, be conjugated at its amino groups (e.g. lysines or the N-terminus) to a PEG reagent functionalized with a succinimidyl group (or other activated ester group). Using this approach, the succinimidyl activated PEG can be attached to amino groups on the IL-15 moiety in an aqueous media 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 different temperatures and/or less than 15 °C) can result in the attachment of the PEG moiety to a different location on the IL-1 moiety.

[00182] A long acting IL-15 R agonist can be prepared as described in Example 1. For example, the long acting IL-15 R agonist may generally be prepared by reacting an interleukin-15, for example, purified IL-15, such as recombinant IL-15, with an activated PEG reagent such as the activated ester, methoxyPEG-succinimidyl butanoate, mPEG-SBA. Other suitable activated PEG reagents include methoxyPEG-succinimidyl propionate, methoxyPEG-succinimidyl pentanoate, and methoxyPEG-succinimidyl hexanoate. While a succinimidyl activating group is typically used, any suitable active ester or activating group may be used, wherein such reacting group is suitable for forming the desired stable amide linkage. Generally, the interleukin-15 is dissolved in a suitable buffer, such as for example, phosphate buffered saline (PBS). The PEG reagent may be added at an equimolar ratio to the IL-15 (relative to the molar amount of interleukin-15), generally in solution in a suitable buffer, or at a molar excess (based upon the molar amount of IL-15), that is up to about a 15-fold molar excess, e.g., a 2-fold molar excess, or a 5-fold molar excess, or a 7-fold molar excess, or a ten-fold molar excess, or even a 12-fold molar excess or more. In some embodiments, the PEG reagent is added at a molar excess of about 5 to 10-fold. The PEG reagent may be added in solid form, or as a solution in a suitable solvent, for example in aqueous acid such as dilute hydrochloric acid.

[00183] In some further embodiments of the method, the interleukin-15 is initially present in solution, i.e., prior to mixing with the methoxyPEG-succinimidyl alkanoate reagent, at a concentration of about 0.5 mg/mL to about 10 mg/mL. Additional illustrative concentration ranges 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 mg/mL, 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 the solution. In some specific, but not 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.

[00184] It will be appreciated that any suitable buffer may be usedor added to the reaction mixture. Some exemplary buffers include sodium phosphate ( aPi), sodium acetate (NaAc), borate, bicine, citrate, and Bis-TRIS buffers.

[00185] In some embodiments, the pH of the IL-15 solution is adjusted to around pH 8 prior to addition of the PEG reagent.

[00186] Following addition of the PEG reagent, the reaction mixture may then be adjusted, if necessary, to a suitable pH, for example, from about 7.0 - 8.5, or to about 8.0. In some embodiments, the reaction mixture is adjusted to a pH of about 7.0-8.0 or to about 7.4-8.5. In some particular embodiments, the reaction mixture is adjusted to a pH of about 8.0. It will be appreciated that the pH may be adjusted both prior to and following addition of the PEG reagent, as necessary to achieve the desired pH.

[00187] Interleukin-15, like many proteins, is subject to deamidation, especially at higher pHs, while lower pH levels may lead to a number is possible disadvantages such as, for example, a lower degree of conjugation at epsilon (ε) amines, and/or increased and/or undesirable

positional isoforms, as well as protein aggregation. Deamidation introduces a negative charge into a protein, which may lead to changes in the protein's activity, structure, function, stability and/or may change the susceptibility of the protein to degradation. Thus, one of the challenges addressed by the instant agonists and related methods was to provide a long acting interleukin-15 receptor agonist that maintains a sufficient degree of activity (i.e., to be therapeutically useful) whilst balancing at least (i) a desired degree of conjugation, (ii) low amounts of deamidation of the interleukin-15 moiety, both before and after conjugation with the subject PEG reagents (which can, for example, lead to decreased interleukin-15 activity), and (iii) protein aggregation (e.g., both before and after conjugation), among other considerations.

[00188] Based on the competing and conflicting challenges associated with reaction parameters for preparing a long-acting interleukin-15 receptor agonist as described herein, it has been discovered by the Applicants that by adjusting the pH of the interleukin-15 solution (prior to or after reaction with the PEG reagent), and/or the IL-15-PEG reagent reaction mixture, one can arrive at optimal (lower levels) of deamidation, while still promoting conjugation of the PEG moieties to the interleukin-15 moiety (e.g., at ε amines as well as the N-terminus) to provide a long acting IL-15 R agonist as described herein. While not being bound by theory, based upon a series of reactions in which numerous reaction parameters were varied, it appears that a pH range of 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, is effective to provide for lower levels of deamidation in the product, while still promoting conjugation of the PEG moieties to form a product as described herein, which also maintains a desirable therapeutic profile.

[00189] For example, the methods described herein are effective to produce PEGylated interleukin-15 that is less than about 35% deamidated, or in some embodiments, is less than about 30% deamidated, or is less than about 25% deamidated, or is less than about 20% deamidated. In some embodiments, the level of deamidation of the product is in a range of from about 20-35%, or is in a range of about 20-25%, or is in a range of about 25-35%, or is in a range of about 25-30%. Alternatively, in some embodiments, PEGylated interleukin-15 having degrees of deamidation less than stated above are contemplated. As shown in Experiment 2 of Example 1, adjusting the pH in a range of about 7.0-8.5 resulted in a level of deamidation of 21.29% (Composition 1) or 33.26% (Composition 2).

[00190] The reactants are generally mixed for up to, and including, about 5 to 10 hours. In some embodiments, the reactants are mixed for up to, and including about 2 to 5 hours. In some embodiments, the reactants are mixed for up to, and including about 2 hours. In some exemplary embodiments, the reactants are 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 hour, 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. The mixing is generally carried out under mild conditions, e.g., from about 20° C to about 65° C, or from about 20° C to about 40° C, or at ambient or room temperature (e.g. about 22 0 C). Lower temperatures may be employed to favor a lower degree of PEGylation. The reaction may be quenched, for example, by addition of an amino acid such as glycine.

[00191] In embodiments, the pH of the composition may further be adjusted in order to mitigate deamidation. In some embodiments, the composition is adjusted to a pH of about 6.5-7.5 or 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.

[00192] The PEGylated rIL-15 reaction product may then generally be purified by any suitable method such as, for example, ion exchange chromatography to obtain the desired product. For example, anion exchange chromatography may be employed. The chromatography product pool may then be concentrated and diafiltered into suitable formulation buffer (for example, sodium acetate buffer with sucrose) using, for example, tangential flow filtration (TFF). Analysis may be conducted by any suitable method, such as for example, SDS-PAGE, reverse phase HPLC or any other suitable analytical method.

[00193] As described previously, amino groups on the IL-15 moiety provide a sie of attachment between the IL-15 moiety and the polyethylene glycol moiety to provide a long acting IL-15 R agonists such as encompassed by Formula (I). For example, considering the exemplary IL-15 amino acid sequences provided herein, it is evident that there are seven lysine residues each having an ε-amino acid that may be available for conjugation. Further, the N-terminal amine of methionine can also serve as a point of attachment to the PEG moiety. It will be appreciated that the polyethylene glycol moiety may be attached at any one or more of the lysine or the N-terminal amine positions. In some embodiments, a polyethylene glycol

moiety attachment site is at one or more of Lys10 and Lys11 (using the numbering as shown in SEQ ID NO: 2 as an example or Lys11 and Lys12 using SEQ ID NO: 1). In some embodiments, a polyethylene glycol moiety is attached at the N-terminal amine. It will be appreciated that any of the lysine sites may be suitable as an attachment site (e.g. Lys37 or Lys42 of SEQ ID NO:l) for the PEG moiety. In some embodiments, the long-acting interleukin-15 receptor agonist comprises a mixture of positional isomers, where covalent attachment of the polyethylene glycol moiety is predominately at the N-terminus (that is to say, of 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).

[00194] If desired, the product pool may be further separated into positional isomers by reverse phase chromatography using a reverse phase-high performance liquid chromatography (RP-HPLC) using a suitable column (e.g., a CI 8 column or C3 column, available commercially from companies such as Amersham Biosciences or Vydac) or by ion exchange chromatography using an ion exchange column, e.g., a Sepharose™ ion exchange column available from

Amersham Biosciences. Either approach can be used to separate PEG-interleukin-15 positional isomers having the same molecular weight (i.e., positional isoforms).

[00195] Gel filtration columns suitable for carrying out this type of separation include Superdex™ and Sephadex™ columns available from GE Healthcare (Buckinghamshire, UK). Selection of a particular column will depend upon the desired fractionation range desired.

Elution is generally carried out using a suitable buffer, such as phosphate, acetate, or the like. The collected fractions may be analyzed by a number of different methods, for example, (i) absorbance at 280 nm for protein content, (ii) dye-based protein analysis using bovine serum albumin (BSA) as a standard, (iii) iodine testing 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).

[00196] The instant long acting IL-15 R agonists have been discovered to possess certain notable and advantageous features. While the features described below are believed to apply generally to compounds as provided herein and encompassed by Formula (I), the following one or more features may be exhibited particularly by compounds in accordance with Formula (lb), and by extension, Formula (lib). The long acting IL-15 R agonist may possess one or more of

the following features. For example, in some embodiments, the long-acting IL-15 receptor agonist exhibits no more than about a 7-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5) when compared to unmodified IL-15. For example, in one or more related embodiments, the long-acting IL-15 receptor agonist exhibits no more than about a 6.5-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5), or no more than about a 6-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5), or no more than about a 5.5-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5), or no more than about a 5-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5), or no more than about a 4.5-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5), or no more than about a 4-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5), or no more than about a 3.5-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5), or even no more than about a 3-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5) when compared to IL-15. Exemplar}' long acting IL-15 R agonists in accordance with the foregoing features are described herein and in the accompanying Examples.

[00197] As described in Example 10, the in vitro activity of the illustrative conjugates (1, 3, and 5) induces IL-15 signaling in huPBMCs, with Conjugate 1 potently inducing such signaling. Further experiments were conducted 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). As shown in Figs. 10A-10B, at least Conjugate 1 induced similar or increased signaling as compared to IL-15 in CD56bright andCD561ow cells. While Conjugate 1 was less potent than IL-15 in engaging CD8 and CD56 bright NK cells (Example 22), it is important to note Conjugate 1 achieved the same maximum response as conventional IL-15 (see Figs. 38A-38B). As described for the mouse model in Example 16, 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 an increase in %pSTAT5 as compared to IL-15. In the mouse models, NK cells were the most sensitive to a single dose of Conjugate, followed by CD8 T cells with CD4 T cells being the least sensitive for the cells tested.

WE CLAIMED:

1. A long acting IL-15 receptor agonist comprising a single linear polyethylene glycol (PEG) moiety stably covalently attached to an amino group of IL-15 via an amide linkage, wherein intervening between the PEG moiety and the amide linkage to the IL-15 amino group is a linear unsubstituted alkylene group (~CH2~)m having from 2 to 5 carbon atoms, and pharmaceutically acceptable salt forms thereof.

2. The long acting IL-15 receptor agonist of claim 1, wherein m is an integer selected from the group consisting of 2, 3, 4, and 5.

3. The long acting IL-15 receptor agonist of claim 1 or claim 2, wherein the unsubstituted alkylene group is selected from (~CH2~)2, (~CH2~)3, (~CH2~)4, or (~CH2~)s.

4. A long acting IL- 15 receptor agonist a structure:

Formula (I)

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, n' is 1, and ~NH~ in the structure represents an amino group of the IL-15 moiety.

5. A long acting IL-15 receptor agonist of any previous claim, wherein n is an integer from about 200 to about 2000, or from about 400 to about 1300, or from about 450 to about

1200.

6. A long acting IL-15 receptor agonist of any previous claim, wherein m is 2 or 3.

7. A long acting IL-15 receptor agonist of any previous claim, wherein m is 3.

8. A long acting IL-15 receptor agonist of any previous claim, wherein n is an integer having a value that corresponds to a polyethylene glycol polymer having a weight average molecular weight selected from the group consisting of 10,000 daltons (-227), 15,000 daltons (-340), 20,000 daltons (-454), 25,000 daltons (-568), 30,000 daltons (-681), 40,000 daltons (909), 50,000 daltons (-1136) and 60,000 daltons (-1364).

A composition comprising a long acting IL-15 receptor agonist of any one of claims 7-11, the composition comprising no more than about 15 mole percent of long-acting IL-15 receptor agonists, when considered collectively, encompassed by the formula:

0

1 - (OCH 2 CH 2 ) n O- (CH 2 ), IL-15

' 2,3 or greater than 3

Formula (II).

10. A composition of any previous claim, comprising no more than about 10 mole percent of long-acting IL-15 receptor agonists, that when considered collectively, are encompassed by Formula (II).

11. A composition in accordance with any previous claim, comprising no more than about 7 mole percent of long-acting IL-15 receptor agonists, that when considered collectively, are encompassed by Formula (II).

12. A composition in accordance with any previous claim, comprising no more than about 5 mole percent of long-acting IL-15 receptor agonists, that when considered collectively, are encompassed by Formula (II).

13. A composition comprising a long-acting IL-15 receptor agonist according to Formula (I),

0

II

CH3-(OCH2CH2)nO-(CH2)mC— NH- 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 n' is 1, and ~ H~ in the structure represents an amino group of the IL-15 moiety, and n' represents the average number of polyethylene glycol moieties covalently attached to IL-15 amino groups in the composition, where n' for the composition is in a range from 1.0 to about 1.3.

14. The composition of any previous claim, wherein n' for the composition is selected from 1.0, 1.1, 1.2 and about 1.3.

15. A long acting IL-15 receptor agonist of any previous claim, wherein the long-acting IL-15 receptor agonist exhibits no more than about a 7-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5) when compared to unmodified IL-15.

16. The long acting IL-15 receptor agonist of any previous claim, wherein the long-acting IL- 15 receptor a reduction in EC50 value (ng/mL, CTLL-2 pSTAT5) selected from the group

consisting of no more than about a 6.5-fold reduction in EC50 value (ng/niL, CTLL-2 pSTAT5), no more than about a 6-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5), no more than about a 5.5-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5), no more than about a 5-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5), no more than about a 4.5-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5), no more than about a 4-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5), no more than about a 3.5-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5), and no more than about a 3-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5) when compared to IL-15.

17. A long acting IL-15 receptor agonist of any previous claim, the long-acting IL-15 receptor agonist exhibiting no more than about a 50% reduction in receptor alpha binding (KD, pM) when compared to IL-15.

18. The long acting IL-15 receptor agonist of any previous claim, wherein the long-acting IL- 15 receptor agonist exhibits no more than about a 45% reduction in receptor alpha binding (KD, pM), or exhibits no more than about a 40% reduction in receptor alpha binding (KD, pM), or exhibits no more than about a 35% reduction in receptor alpha binding (KD, pM), or even exhibits no more than about a 30% reduction in receptor alpha binding (KD, pM) when compared to IL-15.

19. The long acting IL-15 receptor of any previous claim, wherein the long-acting IL-15

receptor agonist exhibits no more than about a 7-fold reduction in EC50 value (ng/mL, CTLL-2 pSTAT5) when compared to unmodified IL-15 and no more than about a 50% reduction in receptor alpha binding (KD, pM) when compared to IL-15.

20. A pharmaceutically acceptable composition comprising the long acting IL-15 receptor agonist of any previous claim, or a composition of any one of claims 12-22, and a pharmaceutically acceptable excipient.

21. A long acting IL-15 R agonist or composition in accordance with any previous claim, that is effective when administered at a therapeutically effective dose to a mammalian subject to stimulate K activation and proliferation.

22. A long acting IL-15 R agonist or composition of any previous claim, that is effective when administered at a therapeutically effective dose to a subject to support CD8 T-cell survival and memory formation.

23. A method of treating a condition that is responsive to treatment with IL-15 by

administering to a subject having the condition a therapeutically effective dose of a long- acting IL-15 R agonist or composition in accordance with any previous claim.

24. A method for treating cancer by administering to a subject having cancer a therapeutically effective dose of a long-acting IL-15 R agonist or composition of any previous claim.