EA043667B1 - LONG-ACTING INTERLEUKIN-15 RECEPTOR AGONISTS, RELATED COMPOSITIONS AND METHODS FOR THEIR PREPARATION AND APPLICATION - Google Patents

Description

Cross references to related applications

This application claims priority under 35 U.S.C. 119(E) under US Provisional Patent Application No. 62/506494, filed May 15, 2017; and US Provisional Patent Application No. 62/536966, filed July 25, 2017; and US Provisional Patent Application No. 62/582186, filed November 6, 2017; and U.S. Provisional Patent Application No. 62/648,240, filed March 26, 2018, the disclosures of which are incorporated herein by reference in their entirety.

Technical field of the invention

The present invention relates to (among other things) a long-acting interleukin-15 (IL-15) receptor agonist, related compositions and methods for its preparation and use, for example, in the treatment of conditions responsive to therapy effective in promoting, for example, the activation of a robust immune response and antitumor activity.

State of the art

Interleukin-15 (IL-15) is a pleiotropic cytokine first reported by Grabstein et al. (Grabstein et al. (1994) Science 264:965-968). Secreted as a 162 amino acid precursor, human IL-15 contains a 29 amino acid leader sequence and a 19 amino acid prosequence; thus, the length of the mature protein is 114 amino acids. Belonging to a family of cytokines with a four α-helical bundle structure, IL-15 binds to a heterotrimeric receptor, where a unique α subunit (IL-15Ra) provides receptor specificity for IL-15, and the β and γ subunits of this receptor share features with one or more other cytokine receptors. Giri et al. (1995) EMBO J. 14:3654–3663.

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). In relation to the innate immune system (which generally protects the host from foreign organisms), IL-15 mediates the development of natural killer cells (NK cells) and natural killer T cells (NK-T cells) and ensures the maintenance of their survival along with with other properties. Given their role in the innate immune system, NK cells do not specifically attack an invading pathogen, but rather these cells destroy defective host cells (such as tumor cells or virus-infected cells). NK T cells produce immunomodulatory cytokines, particularly interferon-γ, which lead to a general activation of the immune response.

With regard to the adaptive immune system (which protects the host from a specific foreign organism after an initial encounter with that specific pathogen), IL-15 is required to maintain helper T cells that produce immunomodulatory cytokines. Importantly, IL-15 also promotes the long-term maintenance of antigen-trained memory T cells, which have the ability to proliferate rapidly, thereby providing a faster and stronger immune response upon repeated exposure to a specific foreign pathogen invading the host.

Finally, despite its specific roles in both the innate and adaptive immune systems, IL-15 has significant and broad effects in both categories of immune systems. Specifically, IL-15 suppresses or reduces apoptosis (or cell death) in a number of cell types (including dendritic cells, neutrophils, eosinophils, mast cells, CD4+ T cells and B cells) associated with both categories of immune systems.

Because it stimulates the proliferation and maintenance of many immune system cells that can fight cells that appear foreign (or non-self) to the host, IL-15 has been proposed for use in treatments for 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 for the treatment of forms of myeloma (Wong et al. (2013) OncoImmunology 2(11), e26442:1-3). In addition, an IL-15-based pharmacotherapeutic agent has been proposed for the treatment of individuals suffering from viral infections such as HIV infection.

Despite their 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, the signal transduction activity of IL-15 in vivo is also short-lived and optimal activity of this molecule requires daily dosing or multi-day continuous infusion, which is inconvenient. In an attempt to overcome these limitations, certain approaches involve the formation of a complex of IL-15 with the alpha subunit of the IL-15 receptor. However, this approach may abolish essential signaling that occurs uniquely through the IL-15 alpha receptor, which is expressed on many cell types. Irreversible PEGylation with a succinimidyl carbonate-terminated polymer with a relatively small molecular weight (5 kDa) has been reported, but this results in a significant change in the biological activity of IL-15. Pettit et al. (1997)J.

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Biol. Chem. 272(4):2312-2318.

However, despite the above approaches, there remains a need for new IL-15 receptor agonists with improved characteristics and profiles, such as, for example, potent immunostimulatory effects, low systemic toxicity, stability and/or improved pharmacokinetic properties. Thus, among other things, the present invention provides a long-acting IL-15 receptor agonist having a number of advantageous features that will be described in more detail below, as well as compositions and kits containing such agonist, and associated methods of preparation and use, described herein are believed to be new and not fully disclosed in the prior art.

Brief description of the invention

In a first aspect, a long-acting IL15 receptor agonist is provided herein, including pharmaceutically acceptable salt forms thereof. The long-acting IL-15 receptor agonist (IL-15-R) contains at least one linear PEG (polyethylene glycol) moiety stably covalently attached to the amino group of IL-15 via an amide bond. Between the linear PEG chain and the stable amide bond with the amino group of IL-15 there may be a linear unsubstituted alkylene group (-CH ₂ -) _m containing from 2 to 5 carbon atoms (ie m=2, 3, 4 or 5).

For example, in some embodiments, the unsubstituted alkylene group is (-CH ₂ -) ₂ ; or in some additional embodiments, the unsubstituted alkylene group is (-CH ₂ -) ₃ ; in some further embodiments, the unsubstituted alkylene group is (-CH ₂ -) ₄ ; in some further embodiments, the unsubstituted alkylene group is (-CH ₂ -) ₅ .

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

ABOUT

[CH ₃ -(OSH ₂ CH ₂ )„O-(CH ₂ ) _t C—NH-}— ^IL - ¹⁵ formula (I), where IL-15 is a fragment representing interleukin-15, n is a whole a number from about 150 to about 3000; m is an integer from 2 to 5 (for example, 2, 3, 4 or 5) and n' is 1. Formula (I) can also be written as [CH3O(CH ₂ CH ₂ O) _n (CH ₂ ) _m C(O)-NH-] _n '-IL15, and these two formulas can be used interchangeably. In Formula I (and similar formulas presented herein), the -NH- in the structure represents the amino group of the IL-15 moiety.

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.

In one or more additional embodiments, m is 2 or 3 such that the linear alkylene group separating the PEG moiety from the stable amide bond with IL-15 is -(CH ₂ ) ₂ - or (CH ₂ ) ₃ -. In some preferred embodiments, m is equal to 3.

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 value 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 (for example, n is -454), 25,000 daltons (for example, n is -568), 30,000 daltons (for example, n is -681), 40,000 daltons (for example, n is -909), 50,000 daltons (for example, n equals -1136) and 60000 daltons (for example, n equals -1364).

In one or more illustrative embodiments, a composition is provided comprising a long-acting IL-15 receptor agonist according to formula (I), including, without limitation, each of its related embodiments presented herein.

In some embodiments, the long-acting IL-15 receptor agonist composition contains no more than about 15 mole percent of the long-acting IL-15 receptor agonists, which when considered collectively are covered by the formula

ABOUT

CH ₃ -(OCH ₂ CH ₂ ) _n O-(CH ₂ ) _m C---NH-l·- ^IL - ¹ 5 ^{1 J} 2, 3 or more 3 formula (II), where the values of n and m are presented in formula (I) above. That is, with respect to the long-acting IL-15 receptor agonist component of such compositions, no more than about 15 mole percent of the long-acting IL-15 receptor agonist contained in the composition is represented by formula (II).

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For example, in some embodiments, the long-acting IL-15 receptor agonist composition contains no more than about 10 mole percent of the long-acting IL-15 receptor agonists considered collectively as covered by formula (II).

In some further embodiments of the foregoing, the composition contains no more than about 7 mole percent of long-acting IL-15 receptor agonists having an n' of 2, 3, or greater than 3 (i.e., polymers with higher numbers of PEG units, also referred to as polymers with a large number of links). In yet other embodiments, the composition contains no more than about 5 mol%, 6 mol%, 9 mol%, or 10 mol% long-acting IL-15 receptor agonists characterized by n' equal to 2, 3, or greater than 3 (t i.e. 2 or more).

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

ABOUT

[ CH3-(OSH ₂ CH ₂ ) _p O-(CH ₂ ) _t C—nh^ ^il - ¹⁵ where n and m are described above and n' represents the average number of polyethylene glycol moieties covalently attached to the amino groups of IL-15 (for the composition ), and n' for the composition ranges from 1.0 to about 1.3. For example, the average number of polyethylene glycol moieties per IL-15 moiety is selected from about 1.0, 1.1, 1.2, and about 1.3.

In yet another aspect, provided herein is a method for preparing a long-acting interleukin-15 receptor agonist such as those described in Formula (I), such as Formulas (Ia), (Ib), (Ic), (Id), and Formula (II). ), for example (IIa), (IIb), (IIc) and IId). In this method, interleukin-15 (typically dissolved in a buffer such as phosphate-buffered saline or any other suitable buffer with a pH value of about 7 (for example, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6)) are reacted with an activated PEG reagent such as methoxy-PEG succinimidyl alkanoate (where n is an integer from about 150 to about 3000) according to the following structure:

for a period of time sufficient to form where n' is 1. Exemplary methoxy-PEG succinimidyl alkanoate reagents for reacting with interleukin-15 include the following:

In some preferred embodiments, the methoxy-PEG-succinimidyl alkanoate reagent is

mPEG-succinimidyl butanoate.

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In some embodiments, the methoxy-PEG 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 dalton (for example, n is -454), approximately 25,000 dalton (for example, n is -568), approximately 30,000 dalton (for example, n is -681), approximately 40,000 dalton (for example, n is -909), approximately 50,000 dalton ( for example, n equals -1136) and approximately 60,000 daltons (for example, n equals -1364).

In one or more embodiments of the method, the methoxy-PEG-succinimidyl alkanoate reagent is added in an equimolar amount (ie, equimolar ratio) relative to interleukin-15.

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

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

In some other embodiments, the methoxy-PEG succinimidyl alkanoate reagent is dissolved in a suitable solvent. In a particular embodiment, the methoxy-PEG succinimidyl alkanoate reagent is dissolved in an aqueous acid solution, such as dilute hydrochloric acid, although any suitable acid can be used.

In some additional embodiments of the method, interleukin-15 is initially present in the solution, i.e. before mixing with the methoxy-PEG succinimidyl alkanoate reagent at a concentration of about 0.5 to about 10 mg/ml. Additional exemplary concentration ranges include, for example, about 0.5 to about 5 mg/mL interleukin-15, about 0.5 to about 3 mg/mL, and about 1.0 to about 4 mg/mL interleukin-15 .

In some further embodiments, the pH of the interleukin-15 solution is adjusted to about 8.0 before adding the methoxy-PEG succinimidyl alkanoate reagent.

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

In some further embodiments, the resulting reaction mixture is stirred (or mixed) for a period of time sufficient to allow reaction between the reactants to occur. In some embodiments, the reactants are mixed for about 15 minutes to about 10 hours, inclusive. In some additional embodiments, the reactants are mixed for about 30 minutes to about 5 hours, or about 30 minutes to about 2 hours.

In some embodiments, the reaction is carried out under ambient conditions, for example, at room temperature, i.e. in the absence of additional heating. Exemplary reaction temperature ranges include, for example, from about 5 to about 50°C, or from about 10 to about 40°C, or from about 15 to about 30°C. In some additional embodiments, the reaction is carried out at a temperature of from about 20 to about 25°C.

In some embodiments of the method, the reaction mixture is quenched by adding an amino acid. In some related embodiments, the reaction mixture is quenched by adding glycine.

In some additional embodiments of the method, the conjugation products, i.e. conjugates based on methoxy-PEG alkanoate and interleukin-15 are isolated from the reaction mixture.

In some additional embodiments, the reaction mixture containing methoxy-PEG alkanoate and interleukin-15 conjugates is purified. In some specific embodiments, the reaction produces

O [cH ₃ O(CH ₂ CH ₂ O) _n (CH ₂ ) _m C--ΝΗ-^ ^ΙΜ5 where n' equals 1.

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

ABOUT

CH ₃ -(OSH ₂ CH ₂ )„O-(CH ₂ ) _t C--NH-— ^IL ' ^{15 LJ} 2, 3 or more 3 formula (II), where the values of n and m are presented in the above formula (I) .

In still further embodiments, the reaction is effective to produce pegylated interleukin-15 that is less than 20-35% or less than about 25% deamidated.

In some embodiments, the long-acting IL-15 receptor agonist has no more than about a 7-fold reduction in EC50 value (ng/ml, pSTAT5 in CTLL-2) compared to unmodified (ie, unconjugated) IL-15. For example, in one or more related embodiments, the long-acting IL-15 receptor agonist is characterized by no more than about 6.5-fold reduction in EC50 value (ng/ml, pSTAT5 in CTLL-2), or no more than about 6-fold reduction in EC50 value (ng/ml, pSTAT5 in CTLL-2), or no more than approximately 5.5-fold decrease in EC50 value (ng/ml, pSTAT5 in CTLL-2), or no more than approximately 5-fold decrease in EC50 value (ng/ml , pSTAT5 in CTLL-2), or no more than approximately 4.5-fold decrease in EC50 value (ng/ml, pSTAT5 in CTLL-2), or no more than approximately 4-fold decrease in EC50 value (ng/ml, pSTAT5 in CTLL -2), or no more than approximately 3.5-fold decrease in EC50 value (ng/ml, pSTAT5 in CTLL-2), or even no more than approximately 3-fold decrease in EC50 value (ng/ml, pSTAT5 in CTLL-2) compared to IL -15.

In some additional embodiments, the long-acting IL-15 receptor agonist has no more than about 50% reduction in alpha receptor binding (KD, pM) compared to unconjugated IL-15, for example, when measured using a methodology suitable for determining the degree of alpha binding -receptor, such as, for example, surface plasmon resonance (SPR). In some related embodiments, the long-acting IL-15 receptor agonist is characterized by no more than about 45% reduction in the degree of alpha receptor binding ( _KD , pM), or is characterized by no more than about 40% reduction in the degree of alpha receptor binding (KD, pM), or is characterized by no more than about 35% reduction in the degree of alpha receptor binding ( _KD , pM), or even characterized by no more than about 30% reduction in the degree of alpha receptor binding ( _KD , pM) compared to unconjugated IL-15.

In still some further embodiments, the long-acting IL-15 receptor agonist has no more than about a 7-fold reduction in EC50 (ng/mL, pSTAT5 in CTLL-2) compared to unmodified IL-15 and no more than about a 50% reduction in the extent of binding alpha receptor ( _KD , pM) compared to IL-15, including any one or more specific combinations of lower EC50 values or KD values described above.

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

In still some further embodiments, a long-acting IL15-R agonist or a composition thereof is effective when administered to a subject at a therapeutically effective dose to promote NK activation and/or proliferation.

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

In another aspect, provided herein is a method of treating a condition that responds 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 a composition containing such an agonist provided herein .

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

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

Brief description of graphic materials

In fig. 1 shows the amino acid sequence of an exemplary recombinant human IL15 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.

Fig. 2 is a chromatogram illustrating the analysis of an exemplary conjugation reaction mixture by RP-HPLC as described in Example 1.

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Fig. 3 is an FPLC purification profile from an anion exchange chromatography column as described in Example 1.

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 represents the indicated molecular weight markers; lane 2 represents unconjugated parent IL-15 molecule, and lane 3 represents mono-mPEG-butanamide-IL-15.

Fig. 5 is an RPHPLC analysis of purified mono-mPEG-buganamide-IL-15 as described in Example 1.

Fig. 6 is a graph showing the concentration of the test article (IL-15, filled circle, or mPEG2-CAC-FMOC-20K-NHS-IL-15, also called N-(2-methoxy-PEGethyl)-7-(4-( (2-methoxy-PEG-ethyl)αmino)-4-oxobubul)-9-ethyl-9H-fluorene-4-carboxamide-carbamate-IL15, or conjugate 2, filled square) in the blood plasma of mice over time after administration of a single dose intravenous dose of the test article as described in Example 6.

Fig. 7 is a graph showing the average concentration of mPEG2-CACFMOC-20K-NHS-IL-15, also called N-(2-methoxy-PEG-ethyl)-7-(4-((2-methoxy-PEG-ethyl)amino )4-oxobutyl)-9-ethyl-9H-fluorene-4-carboxamide-carbamate-IL-15 or conjugate 2, in the blood plasma of rats over time after administration of a single intravenous dose of conjugate 2, with a dose value of 0.3 (), 0.15 (A) and 0.075 mg/kg (·), or a single subcutaneous dose of conjugate 2 of 0.15 mg/kg (♦), as described in example 7.

In fig. 8a and 8B illustrate the extent of STAT5 phosphorylation in various lymphocytes, i.e. CD4 T cells (), CD8 T cells (A) and NK cells (▼), after administration of a single i.v. doses of IL-15 (Fig. 8A) or conjugate 2 (0.3 mg/kg, Fig. 8B) as described in Example 8.

Fig. 9A, 9B and 9C are graphs showing the extent of STAT5 phosphorylation in various lymphocytes, i.e. CD4 T cells, CD8 T cells and NK cells, respectively, in cynomolgus monkeys after administration of a single i.v. doses of conjugate 2 (0.5 mg/kg), as described in example 9.

Fig. 10A and 10B are graphs demonstrating the in-vitro activity of exemplary long-acting IL-15 receptor agonists (Conjugates 1, 3 and 5) as measured by signaling in human PBMC NK subsets, CD56bright (FIG. 10A) and CD56dim ( Fig. 10B) NK cells, respectively, as detailed in example 10.

Fig. 11A and 11B are graphs illustrating NK cell proliferation in mice after 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, filled circles, solid line) or 1 mg/kg (high dose, diamonds), compared to vehicle as described in Example 11. FIG. 11A illustrates Ki67 expression (expressed as a percentage) over time, while FIG. 11B illustrates the numbers of NK cells (cells/μl) as a function of time after administration for each of the groups of samples.

Fig. 12A-D are graphs illustrating the increasing numbers of NK cells of all maturity levels in mice after i.v. administration of conjugate 1 at doses of 0.03 mg/kg (low dose, open squares), 0.3 mg/kg (medium dose, filled circles, solid line) or 1.0 mg/kg (high dose, diamonds) , compared to vehicle medium (filled circles, dotted line) as described in Example 11. NK cell subpopulations were determined by the expression of CD11b and CD27. In fig. 12A shows an increase in the numbers of terminally differentiated effector cells, expressed in cells/μl, from 24 to 120 hours after administration; in fig. 12B demonstrates an increase in the numbers of NK cell precursors, expressed in cells/μl, from 24 to 120 hours after administration; in fig. 12C shows an increase in the numbers of high effector cells, expressed in cells/μl, from 24 to 120 hours after administration, and FIG. 12D demonstrated an increase in the numbers of early NK cells, expressed in cells/μl, from 24 to 120 hours after administration.

Fig. 13A and 13B are graphs illustrating the expression levels of NKG2D (FIG. 13A) and granzyme B (FIG. 13B) by NK cells in mice after i.v. administration of conjugate 1 at doses of 0.03 mg/kg (low dose, open squares), 0.3 mg/kg (medium dose, filled circles, solid line) or 1 mg/kg (high dose, diamonds), according to compared to the carrier medium (filled circles, dotted line), as described in example 11.

Fig. 14 is a graph illustrating CD8 T cell counts in mice, expressed in cells/μl, both predose and from 24 to 120 hours postdose, following i.v. administering conjugate 1 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.

Fig. 15A and 15B are graphs illustrating Ki67 expression levels (expressed as a percentage) over time for effector memory T cells and central memory T cells, respectively, in mice as a function of time post-administration, after i.v. administering conjugate 1 at doses of 0.3 or 1.0 mg/kg compared to vehicle and IL-15 as described in Example 11.

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Fig. 16A and 16B are graphs illustrating NK cell proliferation in cynomolgus monkeys after 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-dose, while FIG. 16B illustrates the numbers of NK cells from pre-dose to 15 days post-dose.

Fig. 17 is a graph illustrating the numbers of CD8 T cells in cynomolgus monkeys (shown for each animal) after 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-dose.

Fig. 18A and 18B are graphs illustrating the numbers of effector memory CD8 T cells (TEM cells) and central memory CD8 T cells ( _TEM ), respectively, in cynomolgus monkeys as a function of time post-dose (from pre-dose to 14 days after administration), after iv administration of conjugate 2 at a dose of 500 μg/kg, as described in example 12.

In fig. 19 illustrates the cumulative amounts of lesions per mouse in the lungs of female Balb/c mice inoculated with CT-26 mouse 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.

In fig. 20 illustrates the cumulative amounts of lesions per mouse in the lungs of female Balb/c mice inoculated with CT-26 mouse 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 detailed in Example 13.

In fig. 21 illustrates the percentage of specific lysis depending on the E:T ratio (effector:target) at doses of 1000 ng/ml (squares), 3000 ng/ml (circles), 300 ng/ml (triangles), 30 ng/ml (upper X ), 3 ng/ml (diamonds) and without stimulation (lower X), as described in Example 14. These data illustrate a dose-dependent increase in NK cell cytotoxicity in vitro after culture with conjugate 1.

Fig. 22A-D are graphs illustrating increasing numbers of NK cells of all maturity levels in mice after 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. In FIG. 22A shows an increase in the numbers of terminally differentiated effector cells, expressed in cells/μl, from 24 to 120 hours after administration; in fig. 22B demonstrated an increase in NK cell precursor numbers expressed in cells/μl from 24 to 120 hours post-administration; in fig. 22C demonstrates an increase in the numbers of high effector cells, expressed in cells/μl, from 24 to 120 hours after administration, and FIG. 22D demonstrated an increase in the numbers of early NK cells, expressed in cells/μl, from 24 to 120 hours after administration.

Fig. 23 is a graph illustrating granzyme B expression as a function of time following treatment with Conjugate 1 at doses of 0.01, 0.03, 0.1, 0.3, 1 and 1.5 mg/kg as described in Example 15. These data indicate that treatment with conjugate 1 increases the level of granzyme B expression in NK cells.

Fig. 24 is a graph illustrating the numbers of CD8 T cells isolated from mouse spleen, expressed in cells/μl, both predose and 24 to 96 hours postdose, in mice following i.v. administration of conjugate 1 at doses of 0.03 mg/kg (filled circles, dotted line) and 0.3 mg/kg (filled circles, solid line) of IL-15 vehicle buffer (open circles, dotted line), as described in example 11.

Fig. 25 is a graph illustrating Ki67 expression levels (expressed as a percentage) for CD49b cells in mice as a function of time post-administration, after i.v. administration of conjugate 1 at doses of 0.03 or 0.3 mg/kg compared to IL-15 vehicle buffer as described in Example 11.

Fig. 26 is a graph illustrating granzyme B expression levels (expressed as a percentage) for CD49b cells in mice as a function of time post-administration, after i.v. administration of conjugate 1 at doses of 0.03 or 0.3 mg/kg compared to IL-15 vehicle buffer as described in Example 11.

Fig. 27 is a graph illustrating in vivo cytotoxicity results following treatment with Conjugate 1 at a dose of 0.3 mg/kg as described in Example 14. Cytotoxicity was assessed at 24, 48 and 72 hours after treatment.

Fig. 28A-D are graphs illustrating increasing amounts of NK

- 7 043667 cells of all levels of maturity in mice after i.v. administration of conjugate 1 at doses of 0.03 mg/kg and 0.3 mg/kg, as a single dose or after the third dose according to the q7dx3 regimen, as described in example 11. FIG. 28A shows an increase in the percentage of Ki67-expressing CD49b cells from 24 to 240 hours post-administration; in fig. 22B demonstrates an increase in the percentage of granzyme B-expressing CD49b cells from 24 to 240 hours post-administration; in fig. 28C demonstrated an increase in the number of CD49b cells, expressed in cells/μl, from 24 to 240 hours after administration; and in fig. 28D demonstrated an increase in the percentage of granB+ MFI CD49b cells from 24 to 240 hours post-administration.

Fig. 29A-C are graphs regarding the study described in Example 16. FIG. 29A is a graph showing plasma concentrations of test article (IL-15 or conjugate 1) in balb/c mice over a period of 144 hours following single intravenous dosing of 0.5 and 0.3 mg test articles. /kg respectively. Conjugate 1 has a half-life of approximately 12 hours, whereas IL-15 is rapidly cleared from plasma with a half-life of less than 1 hour. FIG. 29B is a graph showing the percentage of pSTAT5 positivity in CD8 T cells in mice following a single injection of Conjugate 1 at doses of 0.03 and 0.3 mg/kg. Conjugate 1 at both dose levels induces robust pSTAT5 signaling in CD8 T cells. A 120-hour time period is shown, including the time before dosing. Fig. 29C is a graph showing the percentage of pSTAT5 positivity in mouse NK cells following a single injection of Conjugate 1 at doses of 0.03 and 0.3 mg/kg. Conjugate 1 at both dose levels induces strong and sustained pSTAT5 signaling in NK cells.

Fig. ZOA-C are graphs showing the total numbers of CD8 T cells, central memory CD8 T cells (Tcm) and effector memory CD8 T cells ( _Tem ), respectively, after a single dose of conjugate 1 at doses of 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 induces a significant increase in the total number of CD8 T cells in the blood, as described in Example 17. The lowest dose of 0.01 mg/kg increased CD8 T _cm and CD8 T _em levels. At a dose of 0.3 mg/kg, conjugate 1 provided an increase in the number of CD8 T cells, CD8 T _cm and CD8 T _em by 6.4 times, 37.9 times and 14.5 times, respectively. It should be noted that the numbers of CD8 T cells and memory CD8 T cells did not return to baseline levels 240 hours after injection when Conjugate 1 was administered at a dose of 0.3-1.5 mg/kg, demonstrating the sustained PD effects of Conjugate 1.

Fig. 30D, 30E and 30F are graphs showing the percentage of positivity for Ki-67 in the total populations of CD8 T cells, CD8 Tcm and CD8 Tem, respectively, in mice as described in Example 17. A single dose of conjugate 1 at all dose levels provides an increase Ki-67 positivity in all CD8 T cells and CD8 T cell subsets.

Fig. 31A, 31B and 31C are graphs showing the numbers of CD8 T cells and memory CD8 T cell subsets following administration of Conjugate 1 at doses of 0.03 and 0.3 mg/kg single (dotted lines) or Q7dx3 (solid lines) as described in Example 17. Repeated dosing further increased the size of these populations with increases in CD8, CD8 T _cm , CD8 T _em by 35.3-fold, 183-fold and 73.8-fold, respectively. At the end of this time period (240 hours after the first or last dose of 0.3 mg/kg), cell numbers had not returned to baseline levels in mice.

Fig. 32A and 32B are graphs showing NK cell counts and percent positivity for Ki-67 in mice following administration of a single dose of 0.01 to 1.5 mg/kg of Conjugate 1 as described in Example 17. NK counts -cells were significantly higher than the vehicle control at all dose levels and returned to baseline levels 240 hours after dosing. All dose levels induced a robust increase in the percentage of Ki-67 positivity in NK cells.

Fig. 32C is a graph showing mouse NK cell counts following single (solid lines) or Q7dx3 (dashed lines) administration of Conjugate 1 at doses of 0.03 and 0.3 mg/kg as described in Example 17. Repeated dosing of Conjugate 1 , at 0.3 mg/kg, induced slightly lower, although still significant, numbers of NK cells compared to a single dose. Similar numbers of NK cells were achieved with a single dose compared to repeated doses of 0.03 mg/kg.

In fig. 33A illustrates an in vitro NK cell cytotoxicity assay that measures changes in NK cell-mediated lysis of target cells following test article treatment in mice as described in Example 18. Shows the percentage of specific lysis of YAC-1 cells by spleen NK cells isolated from balb mice /c treated with 0.006, 0.03 or 0.3 mg/kg of 1 or 1 mg/kg IL-15 conjugate at the indicated hours for the time period. Splenic NK cells from vehicle-dosed mice served as controls. Conjugate 1, administered at a dose of 0.3 mg/kg, induced an increase in NK cell cytotoxicity that was superior in magnitude and duration to NK cells from mice receiving a single injection of IL-15 at a dose of 1 mg/kg.

Fig. 33B is a graph showing the percentage of positivity for

- 8 043667 granzyme B in blood NK cells from the same mice used in the in vitro NK cell cytotoxicity assay in FIG. 33A (See example 18). Conjugate 1, administered at doses of 0.03 and 0.3 mg/kg, induced a significant increase in granzyme B expression levels in NK cells, with a strong and sustained increase observed at a dose of 0.3 mg/kg.

In fig. 34A and 34B illustrate the percentage of suppression of pulmonary nodule formation in balb/c mice treated with intravenous injection of CT-26 tumor cells followed by treatment with conjugate 1 at two doses of 0.03 or 0.3 mg/kg, one dose apart. week, as described in example 19. Injection of conjugate 1 at doses of 0.03 and 0.3 mg/kg suppressed the formation of pulmonary nodules by 40 and 80%, respectively. The same mice dosed at 0.3 mg/kg were monitored for 32 days after tumor cell injection to assess survival. Treatment with conjugate 1 significantly increased survival compared to mice treated with vehicle control injected with tumor cells.

Fig. 35 is a graph showing the percentage of suppression of lung nodule formation in mice injected with CT-26 and treated with Conjugate 2 that received antibody-mediated NK cell depletion (olive green), IgG control (blue), or PBS (orange). as described in Example 20. Data are presented as percent suppression of lung nodule formation compared to CT-26-injected mice that were not subjected to NK cell depletion and treated with vehicle control (black). The effectiveness of conjugate 2 in this tumor model was abolished in the absence of NK cells in mice.

Fig. 36A and 36B are graphs illustrating CD8 cell counts and percentage positivity for Ki-67 as a measure of proliferation over a two-week period of time in one male (dotted line) and one female (solid line) cynomolgus monkey following intravenous administration of Conjugate 1 in dose of 0.1 mg/kg as described in Example 21. Conjugate 1 induces a significant increase in CD8 T cells in cynomolgus monkeys with a 7-10-fold increase in cell numbers after a single dose.

In fig. 36C and 36D illustrate the increase in CD8 T _cm and CD8 T _em cell numbers in cynomolgus monkeys following a single injection of Conjugate 1 as described in Example 21. The CD8 T _cm and T _em numbers increased 27-30-fold and 21-33-fold, respectively.

Fig. 37A and 37B are graphs showing the numbers of NK cells and the percentage of positivity for Ki-67 in cynomolgus monkeys after administration of a single dose of 0.1 mg/kg of Conjugate 1 (See Example 21). The number of NK cells increased 9-10 times after treatment with conjugate 1.

Fig. 38A and 38B are EC50 curves for treating human PBMCs with IL-15 (red, filled circle) versus conjugate 1 (green, filled square) and subsequently measuring the percentage of pSTAT5 positivity in CD8 and CD56bright NK cells as described in Example 22. Conjugate 1 is 5.5 and 15 times less effective than IL-15 in activating CD8 and CD56bright NK cells, respectively. However, conjugate 1 achieves the same maximal response as traditional IL-15.

Fig. 39 is a graph showing the concentration of IL-15 administered at a dose of 500 μg/kg (green, filled circles), or conjugate 1 administered at a dose of 10 μg/kg (pink, filled squares), 30 μg/kg (purple, filled triangles pointing up), 100 μg/kg (red, filled triangles pointing down), 300 μg/kg (orange, filled diamonds) or 1000 μg/kg (dark red, open hexagons), in blood plasma over time after administration a single intravenous dose in mice as described in Example 23.

Fig. 40 is a graph showing the concentration of Conjugate 1 administered at a dose of 10 μg/kg (pink, filled squares), 75 μg/kg (purple, filled triangles pointing up), or 150 μg/kg (orange, filled triangles pointing down), in plasma over time after administration of a single intravenous dose in rats as described in Example 24.

Fig. 41 is a graph showing the concentration of IL-15 administered at 50 μg/kg (green, filled circles) or Conjugate 1 administered at 10 μg/kg (red, filled triangles upside down), 50 μg/kg ( blue, filled diamonds) or 100 μg/kg (orange, filled squares), in plasma over time after administration of a single intravenous dose of the test article in cynomolgus monkeys as described in Example 25.

Fig. 42A and 42B are graphs showing CD4 T cell counts and percent positivity for Ki-67, respectively, following administration of a single dose of Conjugate 1 of 0.03 mg/kg (blue, filled circles) or 0.3 mg/kg (orange, filled circles) in mice as described in Example 26. Cell levels during vehicle (black) and pre-dose (open circles) are also shown. At all dose levels, return to baseline occurred 240 hours after dosing. The 0.3 mg/kg dose level induced a sustained increase in the percentage of Ki-67 positivity in CD4 T cells.

Fig. 43 is a graph showing the percentage of pSTAT5 positivity in CD4 T cells in mice following a single injection of Conjugate 1 at a dose of 0.03 mg/kg (orange, filled circles) or 0.3 mg/kg (blue). , filled squares), as described in example 26.

Conjugate 1 at both dose levels induced increased pSTAT5 signaling in CD4 T cells, with 0.3 mg/kg inducing a higher increase. A 120-hour time period is shown, including vehicle (black) and predose (open circles).

Fig. 44A, 44B, and 44C are graphs illustrating cell numbers versus time postadministration for NK cells (FIG. 44A), CD8 T cells (FIG. 44B), and CD4 T cells (FIG. 44C) in cynomolgus monkeys after i.v. administration of vehicle (black) or conjugate 1 at doses of 0.003 mg/kg (blue, filled triangles pointing down), 0.01 mg/kg (green, diamonds) or 0.1 mg/kg (orange, filled squares) ), as described in example 27.

Fig. 45A, 45B and 45C are graphs illustrating the percentage of positivity for Ki-67 after administration of a single dose of conjugate 1 of 0.001 mg/kg (purple, filled squares), 0.003 mg/kg (blue, filled triangles pointing down) or 0 .1 mg/kg (orange, filled squares), in cynomolgus monkeys as described in Example 27. Levels with vehicle (black) are also shown. At all dose levels, return to baseline occurred at least 17 days after dosing. Dose levels of 0.1 mg/kg and 0.003 mg/kg induced a sustained increase in the percentage of Ki-67 positivity in NK cells and CD8 T cells. The 0.1 mg/kg dose level induced an increase in the percentage of Ki-67 positivity in all cell types tested.

In fig. 46A, 46B, and 46C illustrate the extent of STAT5 phosphorylation in lymphocytes such as NK cells (FIG. 46A), CD8 T cells (FIG. 46B), and CD4 T cells (FIG. 46C) following administration of a single i.v. doses of vehicle (black) of either 0.001 mg/kg (purple, filled squares), 0.01 mg/kg (green, diamonds) or 0.1 mg/kg (orange, filled squares) of conjugate 1 in cynomolgus monkeys, as described in example 27.

Fig. 47A-D are graphs illustrating the percentage of positivity for Ki-67 after administration of a single dose of Conjugate 1 of 0.001 mg/kg (purple, filled squares), 0.01 mg/kg (green, diamonds) or 0.1 mg /kg (orange, filled squares), in cynomolgus monkeys as described in Example 27. Levels with vehicle (black) are also shown. In fig. 47A shows results for naïve CD8 T cells, FIG. 47B shows the results for CD8 Tscm cells. In fig. 47C shows results for CD8 Tcm cells. In fig. 47D shows results for CD8 Tem cells.

Fig. 48A, 48B and 48C are graphs illustrating the expression level of granzyme B as a function of time after 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. These data indicate that treatment with Conjugate 1 provides an increase in granzyme B levels in NK cells in non-human primates (NHP).

Fig. 49A, 49B and 49C are graphs illustrating the level of perforin expression as a function of time after 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. These data indicate that treatment with Conjugate 1 provides an increase in perforin levels in NK cells in NHPs.

Detailed description

Before describing in detail one or more aspects or embodiments of the present invention, it should be noted that the present disclosure is not intended to be limited to specific synthesis procedures, fragments representing IL-15, etc., since these may vary, as will be understood by those of ordinary one skilled in the art to which the present invention relates.

In describing and claiming certain features of the present invention, the following terminology will be used in accordance with the definitions described below, unless otherwise indicated.

It should be noted that as used in this specification and the accompanying claims, the singular number includes the plural unless the context clearly indicates otherwise.

In describing and claiming one or more embodiments, the following terminology will be used as defined below.

A physiologically cleavable, or hydrolysable, or cleavable bond is a relatively unstable bond that reacts with water (ie, hydrolyzes) under physiological conditions. The ability of a bond to hydrolyze in water may depend not only on the general type of bond connecting two atoms in a given molecule, but also on the substituents attached to said atoms. Suitable hydrolytically labile or weak bonds include, but are not limited to, those in carboxylic acid esters, phosphoric acid esters, anhydrides, acetals, ketals, acyloxyalkyl ethers, imines, orthoesters, peptides, oligonucleotides, thioesters, and carbonates.

An enzymatically degradable bond means a bond that is broken down by one or more enzymes.

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Stable bonding or bonding refers to a chemical bond that is essentially stable in water, i.e. does not undergo hydrolysis under physiological conditions to any appreciable extent over a long period of time. Examples of hydrolytically stable bonds generally include, but are not limited to, carbon-carbon bonds (eg, in aliphatic chains), bonds in ethers, amides, amines, and the like. Typically, a stable bond is one that exhibits a degree of hydrolysis under physiological conditions of less than about 1-2% per day. Values for the degree of hydrolysis of illustrative chemical bonds can be found in most chemistry reference books.

A covalent release linkage, for example in the context of polyethylene glycol, which is covalently attached to an active moiety, such as interleukin-15, is a linkage that causes the polyethylene glycol polymer to be released or dissociated from the active moiety under physiological conditions, for example, through any suitable mechanism, with at a rate that is clinically applicable, and includes, for example, and without limitation, hydrolyzable bonds and enzymatically degradable bonds.

Essentially or essentially means almost entirely or completely, for example, 95% or more of the specified amount.

Likewise, approximately or approximately, as used herein, means within plus or minus 5% of the stated amount.

Optional or optional means that the circumstance described below may, but need not, occur, so the description includes cases in which the circumstance occurs and cases in which it does not occur.

A pharmaceutically acceptable excipient or pharmaceutically acceptable carrier refers to a component that can be included in the composition described herein and does not cause significant adverse toxic effects in a subject.

The phrases pharmaceutically effective amount and pharmacologically effective amount, as well as therapeutically effective amount and physiologically effective amount are used interchangeably herein and refer to the amount of the long-acting IL-15-R agonist provided herein that is necessary to provide the desired level of the substance in the bloodstream or in the target tissue to provide the desired biological or medical response. For example, such a response could be to destroy targeted cancer cells or slow or delay the progression of cancer in a subject. The term also applies to the dose that will induce a specific response in target cells. The exact amount will depend on a number of factors, such as, for example, the specific condition being treated, the intended patient population, the characteristics of the individual patient, the components and physical characteristics of the therapeutic composition to be administered, and the like.

Reference to the long-acting IL-15-R agonist described herein is intended to include its pharmaceutically acceptable salt forms.

The term patient or subject as used herein refers to a living organism suffering from or susceptible to a condition that can be prevented or treated by administration of a compound or composition provided herein. Subjects include, but are not limited to, mammals (eg, members of the families murine, simian, equine, bovine, porcine, canine, feline, etc.) and are preferably humans.

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 in this document refer to weight average molecular weight. Both definitions of molecular weight, number average and weight average, can be measured using gel permeation chromatography or other liquid chromatography techniques (eg, gel filtration chromatography). The most commonly used are gel permeation chromatography and gel filtration chromatography. Other methods for determining molecular weight include end-group analysis or measurement of colligative properties (such as freezing point depression, boiling point elevation, or osmotic pressure) to determine number-average molecular weight, or the use of light scattering, ultracentrifugation, MALDI TOF, or viscometry techniques to determine weight-average molecular weight. PEG polymers are generally polydisperse (i.e., the number average molecular weight and weight average molecular weight of the polymers are not equal), having low polydispersity values, preferably less than about 1.2, more preferably less than about 1.15, even more preferably less about 1.10, even more preferably less than about 1.05, and most preferably less than about 1.03.

The terms active, reactive or activated when used in combination with a specific functional group refer to a reactive functional group that readily reacts with an electrophile or nucleophile on another molecule. This distinguishes them from groups that require strong catalysts or very difficult reaction conditions to react (ie, a nonreactive or inert group).

As used herein, the term functional group or any synonym thereof is intended to include the protecting group forms as well as the non-protecting group forms.

The terms spacer moiety, linkage, and linker may be used herein to refer to a linkage, or an atom, or set of atoms, optionally used to link interconnecting moieties, such as the end of a polymer reagent and the IL-15 moiety. The spacer moiety may be hydrolytically stable or may include a physiologically hydrolyzable, enzymatically degradable, or otherwise releaseable bond. Unless the context clearly indicates otherwise, the spacer moiety is optionally located between any two compound elements (eg, the IL15 moiety and a water-soluble polymer such as PEG may be attached directly or indirectly by the spacer moiety).

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

Lower alkyl refers to an alkyl group containing from 1 to 6 carbon atoms, and it can be straight chain or branched, exemplified by methyl, ethyl, n-butyl, isobutyl and tert-butyl.

Alkoxy refers to the group -OR, where R represents alkyl or substituted alkyl, preferably C ₁ . ₆ alkyl (eg methoxy, ethoxy, propyloxy, etc.).

The term substituted, e.g., as in substituted alkyl, refers to a moiety (e.g., an alkyl group) substituted with one or more non-interfering substituents, such as, but not limited to, alkyl, _{C3.8cycloalkyl} , e.g., cyclopropyl, cyclobutyl, and the like. .; halogen, such as fluorine, chlorine, bromine and iodine; cyano; alkoxy, lower phenyl; substituted phenyl, etc. Substituted aryl is an aryl containing one or more non-interfering groups as a substituent. With regard to substitutions on the phenyl ring, the substituents can be in any position (ie ortho, meta or para).

Non-interfering substituents are groups which, when present in a molecule, are generally unreactive with other functional groups contained in the molecule.

Aryl means one or more aromatic rings, each with 5 or 6 carbon atoms in the nucleus. Aryl contains several aryl rings, which may be fused, as in naphthyl, or unfused, as in biphenyl. Aryl rings may also be fused to one or more cyclic hydrocarbon, heteroaryl or heterocyclic rings, or unfused. As used herein, aryl includes heteroaryl.

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

Heterocycle or heterocyclic means one or more rings of 5-12 atoms, preferably 5-7 atoms, with or without unsaturated bonds or aromatic properties, and which contain at least one atom in the ring that is not a carbon atom. Preferred heteroatoms include sulfur, oxygen and nitrogen.

Substituted heteroaryl is a heteroaryl containing one or more non-interfering groups as substituents.

A substituted heterocycle is a heterocycle containing one or more side chains formed from substituents that do not influence each other.

An organic radical as used herein will include alkyl, substituted alkyl, aryl and substituted aryl.

A pharmaceutically acceptable excipient or carrier refers to an excipient that may optionally be included in the compositions of the present invention and that does not cause significant adverse toxic effects in the patient.

The term IL-15 fragment as used herein refers to a peptide or protein fragment having human IL-15 activity. In addition, the term IL-15 fragment includes both the IL-15 fragment before conjugation and the remainder of the IL-15 fragment after conjugation. As will be explained in more detail below, one of ordinary skill in the art will be able to determine whether any specified fragment has IL-15 activity. Proteins containing an amino acid sequence corresponding to any of SEQ ID NO: 1-3 are a fragment representing IL-15, as well as any protein or polypeptide substantially homologous thereto. As used herein, the term IL-15 fragment includes those peptides and proteins that are modified intentionally, for example, through site-directed mutagenesis, or randomly through mutations. These terms also include analogues containing from 1 to 6 additional glycosylation sites, analogues containing at least one additional amino acid at the carboxy terminus of the peptide or protein, wherein the additional amino acid(s) include(s) at least one site glycosylation, and analogs having an amino acid sequence that includes at least one glycosylation site. This term includes fragments obtained naturally, recombinantly and synthetically.

The term substantially homologous or substantially identical means that a particular sequence of interest, e.g., a mutant sequence, differs from the reference sequence by one or more substitutions, deletions or additions, the cumulative effect of which does not result in an adverse functional divergence between the reference and the subject sequences. For the purposes of this document, a sequence having greater than 95% homology (identity), equivalent biological activity (though not necessarily an equivalent amount of biological activity), and equivalent expression characteristics with the specified sequence is considered substantially homologous (identical). For purposes of homology determination, truncation of the mature sequence should be neglected. Exemplary IL-15 polypeptides for use herein include sequences that are substantially homologous to SEQ ID NO: 1. SEQ ID NO: 2 is nearly identical to SEQ ID NO: 1 except that at the beginning of the sequence SEQ ID NO: : 2 contains methionine, which is necessary for the initiation of translation in E. coli.

The term component means any protein or polypeptide having the amino acid sequence of a portion or component of a fragment that is IL-15 and which has the biological activity of IL-15 or essentially has the biological activity thereof. The components include proteins or polypeptides formed by proteolytic degradation of the IL-15 moiety, as well as proteins or polypeptides formed by chemical synthesis by methods conventional in the art.

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

Review

The present invention relates to providing a long-acting IL15 receptor agonist. Ideally, such an agonist will have certain advantageous and unanticipated features, such as, for example, at least one, if not more, of the following: (i) the ability to provide sustained IL-15 activity by providing a measurable pharmacodynamic effect without the need for daily dosing, (ii) maintaining significant IL-15 α receptor binding (i.e., relative to IL-15), (iii) promoting NK cell activation and/or proliferation, and/or (iv) maintaining CD8 T survival -cells and/or formation of memory cells and (v) providing suppression of tumor growth. Surprisingly, Applicants have provided a long-acting IL-15-R agonist that has a unique combination of beneficial properties, which will be described in more detail below.

Long-acting IL-15-R agonist and related compositions Typically, a long-acting IL-15 receptor agonist or pharmaceutically acceptable salt form thereof comprises a single linear PEG (polyethylene glycol) moiety stably covalently attached to the amino group of IL-15 via amide bond. Between the PEG moiety and the stable amide bond with the amino group of IL-15 is a linear unsubstituted alkylene group (-CH ₂ -) _m containing from 2 to 5 carbon atoms (ie m=2, 3, 4 or 5).

When considering an IL-15 fragment, the term IL-15 fragment refers to the IL-15 fragment before conjugation, as well as the IL-15 fragment after attachment to a non-peptide water-soluble polymer such as such as poly(alkylene oxide) (eg poly(ethylene glycol) or PEG). Although specific reference is made below to PEG as a non-peptide water-soluble polymer, it should be understood that the present invention generally relates to a non-peptide water-soluble polymer or poly(alkylene glycol). However, it will be appreciated that if the original IL-15 fragment is attached to a polyethylene glycol moiety, there will be little IL-15 fragment

- 13 043667 changes due to the presence of one or more covalent bonds due to binding to the polymer(s).

The IL-15 fragment can be produced by non-recombinant methods and recombinant methods, and the present invention is not limited in this regard. In addition, the IL-15 moiety can be obtained from human sources, animal sources (including insects), fungal sources (including yeast), and plant sources.

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

The IL-15 fragment can be expressed in a bacterial cell-based expression system [eg, E. coli, see, eg, Fischer et al. (1995) Biotechnol. Appl. Biotechnol. 21(3):295-311], mammals [see, for example, Kronman et al. (1992) Gene 121:295-304], yeast [eg Pichia pastoris, see eg Morel et al. (1997) Biochem. J. 328(1): 121-129] and plants [see, for example, Mor et al. (2001) Biotechnol. Bioeng. 75(3):259-266]. Expression can occur as exogenous expression (where the host cell inherently contains the required elements for genetic coding) or as endogenous expression.

Although recombinant-based methods for producing proteins may vary, recombinant methods generally involve constructing a nucleic acid encoding the desired polypeptide or component, cloning the nucleic acid into an expression vector, transforming a host cell (e.g., plant, bacterial, yeast, transgenic animal cell or a mammalian cell, such as a Chinese hamster ovary cell or a newborn hamster kidney cell) and causing the nucleic acid to be expressed to produce the desired polypeptide or component. 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.

To facilitate the identification and purification of a recombinant polypeptide, nucleic acid sequences encoding an epitope tag or other affinity binding sequence may be inserted or added into frame with the coding sequence, thereby producing a fusion protein consisting of the desired polypeptide and the polypeptide suitable for binding. Fusion proteins can be identified and purified by first passing a mixture containing the fusion protein through an affinity chromatography column carrying binding moieties (eg, antibodies) directed to an epitope tag or other binding sequence in the fusion proteins, ensuring that the manner in which the fusion protein binds to the column. The fusion protein can then be isolated by washing the column with a suitable solution (eg acid) to release the bound fusion protein. The recombinant polypeptide can also be purified by lysing host cells, separating the polypeptide, for example, by ion exchange chromatography, affinity binding approaches, hydrophobic interaction approaches, and then identifying by MALDI or Western blotting and collecting the polypeptide. These and other methods for identifying and purifying recombinant polypeptides are known to those of ordinary skill in the art. However, in one or more embodiments, the IL-15 fragment is not in the form of a fusion protein.

Depending on the system used to express proteins having IL-15 activity, the IL-15 moiety may be non-glycosylated or glycosylated, and either may be used. In other words, the IL-15 fragment may be non-glycosylated, or the IL-15 fragment may be glycosylated. In one or more embodiments, the IL-15 fragment is non-glycosylated.

The IL-15 fragment can advantageously be modified to include and/or replace one or more amino acid residues, such as, for example, lysine, cysteine and/or arginine, to allow free attachment of the polymer to an atom in the side chain of the amino acid. An example of a substitution in the IL-15 fragment is described in US Pat. No. 6,177,079. In addition, the IL-15 fragment 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.

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

Exemplary fragments representing IL-15 are described herein, in the literature and, for example, in US Patent Application Publication No. 2006/0104945, Pettit et al. (1997) J Biol. Chem. 272(4):2312-2318 and Wong et al. (2013) OncoImmunology 2(11), e26442:1-3. Preferred IL-15 fragments include those with an amino acid sequence including sequences selected from the group consisting of SEQ ID NOs: 1-3 and sequences substantially homologous thereto (wherein even though SEQ ID NOs: 2 and 3 and sequences substantially homologous to them do not meet the standard of in vitro activity of the IL-15 fragment presented herein, for purposes of the present invention it will be understood that these sequences are also considered to be IL-15 fragments). A preferred IL-15 fragment has the amino acid sequence corresponding to SEQ ID NO: 1. In some embodiments, the IL-15 fragment is a functional homologue of at least about 85% or at least about 90% homologue. identical to any of SEQ ID NO: 1-3. In some embodiments, the fragment representing IL-15 is a functional homolog having at least about 95, 98, or 99% identity with any of SEQ ID NOs: 1-3.

In some cases, the fragment representing IL-15 will be in monomeric form, where one expression product of the corresponding peptide is organized into a discrete unit. In other cases, the fragment representing IL-15 will be in the form of a dimer (eg, a recombinant IL-15 dimer), where two monomeric forms of the protein are linked to each other.

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

Truncated versions, fusions, and peptide mimetics of any of the above sequences may also serve as a fragment representing IL-15. Biologically active components, deletion variants, substitution variants, or addition variants of any of the above that retain at least some degree of IL-15 activity can also serve as the IL-15 fragment.

For any given peptide or protein fragment or conjugate, it can be determined whether the peptide or protein fragment or conjugate has IL-15 activity. Various methods for determining the activity of IL-15 in vitro are described in the prior art. The typical approach is based on pSTAT analysis. Briefly, if an IL-15-dependent CTLL-2 cell is exposed to a test article having IL-15 activity, a signaling cascade is initiated that involves phosphorylation of STAT5 at tyrosine residue 694 (Tyr694), which can be quantified. Protocols and assay kits are known and include, for example, the MSD Phospho(Tyr694)/Total STATa, b Whole Cell Lysate Kit (Meso Scal Diagnostics, LLC, Gaithersburg, MD). For example, using this approach, a proposed fragment that is IL-15 that exhibits a pSTAT5 EC50 value of no more than about 300 ng/ml (more preferably no more than about 150 ng/ml) for at least 5 or 10 minutes is considered a fragment , representing IL-15 in accordance with the present disclosure. However, it is preferred that the IL-15 fragment used be more potent (eg, with a pSTAT5 EC50 value of less than 150 ng/ml for at least 5 or 10 minutes, such as less than about 1 ng/ml, and even more preferably less than 0.5 ng/ml for at least 5 or 10 minutes).

Other techniques known in the art can also be used to assess IL-15 function, including electrometric, spectrophotometric, chromatographic and radiometric techniques. See, for example, Ring et al. (2012) Nat. Immunol. 13(12): 1187-1195 for one such additional type of analysis.

Assays for use in connection with measuring the activity of the IL-15 fragment can also be used to measure the activity of the long-acting IL-15-R agonists described herein. See, for example, the supporting examples presented in this document.

- 15 043667

For purposes of the present disclosure, a compound is considered a long-acting IL-15-R agonist provided that, after administration to a subject, the agonist exhibits IL-15 agonism in vivo for a period of time greater than that of IL-15 administration. Traditional approaches, such as radiolabeling a compound, administering the compound in vivo, and determining its clearance, can be used to assess whether a compound proposed as a long-acting IL-15-R agonist is long-acting (i.e., has clearance that is longer than that of IL-15 administered in the same system in vivo). For the purposes of this document, the long-acting nature of a long-acting IL-15-R agonist can 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 an indicator, the signal is lost by approximately 24 hours for IL-15, but is persistent for a period longer than that for a long-acting IL-15 agonist.

As discussed previously, a preferred long-acting IL-15-R agonist will typically comprise a single linear PEG (polyethylene glycol) moiety stably covalently attached to the amino group of IL-15 via an amide bond. Between the PEG moiety and the stable amide bond to the amino group of IL-15 is a linear unsubstituted alkylene group (-CH ₂ -) _m having from 2 to 5 carbon atoms (ie where m=2, 3, 4 or 5).

For example, in some embodiments, the unsubstituted alkylene group is (-CH ₂ -) ₂ ; or in some additional embodiments, the unsubstituted alkylene group is (-CH ₂ -) ₃ ; in still some additional embodiments, the unsubstituted alkylene group is (-CH ₂ -) ₄ ; in some further embodiments, the unsubstituted alkylene group is (-CH ₂ -) ₅ .

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

O CH ₃ -(OSH ₂ CH ₂ ) _p O-(CH ₂ ) _t C---NH-}—- ^IL ' ¹⁵ formula (I), where IL-15 is a fragment representing interleukin-15, n is an integer from about 150 to about 3000; m is an integer from 2 to 5 (for example, 2, 3, 4, or 5) and n' is 1. In Formula I (and similar formulas presented herein), the -NH- in the structure represents the amino group of the moiety, representing IL-15. Formula (I) can also be depicted as follows, with the brackets shifted to show the terminal PEG methoxy group

O II CH ₃ O(CH ₂ CH ₂ O) _n (CH ₂ ) _m C—NH

and the two formulas can be used interchangeably. Illustrative typical compounds include the following, covered by formula (I):

[ ^eleven

CH ₃ O (CH ₂ CH ₂ O) _P CH ₂ CH ₂ C--NH

[ ^eleven

CH ₃ O (CH ₂ CH ₂ O) _P CH ₂ CH ₂ CH ₂ C--NH

[ ^eleven

CH ₃ O (CH ₂ CH ₂ O) _P CH ₂ CH ₂ CH ₂ CH ₂ C—NH

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

In some additional embodiments, with respect to the structures and formulas described herein, n is an integer from about 200 to about 2000, or from about 400 to about 1300, or from about 450 to about 1200. That is, in some embodiments n is an integer from about 200 to about 2000. In some additional embodiments, n is an integer from about 400 to about 1300. In some additional embodiments, n is an integer from about 450 to about 1200 .

PEGs with a molecular weight corresponding to any of the n value ranges mentioned above are generally preferred.

In one or more embodiments, n is an integer having a value that corresponds to a polyethylene glycol polymer with 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 more.

The following typical weight average molecular weights for the polyethylene glycol portion of the compound, in addition to those mentioned above, include about 11,000 Dalton, about 12,000 Dalton, about 13,000 Dalton, about 14,000 Dalton, about 22,500 Dalton, about 35,000 Dalton, about 45,000 Dalton, about 55,000 Dalton, about 65,000 daltons, approximately 70,000 daltons, and approximately 75,000 daltons.

In some preferred embodiments, the weight average molecular weight of the polyethylene glycol polymer moiety of the compound is approximately 40,000 daltons.

While the PEG moiety is preferably end-capped with a methoxy group as shown in Formula (I) above, the PEG moiety may be end-capped with any lower C _{1 6} alkoxy group, or may be terminated with a hydroxyl group or other suitable end-blocking group.

In some embodiments, the long-acting IL-15 receptor agonist composition contains no more than about 20 mole percent (mol%) long-acting IL-15 receptor agonists (IL-15-containing molecules in the composition), which when considered collectively are encompassed by the formula

ABOUT

[dc ₃ -(dc ₂ dc ₂ ) _p |

2.3 or more than 3 formula (II), wherein the values of n and m are represented in the above formula (I). That is, with respect to the long-acting IL-15 receptor agonist component of such compositions, no more than about 20 mole percent of the long-acting IL-15 receptor agonist contained in the composition is characterized by formula (II).

In some further embodiments, the long-acting IL-15 receptor agonist composition contains no more than about 15 mole percent (mol%) of long-acting IL-15 receptor agonists (IL-15-containing molecules in the composition), which when considered collectively are encompassed formula

O ( CH ₃ -(OCH ₂ CH ₂ ) _n O-(CH ₂ ) _m C—nh-I—— ^il -is , ¹³ formula (II), with the values of n and m presented in the above formula (I). That is, with respect to the long-acting IL-15 receptor agonist component of such compositions, no more than about 15 mole percent of the long-acting IL-15 receptor agonist contained in the composition is characterized by formula (II).In some embodiments, the composition is based on long-acting IL-15 receptor agonist contains no more than about 0.1-20 mol% of the compounds of formula (II).In embodiments, the compositions include 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.% compounds formula (II).

In certain specific embodiments as mentioned above, the long-acting IL-15 receptor agonist composition characterized by formula (Ia) contains no more than about 15 mole percent (mol%) of long-acting IL-15 receptor agonists (containing IL-15). 15 molecules in the composition), which when considered collectively are covered by the formula

ABOUT

CH ₃ O(CH ₂ CH ₂ O) _P CH ₂ CH ₂ C—NH-l— ^IL-15

I 2, 3 or more than 3 formula (Pa), with the values of n and m represented in the above formula (Ia).

In certain other preferred embodiments, the long-acting IL-15 receptor agonist composition of formula (Ib) contains no more than about 15 mole percent (mol%) of long-acting IL-15 receptor agonists (containing IL-15 receptor agonists). 15 molecules in the composition), which, when considered together, are covered by the formula:

О ¹¹ J_IL15

СН3О^НгСНгО^СНгСНгСНгС--NH--11--15

2, 3 or more than 3 formula (lib), wherein the values of n and m are represented in the above formula (Ib).

In certain other embodiments, the long-acting IL-15 receptor agonist composition of Formula (Ic) contains no more than about 15 mole percent (mol%) of long-acting IL-15 receptor agonists (IL-15-containing molecules in the composition) , which, when considered together, are covered by the formula:

ABOUT

CH3O(CH2CH ₂ O) _p CH2CH ₂ CH ₂ CH ₂ C--NH ^[L - ^{15 L} j or more 3

formula (Ps), with the values of n and m presented in the above formula (Ic).

In certain other embodiments, the long-acting IL-15 receptor agonist composition of Formula (Id) contains no more than about 15 mole percent (mol%) of long-acting IL-15 receptor agonists (IL-15-containing molecules in the composition) , which, when considered together, are covered by the formula:

ABOUT

[CH ₃ O(CH ₂ CH ₂ O) _P CH ₂ CH ₂ CH ₂ CH ₂ CH^^---NH or'more than 3 formula (IId), with the values of n and m represented in the above formula (Id).

In some embodiments, the long-acting IL-15 receptor agonist composition contains no more than about 0.1-20 mol% of compounds of formula (II), including compounds of formulas (IIa), (IIb), (IIc), and ( IId). In some further embodiments, the compositions include 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, 510, 10-20, 10-15 or 15-20 mol.% of compounds of formula (II), including compounds of formulas (IIa), (IIb), (IIc) and (IId) . In some embodiments, the compositions include no more than about 0.1, 1, 5, 10, 15, or 20 mole percent of compounds of formula (II), including compounds of formulas (IIa), (IIb), (IIc), and (IId ). It should be appreciated that the compositions can 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) in the composition.

For example, in some embodiments, the long-acting IL-15 receptor agonist composition contains no more than about 12 mole percent or no more than about 10 mole percent of the long-acting IL-15 receptor agonists considered collectively as covered by formula (II), in including compounds of formulas (IIa), (IIb), (IIc) and (IId).

In some additional embodiments of the foregoing, the composition contains no more than about 7 mol.% long-acting IL-15 receptor agonists characterized by n' equal to 2, 3, or greater than 3 (ie, polymers with a higher number of PEG units). In some other embodiments, the composition contains no more than about 5 mol% long-acting IL-15 receptor agonists characterized by n' equal to 2, 3, or greater than 3 (ie, 2 or greater).

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

ABOUT

[ CH ₃ -(OSH ₂ CH ₂ ) _p O-(CH ₂ ) _t C— ^nh 4t ^il - ¹⁵ where n and m are described above, and n' represents the average number of polyethylene glycol moieties covalently attached to the amino groups of IL-15 ( for the composition), and n' for the composition ranges from 1.0 to about 1.3. For example, the average number of polyethylene glycol moieties per IL-15 moiety is selected from about 1.0, 1.1, 1.2, and about 1.3. That is, the preferred long-acting IL-15 receptor agonist according to formula (I) may be referred to herein as mono-PEGylated, it being understood that there is some variability in the degree of PEGylation as described above. In some preferred embodiments, with respect to the formulas described herein, 18 -

mente, m equals 3.

The long-acting IL-15-R agonist composition may comprise one type of molecule where n' is approximately 1 and the PEG moiety is attached at the same position, as is the case for substantially all IL-15 conjugates in the composition, or, alternatively , may contain a mixture of mono-PEGylated conjugate molecules where the attachment of the linear polyethylene glycol moiety occurs at different sites on the interleukin-15 fragment, that is, where the specific sites of attachment are not the same for all mono-PEGylated IL-15 molecules contained in the composition. Thus, such compositions are substantially homogeneous with respect to the number of PEG moieties attached to IL-15 (eg, 1mers), but heterogeneous with respect to the positions of attachment to the amino group on the IL-15 molecule.

While additional PEG architectures and chemical linkages can be used to produce a long-acting IL-15-R agonist, compounds such as those described previously are preferred in one or more embodiments, as will become apparent when considering the supporting examples. However, additional long-acting IL-15-R agonists characterized by the structures presented herein are also contemplated.

In some embodiments, the long-acting IL-15 receptor agonist composition contains at least about 80 mol% long-acting IL-15 receptor agonists (IL-15-containing molecules in the composition) encompassed, when considered collectively, by Formula (I ), including formulas (Ia-d). In one or more embodiments, the long-acting IL-15 receptor agonist composition contains at least about 85 mol%, 90 mol%, 95 mol%, 98 mol%, or 99 mol% long-acting IL-15 receptor agonist -15 formula (I).

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

In some embodiments, the long-acting IL-15 receptor agonist composition contains no more than about 1-5 mole percent free IL-15 protein (IL15-containing molecules in the composition) when considered collectively. In some additional embodiments, the long-acting IL-15 agonist composition contains no more than about 0.5, 1, 2, 3, 4, or 5 mole percent free (ie, unconjugated) IL-15.

To produce a long-acting IL-15 receptor agonist, the IL-15 moiety, for example, can be conjugated at its amino groups (eg, lysine residues or N-terminus) to a PEG reagent functionalized with a succinimidyl group (or other ester activated group). Using this approach, succinimidyl-activated PEG can be attached to the amino groups on the IL-15 moiety in an aqueous environment at a pH of about 7.0 to 9.0, although using different reaction conditions (eg, lower pH such such as 6-7 or 7-8, or other temperatures and/or less than 15°C) may result in attachment of a PEG moiety at a different position on the IL-15 moiety.

A long-acting IL-15-R agonist can be prepared as described in Example 1. For example, a long-acting IL-15-R agonist can typically be produced by reacting interleukin-15, e.g., purified IL-15, such as recombinant IL-15, with an activated PEG reagent such as activated ester, methoxy-PEG succinimidyl butanoate, mPEG-SBA. Other suitable activated PEG reagents include methoxy-PEG-succinimidyl propionate, methoxy-PEG-succinimidyl petanoate and methoxy-PEG-succinimidyl hexanoate. Although a succinimidyl activating group is typically used, any approach

- 19 043667 A reactive ester group or activating group may be used where such a reactive group is suitable to form the desired stable amide bond. Typically, interleukin-15 is dissolved in a suitable buffer, such as, for example, phosphate buffered saline (PBS). The PEG reagent can be added in an equimolar ratio to IL-15 (relative to the molar amount of interleukin-15), typically in solution in a suitable buffer or in a molar excess (based on the molar amount of IL-15), i.e. up to approximately 15-fold molar excess, for example, 2 times molar excess, or 5 times molar excess, or 7 times molar excess, or ten times molar excess, or even 12 times molar excess, or more. In some embodiments, the PEG reagent is added in approximately 5-10 times molar excess. The PEG reagent may be added in solid form or as a solution in a suitable solvent, for example an aqueous acid solution such as dilute hydrochloric acid.

In some additional embodiments of the method, interleukin-15 is initially present in the solution, i.e. before mixing with the methoxy-PEG succinimidyl alkanoate reagent at a concentration of about 0.5 to about 10 mg/ml. Additional exemplary 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, approximately 0.5-1 mg/ml, approximately 1-10 mg/ml, approximately 1-5 mg/ml, approximately 1-4 mg/ml, approximately 1-3 mg/ ml, approximately 1-2 mg/ml, approximately 1-1.5 mg/ml, approximately 1.5-10 mg/ml, 1.5-5 mg/ml, approximately 1.5-4 mg/ml, approximately 1.5-3 mg/ml, approximately 1.5-2 mg/ml, approximately 2-10 mg/ml, approximately 2-5 mg/ml, approximately 2-4 mg/ml, approximately 2-3 mg/ml , approximately 3-10 mg/ml, approximately 3-5 mg/ml, approximately 3-4 mg/ml, approximately 4-10 mg/ml, approximately 4-5 mg/ml or approximately 5-10 mg/ml interleukin- 15 in solution. In some specific, but non-limiting embodiments, the concentration of interleukin-15 in the solution is about 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, or 10 mg/ml.

It should be noted that any suitable buffer may be used or added to the reaction mixture. Some typical buffers include sodium phosphate (NaPi), sodium acetate (NaAc), borate, bicine, citrate and Bis-TRIS buffers.

In some embodiments, the pH of the IL-15 solution is adjusted to approximately pH 8 before adding the PEG reagent.

After adding the PEG reagent, the reaction mixture can then be adjusted to a suitable pH, for example, from about 7.0 to 8.5 or to about 8.0, if necessary. In some embodiments, the reaction mixture is adjusted to a pH of about 7.0-8.0 or about 7.4-8.5. In some specific embodiments, the reaction mixture is adjusted to a pH of approximately 8.0. It should be noted that pH adjustments can be made either before or after the addition of PEG reagent as needed to achieve the desired pH.

Interleukin-15, like many proteins, undergoes deamidation, especially at higher pH levels, while lower pH levels can lead to a number of 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 the protein, which can lead to changes in protein activity, structure, function, stability and/or can change the protein's susceptibility to degradation. Thus, one of the objectives addressed by these agonists and related methods has been to provide a long-acting interleukin-15 receptor agonist that maintains a sufficient degree of activity (i.e., is therapeutically useful) while maintaining a balance in terms of 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 may, for example, result in decreased interleukin-15 activity), and (iii) protein aggregation (eg, before and after conjugation), among others.

Based on the competing and controversial issues associated with the reaction parameters for producing the long-acting interleukin-15 receptor agonist described herein, the applicants have discovered that by adjusting the pH of the interleukin-15 solution (before or after reaction with the PEG reagent) and/or the reaction mixtures of IL-15 with a PEG reagent can achieve optimal (lower levels) deamidation while still promoting conjugation of the PEG moieties to the interleukin-15 moiety (e.g., at the ε-amines as well as the N-terminus) to ensure a long-acting IL-15-R agonist described herein. Without wishing to be bound by theory, based on a series of reactions in which numerous reaction parameters were varied, it appears that the pH range is from about 7.0 to about 8.5, or from about 7.5 to about 8.2, or from about 7. 8 to about 8.2, or at about 8.0 is effective in providing lower levels of deamidation in the product while promoting conjugation of PEG moieties to form the product described herein, which also maintains the desired therapeutic profile.

For example, the methods described herein are effective for producing pegylated interleukin-15 that is less than about 35% deamidated, or in some embodiments less than about 30% deamidated, or less than about 25% deamidated, or deamidated less than approximately 20%. In some embodiments, the level of deamidation of the product is in the range of about 20 to 35%, or is in the range of about 20 to 25%, or is in the range of about 25 to 35%, or is in the range of about 25 to 30%. As an alternative, some embodiments provide pegylated interleukin-15 having lower degrees of deamidation than those noted above. As shown in Experiment 2 of Example 1, adjusting the pH to a range of approximately 7.0 to 8.5 resulted in a deamidation level of 21.29% (Composition 1) or 33.26% (Composition 2).

The reagents are typically mixed for approximately 5-10 hours inclusive. In some embodiments, the reagents are mixed for approximately 2-5 hours inclusive. In some embodiments, the reagents are mixed for approximately 2 hours inclusive. In some exemplary embodiments, the reactants are mixed for about 30 minutes to about 3.0 hours, or about 30 minutes to 2.5 hours, or about 30 minutes to 2 hours, or about 30 minutes to 1.5 hours , or from about 45 minutes to about 3.0 hours, or from about 45 minutes to about 2.5 hours, or from about 45 minutes to about 2.0 hours, or from about 45 minutes to about 1.5 hours, or from about 45 minutes to about 1.0 hour. Mixing is generally carried out under moderate conditions, for example, from about 20 to about 65°C, or from about 20 to about 40°C, or at ambient or room temperature (for example , approximately 22°C). Lower temperatures can be used to promote a lower degree of PEGylation. The reaction mixture can be quenched, for example, by adding an amino acid such as glycine.

In embodiments, the pH of the composition can be further adjusted to reduce deamidation. In some embodiments, the composition is adjusted to a pH of approximately 6.5-7.5 or 6.5-7.0. In some embodiments, the composition is adjusted to a pH of approximately 6.5, 6.8, 7.0, or 7.5.

The reaction product, PEGylated rIL-15, typically can then be purified by any suitable method, such as, for example, ion exchange chromatography, to obtain the desired product. For example, anion exchange chromatography can be used. The pool of chromatography products can then be concentrated and diafiltered in a suitable formulation buffer (eg, sodium acetate sucrose buffer) using, for example, tangential flow filtration (TFF). The analysis can be carried out by any suitable method, such as, for example, SDS-PAGE, reverse phase HPLC or any other suitable analytical method.

As described previously, the amino groups on the IL-15 moiety provide a binding site between the IL-15 moiety and the polyethylene glycol moiety to provide long-acting IL-15-R agonists such as those covered by formula (I). For example, when considering the exemplary amino acid sequences of IL-15 presented herein, it is apparent that there are seven lysine residues, each characterized by the presence of an ε-amino acid that may be available for conjugation. In addition, the N-terminal amine of methionine can also serve as an attachment point to the PEG moiety. It should be noted that the polyethylene glycol moiety may be attached at any one or more lysine or N-terminal amine positions. In some embodiments, the attachment site of the polyethylene glycol moiety is on one or more of Lys ¹⁰ and Lys ¹¹ (using the numbering shown in SEQ ID NO: 2 as an example, or Lys ¹¹ and Lys ¹² using SEQ ID NO: 1). In some embodiments, the polyethylene glycol moiety is attached at the N-terminal amine. It should be noted that any of the lysine positions may be suitable as an attachment site (eg Lys ³⁷ or Lys ⁴² of SEQ ID NO: 1) for the PEG moiety. In some embodiments, the long-acting interleukin-15 receptor agonist comprises a mixture of positional isomers, wherein the covalent attachment of the polyethylene glycol moiety occurs predominantly at the N-terminus (i.e., of the totality of positional isomers, the isomer with the PEG moiety attached at the N-terminus is present in the greatest proportion amount compared to other positional isomers).

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

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

These long-acting IL-15-R agonists have been found to have certain significant and beneficial properties. Although the features described below are believed to apply generally to the compounds provided herein and covered by formula (I), the following one or more features may be exhibited in particular by compounds of formula (Ib) and, accordingly, formula (IIb ). A long-acting IL-15-R agonist may have 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, pSTAT5 in CTLL-2) compared to unmodified IL-15. For example, in one or more related embodiments, the long-acting IL-15 receptor agonist is characterized by no more than about 6.5-fold reduction in EC50 value (ng/ml, pSTAT5 in CTLL-2), or no more than about 6-fold reduction in EC50 value (ng/ml, pSTAT5 in CTLL-2), or no more than approximately 5.5-fold decrease in EC50 value (ng/ml, pSTAT5 in CTLL-2), or no more than approximately 5-fold decrease in EC50 value (ng/ml , pSTAT5 in CTLL-2), or no more than approximately 4.5-fold decrease in EC50 value (ng/ml, pSTAT5 in CTLL-2), or no more than approximately 4-fold decrease in EC50 value (ng/ml, pSTAT5 in CTLL -2), or no more than approximately 3.5-fold decrease in EC50 value (ng/ml, pSTAT5 in CTLL-2), or even no more than approximately 3-fold decrease in EC50 value (ng/ml, pSTAT5 in CTLL-2) compared to IL-15. Exemplary long-acting IL-15-R agonists consistent with the above features are described herein and in the accompanying Examples.

As described in Example 10, the in vitro activity of exemplary conjugates (1, 3 and 5) induces IL-15 signaling in huPBMC, with conjugate 1 effectively inducing such signaling. Additional experiments were performed to examine the in vitro activity of Conjugate 1 against human CD8 T cells, NK cells and CD4 T cells (Examples 16, 22 and 26-27). As shown in FIG. 10A-10B, at least conjugate 1 induced similar or enhanced signaling compared to IL-15 in CD56bright and CD561ow cells. Although conjugate 1 was less effective than IL-15 in recruiting CD8 and CD56bright NK cells (Example 22), it is important to note that conjugate 1 achieved the same maximal response as conventional IL-15 (see Fig. 38A- 38B). As described for the mouse model in Example 16, a single injection of conjugate 1 at two different doses induced robust pSTAT signaling in CD8 and NK cells. As described for the mouse model in Example 26, a single injection of conjugate 1 resulted in an increase in % pSTAT5 compared to IL-15. In mouse models, NK cells were the most sensitive to a single dose of the conjugate, followed by CD8 T cells, and CD4 T cells were the least sensitive of the cells tested.

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

In some additional embodiments, the long-acting IL-15 receptor agonist exhibits no more than about 50% reduction in alpha receptor binding (KD, pM) compared to IL-15. That is, in certain related embodiments, the long-acting IL-15 receptor agonist exhibits no more than about 45% reduction in alpha receptor binding (KD, pM), or exhibits no more than about 40% reduction in alpha receptor binding (KD, pM) , or exhibits no more than about a 35% reduction in alpha receptor binding (KD, pM), or even exhibits no more than about a 30% reduction in alpha receptor binding (KD, pM) compared to IL-15.

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

- 22043667 pM) compared to IL-15, including any one or more specific combinations of reductions in EC50 values or _KD values described above.

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

A carbohydrate such as a sugar, a derivatized sugar such as an alditol, an aldonic acid, an esterified sugar and/or a sugar-based polymer may be present as an excipient. Specific carbohydrate excipients include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose and the like; polysaccharides such as raffinose, melicitose, maltodextrins, dextrans, starches and the like; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucite), pyranosyl sorbitol, myoinositol, cyclodextrins and the like.

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

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

An antioxidant may also be present in the composition. Antioxidants are used to prevent oxidation, thereby preventing the decomposition of the conjugate or other components of the drug. Suitable antioxidants for use in one or more embodiments of the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

A surfactant may be present as an excipient. Typical surfactants include polysorbates such as Tween 20 and Tween 80, and pluronics such as F68 and F88 (both of which are available from BASF, Mount Olive, NJ); sorbitan esters; lipids such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (but preferably not in liposomal form), fatty acids and fatty acid esters; steroids such as cholesterol; and IL-15 chelating agents such as EDTA, zinc and other such suitable cations.

Acids or bases may be present in the composition as excipients. Non-limiting examples of acids that can be used include acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid and combinations thereof. Examples of suitable bases include, but are not limited to, those selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate and combinations thereof.

One or more amino acids may be present as an excipient in the compositions described herein. Exemplary amino acids include arginine, lysine and glycine. Additional suitable pharmaceutically acceptable excipients include those described, for example, in Handbook of Pharmaceutical Excipients, ^7th ed., Rowe, RC, Ed., Pharmaceutical Press, 2012.

The amount of long-acting IL-15-R agonist contained in the composition will vary depending on a number of factors, but will optimally represent a therapeutically effective dose if the composition is stored in a unit dose container (eg, an ampoule). In addition, the pharmaceutical preparation can be placed in a syringe. The therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the long-acting IL-15-R agonist to determine the amount that produces the clinically required expected result described herein. The amount of any individual excipient in the composition will vary depending on the activity of the excipient and the specific requirements of the composition. Typically, the optimal amount of any particular excipient is determined by routine experimentation, i.e. by preparing compositions containing varying amounts of excipient (ranging from low to high), testing stability and other parameters, and then determining the range at which optimal effectiveness is achieved without significant adverse effects.

A long-acting IL-15-R agonist is suitable for administration to a patient suffering from a condition that responds to treatment with interleukin-15. The method involves administering to a patient, typically parenterally, a therapeutically effective amount of a long-acting IL-15-R agonist (preferably provided as part of a pharmaceutical composition). As described previously, a long-acting IL-15-R agonist can be administered parenterally (eg, intramuscularly, subcutaneously, intravenously, or intraperitoneally). Suitable types of formulations for parenteral administration include, but are not limited to, solutions ready for injection, dry powders for combination with a vehicle before use, suspensions ready for injection, dry insoluble compositions for combination with a vehicle before use, and emulsions and liquid concentrates for dilution before administration. . In some specific embodiments, the long-acting IL-15 receptor agonist is provided in a formulation suitable for intravenous administration and is administered intravenously. In some other embodiments, the long-acting IL-15 receptor agonist is provided in a formulation suitable for subcutaneous administration and is administered subcutaneously.

The route of administration of a long-acting IL-15 receptor agonist (eg, provided as part of a pharmaceutical composition) may not necessarily be designed to limit the localization of the agonist to a specific region. For example, liquid, gel and solid formulations containing an agonist can also be surgically implanted into an affected area (such as into a tumor, peritumorally, into an area of inflammation and around an area of inflammation). It is advisable that organs and tissue can also be visualized to ensure that the desired location is better exposed to the conjugate.

The route of administration can be used to treat any condition that can be treated or prevented by administration of a long-acting IL-15-R agonist, such as, for example, cancer. For example, a long-acting agonist may be used either alone or in combination with another pharmacotherapeutic agent to treat patients suffering from a condition that may respond to IL-15 therapy, such as, for example, cancer.

As used herein in relation to the treatment of a subject having cancer, the terms treatment, treat and treating include the full range of effects applied to the cancer that the subject has, such as administering a combination to alleviate, delay, stop or reverse one or more symptoms of cancer or to delay the progression of cancer, even if the cancer is not actually eliminated. Treatment may include, for example, reducing the severity of a symptom, the number of symptoms, or the frequency of relapses, such as inhibiting tumor growth, arresting tumor growth, or regressing existing tumors.

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

The terms cancer and cancerous refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth.

Tumor and solid tumor, as used herein, refer to all lesions, as well as the growth and proliferation of neoplastic cells, whether malignant or benign, as well as all precancerous and cancerous cells and tissues.

Typical conditions are cancers such as, for example, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, carcinoma colon cancer, pancreatic cancer , breast cancer, ovarian cancer, colon cancer, prostate cancer, squamous cell cancer, basal cell cancer, head and neck cancer, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary cancer, papillary adenocarcinomas, cystadenocarcinoma, medullary cancer, bronchogenic cancer, renal cell cancer, hepatoma, bile duct cancer, choriocarcinoma, seminoma, embryonal cancer, Wilms tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma (including, for example, uveal melanoma, mucosal melanoma and leptomeningeal melanoma), neuroblastoma, retinoblastoma and leukemias.

In one particular method, a long-acting IL-15-R agonist is used to treat a hematologic malignancy such as leukemia or lymphoma. In yet another method, a long-acting IL-15-R agonist is used to treat solid cancer.

In some 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.

In an illustrative mouse model, as described in Example 11, Conjugate 1 was effective in inducing proliferation and sustained increases in NK cell numbers, as evidenced by increased cell counts (cells/μL, as shown in Figures 12B-12C), as well as increase in % Ki67 (eg Fig. 11A). % Ki67 is used as a marker for proliferating cells. As can be seen in FIG. 12A-12D, increased cell numbers were observed in NK cells of all levels of maturity (end effector cells, NK progenitor cells, high effector cells and early NK cells). As shown in Example 17, the increase in NK cell counts was sustained at all dose levels for at least 96 hours, with medium and high dose levels maintained for at least 144 hours. Administration of Conjugate 1 in a mouse model induced an increase in % Ki67 at all dose levels compared to vehicle, which was maintained for at least 120 hours. For % Ki67, at least the intermediate dose ranges (eg 0.1 and 0.3 mg/kg) induced an increase in % Ki67, which was maintained for at least 144 h.

The effects of Conjugate 1 can be induced and can be maintained with a single dose. In an exemplary mouse model, as described in Example 11, an increase in cell number was induced and maintained upon administration of a single dose of Conjugate 1. With a single dose, %Ki67 levels were induced and sustained in murine CD49b cells compared to repeated (eg Q7dx3) dosing at the same level (see FIG. 28A).

In some additional embodiments, the long-acting IL-15-R agonist or composition, when administered at a therapeutically effective dose, is effective in increasing NK cell activation, as evidenced by increased expression of a cytotoxic protease by NK cells. In an illustrative non-human primate model as described in Example 28, NK cell expression of the cytolytic enzymes granzyme B and perforin was induced and enhanced by a single dose of conjugate 1. As seen in FIG. 48A-48C, all dose levels increased granzyme B expression, and intermediate/higher doses at least tripled expression (MFI) compared to pre-dose levels. As can be seen in FIG. 49A-49C, all dose levels increased perforin expression, and intermediate/higher doses doubled expression (MFI) compared to pre-dose levels. Thus, conjugate 1 is effective in enhancing the cytotoxicity of NK cells.

In still some further embodiments, the long-acting IL15-R agonist or composition is effective, when administered at a therapeutically effective dose to a subject, to support CD8 T cell survival and memory formation.

In an exemplary mouse model, a single dose at all dose levels of Conjugate 1 enhanced cellular proliferation as evidenced by increased % positivity for Ki-67 in CD8 cells (Figure 30D), as well as in the central memory cell and effector memory CD8 cell subpopulations (Figure 30D). .30E-30F). As shown in Example 17, administration of Conjugate 1 in a mouse model induced a significant increase in the total number of CD8 T cells in the blood (see FIG. 30A). The lowest dose increased CD8 T _cm and CD8 T _em (see Fig. 30B-30C). In an exemplary mouse model, a single iv injection of conjugate 1 maintained increased cell numbers compared to vehicle administration for at least 240 hours (see FIG. 30A, for example). It should be noted that the numbers of CD8 T cells and memory CD8 T cells do not return to baseline levels 240 hours after injection with conjugate 1 at some dose levels. Thus, conjugate 1 maintains memory CD8+ T cell populations over an extended period. A single dose of conjugate 1 at all dose levels also enhanced Ki-67 positivity in all CD8 T cells and CD8 T cell subsets, indicating increased proliferation of these cells. Repeated dosing of conjugate 1 resulted in further increases in the CD8, CD8 T _cm and CD8 T _em populations (see FIGS. 31A-31C). Repeated dosing also resulted in long-term increases in cell proliferation for at least 240 hours for each of the CD8 cell populations, CD8 T _cm and CD8 T _em in mice.

Conjugate 1 also induced proliferation and sustained increases in NK cells and CD8 T cells in a non-human primate model (cynomolgus model in Example 27) compared to vehicle administration. Each dose level of Conjugate 1 induced and maintained an increase in NK cell counts for at least 14 days (see FIG. 44A). Conjugate 1 induced and sustained increases in CD8 T cells for at least 10 days at each dose level.

In one or more additional embodiments, the IL-15-R agonist is administered intravenously. In still further embodiments, the IL-15-R agonist is administered subcutaneously.

- 25 043667

In still some further embodiments, when administered, the IL-15-R agonist is effective in inducing sustained signaling in lymphocytes that results in proliferation of CD8 T cells and/or preferential expansion of the central memory CD8 cell population.

The actual dose to be administered will vary depending on the age, weight and general condition of the subject, as well as the severity of the condition being treated, the judgment of the attending physician and the conjugate to be administered.

Therapeutically effective amounts are known to those skilled in the art and/or are described in the reference texts and literature on the subject. In general, a therapeutically effective amount will range from about 0.001 to 100 mg, preferably in doses from 0.01 to 75 mg/day, and more preferably in doses from 0.10 to 50 mg/day. This dose may be administered periodically until, for example, the physician determines that the appropriate expected result (eg, cure, regression, partial regression, etc.) has been achieved.

In some embodiments, the therapeutically effective dose ranges from about 0.25 to 25 μg/kg. In other embodiments, the therapeutically effective dose ranges from about 0.25 μg/kg to about 0.1 mg/kg, from about 0.01 to about 0.1 mg/kg per day, or from about 0.03 to about 0. 1 mg/kg per day. In other embodiments, the therapeutically effective dose ranges from about 1 to 10 μg/kg, from about 0.03 to about 0.1 mg/kg. In some specific, but non-limiting embodiments, the therapeutically effective dose is approximately 0.25 μg/kg, 0.3 μg/kg, 0.5 μg/kg, 1 μg/kg, 2 μg/kg, 3 μg/kg, 5 mcg/kg, 6 mcg/kg, 7 mcg/kg, 10 mcg/kg, 15 mcg/kg, 20 mcg/kg, 25 mcg/kg, 0.01 mg/kg, 0.03 mg/kg, 0 .05 mg/kg or 0.1 mg/kg. With reference to the doses indicated in the examples provided herein, one skilled in the art can convert animal (e.g., mouse) doses to the corresponding human dose using conversions known in the art (e.g., Nair et al., J. Basic and Clin Pharmacy (2016) 7:27–31).

A dosage unit of any said conjugate (here also preferably provided as part of a pharmaceutical preparation) may be administered in a variety of dosage regimens depending on the judgment of the clinician, the needs of the patient, etc. The specific dosage schedule will be known to one skilled in the art or can be determined experimentally using conventional methods. Exemplary dosage regimens include, but are not limited to, once daily, three times weekly, twice weekly, once weekly, twice monthly (eg, every 14 days), once monthly (eg, every 30 or 31 day or every 21 days) and any combination thereof. Once the desired clinical result is achieved, the dosage of the composition is stopped or reduced. In some embodiments, a unit dose of any given conjugate can be administered once with sustained effect.

It should be understood that although the present invention has been described in connection with its preferred specific embodiments, the foregoing description, as well as the examples that follow, are provided to illustrate and not limit the scope of the present invention. Other aspects, advantages and modifications within the scope of the present invention will be apparent to those skilled in the art to which the present invention relates.

All articles, books, patents and other publications referenced herein are hereby incorporated by reference in their entirety. In the event of a discrepancy between the teachings of this specification and the prior art incorporated by reference, the meaning of the teachings and definitions in this specification shall control (especially with respect to the terms used in the claims appended herein). For example, if this application and the incorporated publication define the same term differently, then the definition of that term should be kept within the scope of the document in which the definition is found.

Examples

It should be understood that the above description, as well as the following examples, are intended to illustrate and not to limit the scope of the present invention presented herein. Other aspects, advantages and modifications will be apparent to those skilled in the art to which the present invention relates.

In the following examples, efforts have been made to ensure accuracy with respect to the numbers used (eg quantities, temperatures, etc.), but some experimental error and variation should be taken into account. Unless otherwise noted, temperature is expressed in degrees C and pressure is atmospheric pressure at or approximately sea level. Each of the following examples is intended to provide instruction to one skilled in the art regarding the performance of one or more embodiments described herein.

Materials and methods

Recombinant IL-15 (rIL-15) of SEQ ID NO: 1 (as shown in FIG. 1), prepared using conventional techniques, was used in the following examples, although any suitable

- 26 043667 fragment representing IL-15 can be used in a similar way. SEQ ID NO: 1, which is a recombinant human IL-15 derived from E. coli, is a single non-glycosylated polypeptide chain containing 115 amino acids, with a molecular weight of 12.9 kDa.

The reactive polymer reagent linear mPEG-succinimidylbutanoate weighing 40 kDa (mPEG-SBA) is characterized by the following structure:

Oh Chh

CH ₃ O-(CH2CH2O) _n -CH2CH2CH ₂ CON ^/ | where n corresponds to the number of monomer subunits providing a polymer with a weight average molecular mass of approximately 40 kilodaltons, i.e. where n equals ~909. Additional mPEG succinimidyl butanoate reagents suitable for use include those having weight average molecular weights, for example, about 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 40 kDa, 50 kDa, or 60 kDa. This activated polymer reagent, when reacted with amino groups of IL-15 (eg, lysines or N-terminus), effectively forms a stable amide bond between the IL-15 moiety and the polyethylene glycol moiety.

The reactive fluorenyl-PEG reagent, PEG2-CAC-FMOC-20kD-NHS, is characterized by the following structure:

where mPEG is methoxy(polyethylene glycol), and the weight average molecular weight of the polymer reagent is approximately 20 kDa (ie, with each mPEG fragment having a weight average molecular weight of approximately 10 kDa). Additional PEG2-CAC-FMOC reagents having different molecular weights are named accordingly, for example, PEG2-CAC-FMOC-10kDNHS, PEG2-CAC-FMOC-15kD-NHS, PEG2-CAC-FMOC-30kD-NHS, PEG2-CAC- FMOC-40kD-NHS, wherein such reagents have the structure shown above, and differ only in the molecular weight of the mPEG fragments attached to the FMOC core.

SDS-PAGE Analysis

Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using an Invitrogen gel electrophoresis system (XCell SureLock Mini-Cell). Samples were mixed with sample buffer. The resulting samples were then loaded onto a preformed NuPAGE Novex gel and run for approximately 30 min.

RP-HPLC Analysis

Reverse-phase chromatography (RP-HPLC) analysis was performed on an Agilent 1200 HPLC system (Agilent). Samples were analyzed using a Poroshell 300SB-C3 column (2.1 x 75 mm, Agilent) at 60°C. The mobile phases used were 0.1% TFA/H2O (A) and 0.1% TFA/CH ₃ CN (B). The column flow rate was 0.5 mL/min. Eluted protein and PEG-protein conjugates were detected using UV at 280 nm.

Bioassays

Efficacy assay (mouse T cell) based on STAT5 phosphorylation in CTLL-2 cells

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

One day prior to analysis, CTLL-2 cells were selected and placed in fresh growth medium [RPMI 1640 supplemented with 10% FBS, 10% T Cell Culture Supplement (cat. no. 354115, Corning, Inc., Tewksbury, MA), 2 mM L-glutamate and 1 mM sodium pyruvate]. On the day of assay, cells were preincubated in assay media (RPMI 1640 supplemented with 1% FBS, 2 mM L-glutamate, and 1 mM sodium pyruvate) for at least 4 h and then plated in assay media in a 96-well plate at 50,000 cells. /hole. Dilutions of the test article were prepared in the appropriate buffer immediately prior to analysis. Stimulation of CTLL-2 cells was initiated by transferring 25X solutions of the test article into wells containing CTLL-2 cells in triplicate. The plates were incubated at 37°C, 5% CO2 for 10 minutes, and the reaction was stopped by cell lysis. Detection of phospho-STAT5 and total STAT5 protein levels in cell lysates was performed using the MSD Phospho(Tyr694)/Total STATa, b Whole Cell Lysate Kit (Meso Scale Discovery, Maryland). After 10 minutes of treatment, recombinant human IL-15 obtained from PeproTech exhibited IL-15 activity by inducing STAT5 phosphorylation in CTLL-2 cells with an average EC50 value of 0.27 ng/ml, which served as a control.

HuPBMC-pStat5 Assay

The efficacy of IL-15 or long-acting IL-15-R agonists on various human lymphocyte subsets was determined using a phosphoSTAT5(Y694) dose response assay. Frozen human PBMCs from multiple donors were obtained from AllCells. 1 x 10 ⁶ cells/100 μl were cultured in complete RPMI medium for 2 h and then incubated with the indicated concentrations (serial dilutions from 10,000 ng/ml to 0.001 ng/ml) of IL-15 or conjugates for 20 min at 37°C C. Cells were then fixed (using BD Cytofix), permeabilized (using 100% pre-chilled methanol) and stained with antibodies to CD3, CD4, CD8, CD4 regulatory T cells (CD4+ CD25+ Foxp3+), CD56 and phosphorylated STAT5 (Y694) before analysis by the method flow cytometry. Concentration-response relationships were used to calculate EC50 values.

Receptor affinities of long-acting rIL-15 receptor agonists for IL-15Ra

The affinities of IL-15 and exemplary long-acting rIL-15 receptor agonists were measured using surface plasmon resonance (SPR) using the BIAcore™ SPR system. Briefly, the surface of the touch chip

Biacore CM5 was activated using a 1:1 mixture of NHS:EDC to form active NHS esters. Goat anti-human immunoglobulin Fc antibody was covalently attached to the surface by injecting it for five minutes in 10 mM sodium acetate (pH 4). Approximately 8000 EU of antibodies bound to the surface. Any remaining NHS ester was then quenched with ethanolamine.

At the initiation of each injection cycle, IL-15-Ra-Fc was captured in the sensor chip channel through a five-minute injection step in PBSP. Typically, 150-200 EO of receptors were bound to the surface.

The long-acting rIL-15 receptor agonist test articles were diluted to 10 μM in PBS (containing 0.05% Tween 20 and 0.1 mg/ml BSA). A series of 3-fold dilutions were prepared and injected into a sensor chip that was coated with IL-15Ra. Affinities were measured by determining the rates k _a and kd separately, and the ratio of kd to k _a was used to calculate Kd values.

Example 1: Preparation of a long-acting IL-15 receptor agonist

O II H ₃ C-(OCH2CH2)p-O-CH2CH ₂ CH2-C—NH--(IL-15) mono(methoxyPEO-M-butanamide) _{40 k} daInter leukin-15.

Experiment 1.

2.7 ml of IL-15 solution (1.23 mg/ml in PBS buffer, pH 7.4) was transferred to a small reaction vial. 300 μl of 0.6 M borate buffer pH 8 was added to adjust the pH to pH 8. mPEG-SBA, 40 kDa, stored at -20°C under nitrogen, was warmed to ambient temperature. A tenfold excess (relative to the molar amount of IL-15) of mPEG-SBA-40K was dissolved in 2 mM HCl to form a 10% PEG reagent solution. Quickly add the 10% PEG reagent solution to the IL-15 solution and mix well. After addition of mPEG-SBA-40K, the pH of the reaction mixture was determined and adjusted to pH 8 using conventional techniques. To ensure binding of mPEG-SBA-40K to IL-15 (i.e., via formation of a stable amide bond), the reaction solution was placed on a low-speed laboratory rotator for 1.5 h to promote conjugation at room temperature. The reaction was quenched by adding a glycine solution.

Fig. 2 shows the chromatogram after RP-HPLC analysis of the conjugation reaction mixture. The compounds produced in the reaction were 40% monoconjugate (i.e., having one PEG moiety attached to IL-15), 24% diconjugate (having two PEGs attached to IL-15), and 6% triconjugate (having three PEGs , attached to IL-15). Approximately 30% of unreacted IL-15 remained in the reaction mixture, although reaction conditions were not optimized.

The desired monoconjugate was separated/isolated by anion exchange chromatography using a Q Sepharose High Performance column and sodium phosphate buffers as the eluent phase. Purified mono-mPEG-SBA40K-IL-15 conjugate (also referred to herein as mono-mPEGbutanamide-40K-IL-15, or mono(methoxy-PEG-N-butanamide) _{40 kda} interleukin-15, or mono-mPEG _40K - C4amide-IL-15) was characterized by HPLC and SDS-PAGE. In other examples, purified mono

- 28 043667 mPEG-SBA40K-IL-15 is called conjugate 1.

Fig. 3 is an FPLC purification profile from an anion exchange chromatography column. Fig. 4 shows an SDS gel of the purified mono-mPEG-SBA-40K-IL-15 conjugate. As shown by the gel, the purified conjugate is of high purity and contains no unreacted IL-15. Fig. 5 shows RP-HPLC analysis of purified mono-mPEG-SBA-40K-IL-15. As can be seen from the HPLC result, the purified mono-mPEG-SBA-40K-IL-15 contains less than approximately 10% (molar amount) of the di-conjugate or higher level conjugates.

Using this approach, conjugates such as mono-mPEG-SBA-10K-IL-15, mono-mPEG-SBA-15K-IL-15, mono-mPEG-SBA-20K-IL-15, mono-mPEG-SBA are obtained -25K-IL-15, mono-mPEGSBA-30K-IL-15, mono-mPEG-SBA-40K-IL-15, mono-mPEG-SBA-50K-IL-15 and mono-mPEG-SBA-60K-IL -15, using mPEG-SBA having different weight average molecular weights.

Experiment 2.

A solution of approximately 2 mg/ml IL-15 in buffer (50 mM sodium phosphate, 100 mM sodium chloride, 10% sucrose, pH 7.4) was transferred to each of two different reaction vessels (referred to herein as Composition 1 and Composition 2 ). To adjust the pH to 8.0, a borate buffer (0.4 M or 0.6 M) having a pH of 8 was added. A tenfold excess (relative to the molar amount of IL-15) of mPEG-SBA-40K (mPEG-SBA, 40 kDa) was added. , diluted in 2 mM HCl, into each of the IL-15 solutions and mixed well. After addition of mPEG-SBA-40K, the pH of the reaction mixture was determined to be pH 8 or, if necessary, adjusted by using additional borate buffer. The final concentration of IL-15 in the reaction was adjusted to 1 g/L, using additional diluent if necessary (a buffer containing 50 mM sodium phosphate, 100 mM sodium chloride, 10% sucrose, pH 7.4 was used for composition 1 and water was used for compositions 2). To ensure binding of mPEG-SBA-40K to IL-15 (ie, through the formation of predominantly stable amide bonds), the reaction solution was stirred for 45 or 60 minutes to promote conjugation at room temperature for composition 1 or composition 2, respectively. The reaction was quenched by adding 71-fold excess of glycine (relative to the molar amount of PEG) initially added to the reaction mixture at pH 8.0 for 30 min. For the composition, pH 1 was adjusted by titration to pH 7.0 using 0.2 M phosphoric acid.

The resulting composition was characterized by reverse phase HPLC (RP-HPLC), SDSPAGE and ion exchange HPLC (IEX-HPLC). The results of the RP-HPLC analysis are presented in Table. 1A below.

Table 1A

Component Average % area Composition 1 IL-15 0.57 Monopegylated IL-15 82.66 RRTl=nHK T1 (not characterized) 8.05 RRT2=nHK T2 (not characterized) 5.17 RRT3=nnK TK (not characterized) 3.57 Composition 2 IL-15 0.36 Monopegylated IL-15 92.17 RRTl=nHK T1 (not characterized) 2.26 RRT2=nHK T2 (not characterized) 3.28 RRT3=nHK TK (not characterized) 1.93

RRT represents relative retention time. The results of the SEC-HPLC analysis are presented in table. 1B below.

Table 1B

Component Average % area Composition 1 Connections to HMW 5.56 Di-mPEG IL-15 (dimer) 15.46 Mono-mPEG IL-15 (monomer) 78.98 Composition 2 mPEG connections to HMW 5.25 Di-mPEG IL-15 6.62 Mono-mPEG IL-15 88.14

HMW=high molecular weight mPEG-IL-15 conjugates containing 3 or more PEGs covalently attached to interleukin-15.

The results of the IEX-HPLC analysis are presented in Table. 1B below.

- 29 043667

Table 1C

Average % area Composition 1 Main area 10.20 Main Peak 68.53 Acidic region 21.29 Composition 2 Main area 10.43 Main Peak 56.32 Acidic region 33.26

The resulting compositions predominantly contained mono-PEGSBA-40K compounds with less than 10% PEG dimer (i.e., containing 2 PEG moieties attached to IL-15) and even lower amounts of higher PEG content compounds (i.e., with 3 or more PEG fragments attached to IL-15).

In addition, the compositions had a relatively low degree of deamidation (indicated as the acidic region in Table 1C). Composition 1 was 21.29% deamidated and Composition 2 was 33.26% deamidated.

Example 2: Preparation of a long-acting IL-15 receptor agonist

mPEG2-CAC-fmoc-20K-NHS, which was stored at -80°C under nitrogen, was heated to ambient temperature with a nitrogen purge. A stock solution (200 mg/ml) of mPEG2-CAC-fmoc-20K-NHS was prepared in 2 mM HCl and mPEG2-CAC-fmoc-20K-NHS was added to rIL-15 at the molar ratios of mPEG2-CACfmoc-20K-NHS to rIL- 15, ranging from 5:1 to 100:1. The final concentration of rIL-15 in the mixture was 0.5 mg/ml (0.031 mM). Sodium bicarbonate buffer (1 M, pH 8.0) was added to the mixture to achieve a final concentration of 100 mM, and conjugation was allowed to proceed for 30 min to obtain the conjugate [mPEG2-CAC-fmoc-20K-NHS]-[rIL-15 ] (wherein the informal name of the conjugate reflects the polymer reagent used to prepare the conjugate, with the understanding that for the resulting product(s), the reactive moiety in the polymer reagent has been replaced by a bond to, for example, IL-15). After 30 minutes, the reaction was quenched by adding 1 M glycine (pH 6.0) to the reaction mixture to achieve a final concentration of 100 mM. The pH of the quenched reaction mixture was then adjusted to 4.0 using glacial acetic acid before purification by column chromatography and characterization.

The reaction mixture was analyzed by RP-HPLC analysis. By SDS-PAGE, the reaction mixture contained approximately 10-20% monoconjugates, approximately 50-70% diconjugates and approximately 20-30% triconjugate, that is, the reaction mixture contained predominantly diconjugate compounds. The conjugate mixture was separated/isolated by anion exchange chromatography using a Q Sepharose High Performance column and sodium phosphate buffers as the eluent phase to obtain purified [mPEG2-CAC-FMOC-20kD-NHS]-IL-15 with an average degree of PEGylation of approximately 2 ( the degree of PEGylation varied from approximately 1.7 to 2.5), resulting in an n' value in the structure shown above for the purified composition of approximately 2.

In the remaining examples, purified [mPEG2-CAC-FMOC-20kD-NHS]-IL-15 will be referred to as conjugate 2.

Example 3: Preparation of long-acting IL-15 receptor agonist [moho-PEG2-RUButryALD-40K]-IL15

Branched PEG reagent mPEG-butyraldehyde, which is moho-PEG2-RUButryALD-40K

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H ₃ C-(-OCH2CH2)-NH-CO— o ^η o —OCH ₂ CH2CH2-C-YN-^CH2CH ₂ o)-CH2CH2CH2CHO

Η ₃ θ4θΟΗ ₂ ΟΗ ₂ )-ΝΗ-Ο-Ο—I ⁴ η

was used to prepare the claimed long-acting IL-15-R agonist, wherein the weight average molecular weight of the PEG reagent used to produce the agonist was approximately 40,000 Da.

2.7 ml of IL-15 (1.23 mg/ml in PBS buffer, pH 7.4) was transferred into a small reaction vessel, 0.3 ml of 1 M sodium acetate buffer (pH 5) was added to adjust the pH to pH 6 mPEG2-ru-ButyrALD, 40 kDa, stored at -20°C under nitrogen, was warmed to ambient temperature. A fifteenfold excess (relative to the amount of IL-15) of mPEG2-ru-ButyrALD was dissolved in MilliQ H ₂ O to obtain a 10% reagent solution. The 10% reagent solution was quickly added to the IL-15 solution, mixed well, and placed in the RotoMixer for 15 minutes. Then, 1/100 volume of 1 M NaCNBH ₃ /H ₂ O was added to the reaction mixture. To ensure the binding of mPEG2-ru-ButyrALD to IL-15 via a secondary amine bond, the reaction solution was placed in a RotoMixer at 4°C for 17 hours and then quenched glycine solution. Because the PEGylation reaction was performed at an acidic pH, the attachment of the PEG derivative to IL-15 was more selective at the N-terminus. Anion exchange chromatography using a Q Sepharose High Performance column and sodium phosphate buffers was also developed to purify the conjugate. The purified mono-PEG2-ru-ButyrALD-40K-IL-15 conjugate was characterized by HPLC and SDS-PAGE.

For the rest of the examples and in the accompanying description, purified [mono-PEG2-RU-ButryALD40K]-IL-15, i.e. containing one PEG fragment with the structure shown above covalently attached to IL-15 via an amine linkage will be referred to as conjugate 3.

Using the same approach, conjugates using PEG2-RU-ButryALD with other weight average molecular weights were prepared. For example, mono-PEG2-RU-ButryALD-20K]-IL-15 was prepared as described above using a 20 kDa polymer reagent (referred to herein as conjugate 4).

Example 4. Preparation of long-acting IL-15 receptor agonist mono-mPEG-ButyrALD40K-IL-15

H ₃ C-(OCH2CH ₂ )nOC NH—(CH ₂ CH ₂ O) ₄ —CH2CH2CH2CH2—NH--IL-15

PEG reagent, which is a linear mPEG-butyraldehyde, 40 kDa (mPEG-ButyrALD) with the structure

O CH ₃ o-^CH ₂ CH ₂ O^—c-nh-^ch ₂ ch ₂ o^—CH ₂ CH ₂ CH ₂ CHO was used to obtain the claimed long-acting IL-15 receptor agonist, while the weight-average molecular weight of the reagent The PEG used to produce the agonist was approximately 40,000 Da.

2.7 ml of IL-15 (1.23 mg/ml in PBS buffer, pH 7.4) was transferred into a small reaction vessel, 0.3 ml of 1 M sodium acetate buffer (pH 5) was added to adjust the pH to pH 6 mPEG-ButyrALD, 40 kDa, stored at -20°C under nitrogen, was warmed to ambient temperature. A tenfold excess (relative to the amount of IL-15) of mPEG-ButyrALD was dissolved in MilliQ H2O to obtain a 10% reagent solution. The 10% reagent solution was quickly added to the IL-15 solution, mixed well, and placed in the RotoMixer for 15 minutes. Then, 1/100 volume of 1 M NaCNBH ₃ /H ₂ O was added to the reaction mixture. To ensure the binding of mPEG-ButyrALD to IL-15 via a secondary amine linkage, the reaction solution was placed in a RotoMixer at 4°C for 17 hours and then quenched with glycine solution . Because the PEGylation reaction was performed at an acidic pH, the attachment of the PEG derivative to IL-15 was more selective at the N-terminus. Anion exchange chromatography using a Q Sepharose High Performance column and sodium phosphate buffers was also developed to purify the conjugate. The purified mono-mPEG-ButyrALD-40K-IL-15 conjugate was characterized by HPLC and SDS-PAGE.

In the remaining examples, mono-mPEG-ButryALD-40k-IL-15 will be referred to as conjugate 5.

Example 5 Assessing whether long-acting IL-15 receptor agonists display biased activity at the IL-15Ra receptor

The affinities of exemplary long-acting IL-15 receptor agonists (test products) for the IL-15a receptor were measured and compared with those for IL-15. Affinity was measured by BIAcore using IL-15Ra:Fc captured with immobilized anti-Fc antibody.

Test items were diluted to 10 µM in PBS (containing 0.05% Tween 20 and 0.1 mg/ml BSA). A series of 3-fold dilutions were prepared and injected into a sensor chip that was coated with IL-15Ra.

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Affinities were measured by determining the rates k _a and kd separately, and the ratio of kd to k _a was used to calculate Kd values.

Preferred conjugates are generally those that retain as much affinity for IL-15-Ra as possible after PEGylation compared to unmodified IL-15. In other words, the preferred conjugates are generally those whose affinity for IL-15-Ra is reduced to the least extent compared to unmodified IL-15.

For example, in some embodiments, a preferred conjugate exhibits no more than about a 7-fold reduction in EC ₅₀ value (ng/mL, pSTAT5 in CTLL-2) and no more than about a 50% reduction in alpha receptor binding (KD, pM) compared with IL-15. For example, conjugate 1 has approximately 2-fold reduction in potency compared to IL15 and retains approximately 80% alpha receptor affinity compared to IL-15.

Table 2A

Testable product k _a (MV ¹ ) KDS- ¹ ) K _D (pm) IL-15 5.78x10 ⁵ 1.49x10^ 258 Conjugate 1 4.92x10 ⁵ 1.03x10^ 209

As shown in the table above, Conjugate 1 retains its high affinity for the IL-15a receptor (ie, relative to IL-15), which is a characteristic that is particularly advantageous for a long-acting IL-15 receptor agonist. Affinity constants (KD) (in pM) are given for additional conjugates below.

Table 2B. Additional characteristics of illustrative long-acting

Testable product re1 agonists EU 50 (ng/ml) CTLL-2 j, eptora IL-15 IL-15Ra K _D (pm) PK (MRT), h. pSTAT5 IL-15 0.27 258 Conjugate 1 0.62 201/209 21 Conjugate 3 16.80 411 thirty Conjugate 4 6.21 215 Conjugate 5 1.45 144 25

Example 6 In Vivo Study: Single Dose PK Study in Mice

C57BL/6 mice (n=3/group) were intravenously administered a single dose of IL-15 (control) at 0.3 mg/kg) or conjugate 2 at a dose of 0.3 mg/kg. After administration, blood samples were collected at different time points after administration (24, 48, 78, 96 h). Samples were pooled and evaluated in a pharmacodynamic analysis of drug effects on lymphocyte populations using flow cytometry, with results expressed as fold change relative to vehicle control (results described in the following examples). In addition to changes in cell number, functional and activity markers were quantified. Finally, the plasma drug concentration was determined at each time point (see Fig. 6).

As shown in FIG. 6, conjugate 2 maintained measurable plasma concentrations for an extended period of time, e.g., over 1 week (filled squares), at a T _1/2 of approximately 20 to 30 hours, in contrast to a rapid fall in plasma levels observed after administration for IL-15, which is not characterized by long-lasting action (filled circles).

Example 7 In Vivo Study: Single Dose PK Study in Rats

Rats (n=3/group) were administered a single dose of conjugate 2 at dosages of 0.3, 0.15 and 0.075 mg/kg intravenously or a single dose of conjugate 2 at 0.15 mg/kg subcutaneously. Post-administration, blood was collected on days 1-7 post-administration (with several samples collected during the first 24 hours post-administration). At each time point, the concentration of the drug in the blood plasma was determined (see Fig. 7).

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As shown in FIG. 7 and similar to the results shown in FIG. 6 for mice, administration of conjugate 2 resulted in prolonged and dose-proportional drug exposure.

Example 8 In vivo study of IL-15 signaling in mice

Mice were dosed as described in Example 6 above to assess in vivo signaling as assessed by the extent of STAT5 phosphorylation. The extent of STAT5 phosphorylation in various lymphocytes (CD4, CD8 and NK cells) was assessed by staining whole blood for leukocyte surface markers and pSTAT5, followed by measurement by flow cytometry. The results are shown in Fig. 8A and 8B for IL-15 and conjugate 2, respectively.

Phosphorylation of STAT5 is an early and transient event in IL-15/IL-2 receptor signaling. As can be seen in FIG. 8A, although in vivo signaling activity is extremely transient for IL-15, exemplary conjugate 2 induces long-lasting phosphorylation of STAT5, which is most prominent in NK cells (inverted filled triangles ▼) and in CD8 cells (normal filled triangles A) also, wherein measurable STAT5 phosphorylation activity was observed in NK cells and CD8 cells after 72 hours. STAT5 phosphorylation activity for CD4 cells is also shown (filled squares).

Example 9 In Vivo Study of IL-15 Signaling in Non-Human Primates

In a study using cyno monkeys, one female and one male were administered a single dose of conjugate 2 (0.5 mg/kg) intravenously. Serial blood samples were collected from each animal both before treatment (days -6 and -1) and at several intervals after treatment to assess STAT5 phosphorylation in different lymphocyte types (CD4, CD8 and NK cells) by flow cytometry. The results are presented in Fig. 9A (CD4), 9B (CD8) and 9C (NK).

As shown in FIG. 9A-9C, results are similar to those observed in mice (Example 7), although in non-human primates STAT5 phosphorylation was also observed in CD4 cells (Fig. 9A). Phosphorylation of STAT5 in each of the three cell types increased significantly following administration, reaching a maximum level between approximately 3 and 4 days after administration of an exemplary long-acting IL-15 agonist and returning to levels similar to those on day -1 (i.e., before administration dose) by approximately days 5-10. Similar to example 7, these results indicate the long-term presence of active IL-15 molecules.

Example 10 In vitro IL-15 activity of exemplary long-acting IL-15 receptor agonists in human peripheral blood mononuclear cell (PBMC) NK cell subsets

Assessing the in vitro activity of exemplary long-acting IL-15 receptor agonists (eg, conjugates 1, 3, and 5) was accomplished by examining signaling in human PBMC NK cell subsets as shown in FIG. 10A (CD56bright cells) and 10B (CD56dim cells). STAT5 phosphorylation was assessed as previously described to assess IL-15 signaling activity exhibited by long-acting IL-15 receptor agonists.

The results are shown in Fig. 10A (CD56bright) and 10B (CD56dim).

As can be seen, each of the illustrative conjugates induces IL-15 signaling in huPBMC, while conjugate 1 effectively induces such signaling. Of the conjugates tested (not all data shown), conjugate 1 showed the highest potency/activity against huPBMC. Data show that even when maintaining the same degree of PEGylation (i.e., number of PEG fragments) and the same size of PEG fragments in the IL-15 protein, different PEG architectures and linkers can cause very different effects on bioactivity in the resulting conjugates.

A second study was conducted to examine/compare the response of pStat5 in human PBMCs derived from two donors (CD3, CD4, CD8, CD56 (bright and dim), and CD4 regulatory T cells (CD25+Foxp3+)) to IL-15 conjugate 1 and conjugate 5. An 11-point dose response was examined using a 10-fold dilution with a dose range of 0.001-10,000 ng/mL and a 20-minute stimulation. Each of the test products was diluted in IL-15 buffer + 0.1% BSA. The results are presented in the following tables.

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Table 4. %pSTAT5 and pSTAT5 MFI in CD3 and CD4 cells

Compound EC50 (ng/ml) - %pSTAT5 EC50 (ng/ml) - MFI CD3 CD4 CD3 CD4 donor 1 donor 2 donor 1 donor 2 donor 1 donor 2 donor 1 donor 2 IL-15 0.17 0.17 0.177 0.20 4.0 5.22 3.6 5.8 Conjugate 1 0.75 1.1 0.75 0.83 22.1 108.8 23.54 106.5 Conjugate 5 1.3 1.7 1.3 1.14 84.73 154.2 84.21 98.18

Based on the data in table. 3 above, IL-15 appears to be approximately 4-6 times more potent than conjugate 1 in inducing both CD3 and CD4; the efficacies of conjugate 1 and conjugate 5 appear to be similar in inducing CD3 and CD4.

Table 5. %pSTAT5 and pSTAT5 MFI in CD4 regulatory T cells and CD8 cells

Compound EC50 (ng/ml) - %pSTAT5 EC50 (ng/ml) - MFI Regulatory Cells CD8 Regulatory T cells CD8 donor 1 donor 2 donor 1 donor 2 donor 1 donor 2 donor 1 donor 2 IL-15 0.08 0.084 0.17 0.15 0.6 0.71 3.5 5.5 Conjugate 1 0.23 0.37 0.76 1.54 2.5 2.9 16.73 99.76 Conjugate 5 0.44 0.45 1.4 2.5 3.5 5.4 53.7 204.6

Based on the data in table. 5 above, IL-15 appears to be approximately 3-5 times more effective than conjugate 1 in inducing regulatory T cells and CD8; the efficacies of conjugate 1 and conjugate 5 appear to be essentially the same in inducing CD4 and CD8.

Table 6. %pSTAT5 and pSTAT5 MFI in CD56bright and CD56dim cells

Compound EC50 (ng/ml) - %pSTAT5 EC50 (ng/ml) - MFI CD56bright CD56dim CD56bright CD56dim donor 1 donor 2 donor 1 donor 2 donor 1 donor 2 donor 1 donor 2 IL-15 oh and 0.19 0.26 0.54 0.49 0.96 1.4 3.7 Conjugate 1 1.4 1.9 2.9 5.9 7.8 12.4 23.25 73.05 Conjugate 5 5.1 4.5 16.48 27.12 24.86 56.9 97.51 283.4

Based on the data in table. 6, IL-15 appears to be ∼10-fold more potent than conjugate 1 in inducing CD56. However, conjugate 1 appears to be more effective than conjugate 5 in inducing CD56bright and CD56dim.

Based on previous data, IL-15, conjugate 1, and conjugate 5 demonstrated similar induction of pSTAT5 in all cell populations, with the maximal response appearing to be higher in CD56bright and regulatory T cells.

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Table 7. Summary table

Compound EC50 (ng/ml) - %pSTAT5

CD3 CD4 Regulatory T cells CD8 CD56bright CD56dim IL-15 0.17 0.177 0.08 0.17 oh and 0.26 Conjugate 1 0.75 0.75 0.23 0.76 1.4 2.9 Conjugate 5 1.3 1.3 0.44 1.4 5.1 16.48

Based on the above data, IL-15 is approximately 3-4 times more effective than conjugate 1 and approximately 5-8 times more effective than conjugate 5 in inducing CD3, CD4, CD8 and regulatory T cells, then how IL-15 is approximately 12 times more effective than conjugate 1 and approximately 40-60 times more effective than conjugate 5 in inducing CD56bright and CD56dim cells, indicating some unanticipated and particularly beneficial characteristics of conjugate 1.

Example 11 In Vivo Study: Single Dose PD Study in Mice - Cell Proliferation and Activation

Balb/c mice (n=3/group) were intravenously administered a single dose of vehicle (50 mM sodium phosphate, 100 mM sodium chloride, 10% sucrose, pH 7.4) or conjugate 1 at a dose rate of 0.03 mg/kg (Fig. 11, low dose), 0.3 mg/kg (Fig. 11, medium dose) or 1 mg/kg (Fig. 11, high dose). After administration, blood samples were collected at different time points after administration (24, 48, 78, 96, 120 h). Samples from each mouse were subjected to pharmacodynamic analysis of drug effects on lymphocyte populations using flow cytometry. In addition to changes in cell number, functional and activity markers were examined.

Additional administrations of conjugate 1 were carried out in doses of 0.01, 0.1 and 1.5 mg/kg.

Results illustrating NK cell proliferation in mice administered each of 0.03 mg/kg (FIG. 11, low dose), 0.3 mg/kg (FIG. 11, medium dose), and 1 mg/kg kg (Fig. 11, high dose) of conjugate 1 are presented in Fig. 11A and 11B.

NK cells and their proliferation were determined using combinations of CD45+CD3CD49b+ and CD45+CD3-CD49b+Ki67+ markers. After dosing, blood samples were analyzed on a Fortessa flow cytometer with FACS DIVA software. Flowjo software was used for analysis, and absolute NK cell values and % Ki67 positivity for NK cells were plotted using Prism.

Fig. 11A is a graph of Ki67 expression (percentage) over time; in fig. 11B shows the numbers of NK cells over time. The graphs illustrate the ability of conjugate 1 to induce sustained NK cell proliferation in mice.

The effect of exemplary long-acting IL-15 receptor agonists was studied on NK cells of all levels of maturity. The peripheral NK cell pool can be determined by the expression of CD27, with CD27 ^low NK cells being more cytotoxic and producing more cytokines than CD27 ^high NK cells (Hayakawa Y, et al., J Immunol. 2006; 176:1517- 1524). Mature peripheral NK cell populations have been further classified into four stages of maturation, defined by the sequential upregulation of CD11b expression followed by down-regulation of CD27, with the most immature NK cells being CD27-CD11b- and the most mature NK cells being CD27-D11b+ (Chiossone L ., et al., Blood, 2009;113:5488-5496).

In mice, four different stages of NK cell maturation were determined by the expression of CD27 and CD11b. After identifying triple positive cells for NK marker (CD49b+), natural NK activating receptor (NKp46+) and IL-15/IL-2RB (CD122+), immature (CD11b-CD27-), early (CD11b-CD27+), high-effector (CD11b+CD27+) and terminal effector (CD11b+CD27-) NK cells were quantified by flow cytometry.

NK cells at various stages of maturation were quantified in mice receiving a single dose or after receiving a third dose of q7dx3 conjugate 1 at 0.01, 0.03, 0.1, 0.3, 1 .0 and 1.5 mg/kg, as described above. Using flow cytometry, the NK population of interest was identified by positivity for CD49b, NKp46, and CD122. CD11b and CD27 were then used to further differentiate the NK population into subpopulations of immature (CD11b-CD27-), early (CD11b-CD27+), high effector (CD11b+CD27+) and terminal effector (CD11b+CD27-) cells. Peripheral blood was analyzed using a Fortessa flow cytometer, and absolute values for each population were determined using counting beads during sampling using BD FACS DIVA software. Flow cytometry analysis was performed using Flowjo software and data were plotted in Prism.

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The results are shown in Fig. 12A-D. Additional results are shown in FIG. 22A-D. The results for q7dx3 are shown in Fig. 28A-D. As can be seen from the graphs, conjugate 1 was effective in increasing the numbers of NK cells of all levels of maturity (terminal effector cells, NK cell precursors, high effector cells and early NK cells). A dose-dependent increase in NK cell numbers was observed in subsets of all maturation stages, with this effect lasting for at least 120 hours.

Determination of surface expression of NKG2D was performed using flow cytometry analysis of the anti-NKG2D antibody signal expressed as mean fluorescence intensity (MFI) in NK cells. Similarly, intracellular granzyme B levels were determined using flow cytometry to detect anti-granzyme B antibody signal, also expressed as MFI in NK cells. After detection of NKG2D and granzyme B signal using a Fortessa flow cytometer and FACS Diva software, analysis was performed using Flowjo software. MFI values were plotted using Prism. The results are shown in Fig. 13A and 13B, which further illustrate the ability of Conjugate 1 to enhance NK cell activation, as evidenced by its ability to provide a sustained increase in the expression of both NKG2D and granzyme B (a pro-apoptotic serine protease) by NK cells compared to vehicle, most notably for medium and high dose sizes. A dose-dependent increase in both markers of NK activation was observed after a single dose of conjugate 1.

In mice, CD8 T cells were defined as CD45+CD3+CD4-CD8+. Blood and spleens from mice were immunophenotyped using a Fortessa flow cytometer and analyzed using Flowjo software. Absolute CD8 cell counts were plotted in Prism as shown in FIG. 14 (blood) and fig. 24 (spleen). In fig. 14 and 24 illustrate the ability of Conjugate 1 to induce proliferation and sustained expansion of CD8 T cells after a single i.v. administration in mice at each of the dosage amounts described above. The effects are most noticeable at medium (0.1 mg/kg, 0.3 mg/kg) and high (1.0 mg/kg, 1.5 mg/kg) dosage amounts.

In mice, CD8 effector memory T cells (T _em ) and CD8 central memory T cells (T _cm ) were identified as CD45+CD3+CD4-CD8+CD44+CD62- and CD45+CD3+CD4-CD8+CD44+CD62L+. The proliferation of these memory T cell populations was determined using Ki-67 positivity. After a single dose of conjugate 1 or IL-15, blood and spleen were subjected to immunophenotyping using a Fortessa flow cytometer, DIVA acquisition software, and Flowjo analysis software. Graphs were created in Prism. Conjugate 1 induced a dose-dependent increase in both effector memory CD8 T cells and central memory CD8 T cells, whereas a single dose of IL-15 did not, as shown in FIG. 15A and 15B for blood. Conjugate 1 induced a dose-dependent increase in both Ki67 and granzyme B, whereas a single dose of IL-15 did not, as shown in FIG. 25 and 26 for the spleen. Both effector and central memory cell populations proliferate in response to administration of conjugate 1, an exemplary long-acting IL-15 agonist.

Example 12 In Vivo Study: Single Dose PD Study in Nonhuman Primates

In a study using cynomolgus monkeys, one female and one male were intravenously administered 500 μg/kg of Conjugate 2. Serial blood samples were collected from each animal before treatment (days 6 and -1) and at several intervals after single-dose treatment for evaluation by flow cytometry. lymphocyte counts (NK cells, CD8 T cells, etc.) and activation.

NK cell numbers were determined to evaluate the ability of Exemplary Conjugate 2 to induce sustained NK cell proliferation in non-human primates; the results are shown in Fig. 16A and 16B. NK cells and their proliferation in the blood of cynomolgus monkeys were identified using flow cytometry. Collection and analysis of NK cells (CD45+CD3-CD16+) and their proliferation status (CD45+CD3-CD16+Ki67+) were performed using BD FACS DIVA software. Absolute values for NK cells and proliferating NK cells were used to calculate % Ki67 positivity in the NK population. The values obtained before and after treatment were plotted using Prism.

As shown herein, administration of a single dose of conjugate 2 was effective in inducing sustained proliferation of NK cells in non-human primates.

CD8 T cell numbers were also determined as shown in FIG. 17, for each animal from pre-dose to 14 days post-dose. Specifically, CD8 T cells were defined as CD45+CD3+CD4-CD8+. Blood from the monkeys was also subjected to immunophenotyping as previously described. In monkeys, the number of CD8 T cells increased in a sustained manner, with the effect lasting for at least 10 days. The graph further illustrates the ability of an exemplary long-acting IL-15 receptor agonist, Conjugate 2, to induce proliferation and sustained expansion of CD8 T cells following administration.

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In monkeys, CD8 TEM cells were defined as CD45+CD3+CD4-CD8+CD45Ra-CD197-, and CD8 T _CM were defined as CD45+CD3+CD4-CD8+CD45Ra-CD197+. Immunophenotyping was performed using flow cytometry with sample data acquisition using DIVA software and data analysis using Flowjo software. Graphs were generated using Prism. The numbers of CD8 effector memory T cells (TEM cells) and CD8 central memory T cells ( _TCM ) were determined for each animal from predose to 14 days postdose, as shown in FIG. 18A and B respectively. Conjugate 2 induces significant and sustained increases in CD8 effector and central memory T cell populations in cynomolgus monkeys. The figures show that both CD8 effector and central memory T cell populations proliferate in response to exemplary conjugate 2.

Example 13. Evaluation of antitumor activity in a model of CT26-induced subcutaneous metastatic lung tumor in Balb/c mice

On day 0, 6-8 week old female Balb/c mice were inoculated with 1x105 murine CT-26 cells by tail vein injection. On day 1, 24 hours after injection of CT-26 cells, mice were divided into ten groups. Each group consisted of 6-9 animals (for conjugate 2) or 9-12 animals (for conjugate 1). (Two separate studies were conducted to administer Conjugate 1 and Conjugate 2, although the study protocol was essentially the same in both studies.) Each group was assigned to the following single intervention: vehicle medium, 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); for conjugate 1: carrier medium, which is a phosphate buffered saline solution (group H); conjugate 1 at a dose of 0.03 mg/kg (group I) and conjugate 1 at a dose of 0.3 mg/kg (group J). Animals were dosed on days 1, 5, and 10.

13 days after the date of injection of CT-26 tumor cells, mice were anesthetized and blood and spleen cells were collected for further analysis of immunophenotype markers, while the lungs were fixed for 24-48 hours in Bouin's solution containing picric acid and formaldehyde.

The number of tumor nodes in the lung was counted during dissection for each lung, and the average number of nodes in the lung was determined for each group. Statistical significance was also determined by comparing the vehicle group with the experimental groups using an unpaired Student's t test.

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

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

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Group Product under test Table 8 Dose Scheme (mg/kg) Path Number of animals A Control with media environment- one-time i.v. 6 IN IL-15 o, s days 1, 5 and 10 i.v. 9 WITH Conjugate 2 0.03 days 1, 5 and 10 i.v. 8 D Conjugate 2 0.1 days 1, 5 and 10 i.v. 8 E Conjugate 2 o, s days 1, 5 and 10 i.v. 9 F Conjugate 2 1.0 days 1, 5 and 10 i.v. 9 G Conjugate 2 h,o days 1, 5 and 10 i.v. 9 N Control with media environment- one-time i.v. 9 I Conjugate 1 0.03 days 1, 5 and 10 i.v. 12

J Conjugate 1 o, s two doses) i.v. 9

Example 14. In vitro and in vivo cytotoxicity of NK cells after treatment with conjugate 1

NK cell-mediated cytotoxicity toward targeted tumor cells was assessed in vitro using a flow cytometry-based assay. NK cells were isolated from the spleens of Balb/c mice using magnetic negative cell selection isolation (Mouse NK Cell Enrichment Kit, Stemcell Technologies) and used as effector cells. For in vitro studies, isolated NK cells were stimulated with conjugate 1 at concentrations of 3000, 1000, 300, 30, 3 or 0 (unstimulated) ng/ml overnight in a humidified incubator at 37°C, 5% CO2 before use in a cytotoxicity assay . For in vivo studies, mice were dosed with 0.3 mg/kg of conjugate 1, and splenic NK cells were isolated at 24, 48, and 72 h postdose and used directly in the cytotoxicity assay.

YAC-1 T cells labeled with PKH26 were used as target cells. To monitor NK cell cytotoxicity, NK cells and YAC-1 cells were co-cultured at different effector:target ratios (50:1, 25:1 and 12.5:1) for 4 h at 37°C, 5% CO ₂ , then stained with 7-AAD for 10 min to label dead cells. Cells were immediately analyzed using flow cytometry. Lysed target cells were identified as PKH26+7-AAD+.

In vitro results. After co-culture for 4 h, cytotoxicity was assessed by flow cytometry. The results are presented in Fig. 21.

In vivo results. After treatment at 0.3 mg/kg, cytotoxicity was assessed at 24, 48 and 72 hours. The results are presented in FIG. 27.

These data illustrate dose-dependent enhanced NK cell cytotoxicity in vitro and in vivo following treatment with conjugate 1.

Example 15 Induction of granzyme B by conjugate 1

Granzyme B expression by NK cells as a function of time was measured after treatment with conjugate 1 at doses of 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg and 1 .5 mg/kg. Whole blood was collected from 24 to 240 hours post-dose for immunophenotyping. After red blood cell lysis, leukocytes were labeled with a dye to determine viability and markers specific for CD45, CD3, and CD49b to identify living NK cells. Cells were then simultaneously fixed and permeabilized for intracellular granzyme B staining. Stained blood was analyzed on a Fortessa flow cytometer, data acquired using DIVA software, and analysis performed using Flowjo software. Data are presented as the percentage of NK cells that were positive for granzyme B expression.

The results are presented in Fig. 23. These data indicate that treatment with conjugate 1 increases the level of granzyme B expression in NK cells.

Example 16 In Vivo Study: Single Dose PK Study of IL-15 and Conjugate 1 and JAK/STAT Signaling in Mice

For the PK assay, conjugate 1 was administered as a single intravenous dose of 0.3 mg/kg to balb/c mice (n=3). After administration, mice were humanely sacrificed and plasma was collected at 24, 48, 72, 96, 120, and 144 h posttreatment. In a separate study, mice were given a single dose of IL-15 (0.5 mg/kg) intravenously. Samples from these mice were collected at the indicated time points within 6 h of treatment.

- 38 043667

[Methods for determining PK are previously described in this document]. For the pharmacodynamic study, balb/c mice (n = /group) were given i.v. injection of conjugate 1 in an amount of 0.03 or 0.3 mg/kg or vehicle medium (50 mM sodium phosphate, 100 mM sodium chloride, 10% sucrose, pH 7.4) and blood was collected before dosing and after 15 min, 1, 24, 48, 72, 96 and 120 hours after treatment. Samples were analyzed individually by flow cytometry and expressed as percentage positivity for pSTAT5 in CD8 and NK cells.

Fig. 29A is a graph showing the plasma concentration of test article (IL15 or conjugate 1) in balb/c mice over a period of 144 hours after administration of a single intravenous dose of 0.5 and 0.3 mg/kg test articles respectively.

Results. Conjugate 1 has a half-life of approximately 12 hours, whereas IL-15 is rapidly cleared from plasma with a half-life of less than 1 hour.

Fig. 29B is a graph showing the percentage of pSTAT5 positivity in CD8 T cells in mice following a single injection of Conjugate 1 at doses of 0.03 and 0.3 mg/kg.

Results. Conjugate 1 at both dose levels induces robust pSTAT5 signaling in CD8 T cells. A 120-hour time period is indicated, including before dosing.

Fig. 29C is a graph showing the percentage of pSTAT5 positivity in mouse NK cells following a single injection of Conjugate 1 at doses of 0.03 and 0.3 mg/kg.

Results. Conjugate 1 at both dose levels induces strong and sustained pSTAT5 signaling in NK cells.

Example 17 In vivo pharmacodynamics study in mice with a single dose and Q7dx3 - cell numbers and proliferation

balb/c mice (n=3/group) were treated with a single dose or three weekly doses of 0.01, 0.03, 0.1, 0.3, 1 or 1.5 mg/kg of conjugate 1 or vehicle . Mice were sacrificed and blood was collected at various time points after administration (24, 48, 72, 96, 120, 144, 240 h). Samples from each mouse were subjected to flow cytometry analysis to examine pharmacodynamic effects in lymphocyte populations and a functional marker of interest (numbers of CD8 T cells, memory CD8 T cells, and NK cells, and percentage of Ki-67 positivity in each population). The results are shown in Fig. 30A-F, 31A-C and 32A-C.

Fig. ZOA-C are graphs of the total numbers of CD8 cells, CD8 central memory cells (T _cm ) and CD8 effector memory cells (T _em ), respectively, after a single injection of conjugate 1 in amounts of 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 induces a significant increase in the total number of CD8 T cells in the blood, as described in Example 17. Lowest the 0.01 mg/kg dose increased CD8 T _cm and CD8 T _em levels. At 0.3 mg/kg, conjugate 1 increased CD8, CD8 T _cm , and CD8 T _em levels by 6.4-fold, 37.9-fold, and 14.5-fold. Of note, the numbers of CD8 and memory CD8 T cells did not return to baseline levels at 240 hours post-injection when conjugate 1 was administered at doses of 0.3-1.5 mg/kg, demonstrating the sustained PD effects of conjugate 1.

Fig. 30D, 30E and 30F are graphs showing the percentage of Ki-67 positivity in the total populations of CD8 T cells, CD8 T _cm and CD8 T _em , respectively, in mice as described in Example 17. Single dose of conjugate 1 at all dose levels provides an increase in Ki-67 positivity in all CD8 T cells and CD8 T cell subsets.

Fig. 31A, 31B and 31C are graphs of the numbers of CD8 T cells and memory CD8 T cell subset following administration of a single dose of Conjugate 1 (dotted lines) or Q7dx3 (solid lines) at 0.03 and 0.3 mg/kg, as described in example 17. Repeated dosing further increased the size of these populations with an increase in CD8, CD8 T _cm , CD8 T _em by 35.3 times, 183 times and 73.8 times, respectively. At the end of this time period (240 hours after the first or last dose of 0.3 mg/kg), cell numbers had not returned to baseline levels in mice.

Fig. 32A and 32B are graphs of NK cell counts and percent positivity for Ki-67 following a single dose of 0.01 to 1.5 mg/kg of Conjugate 1 in mice as described in Example 17. NK Cell Counts were significantly higher than the vehicle control at all dose levels and returned to baseline levels 240 hours post-dose. All dose levels induced a robust increase in the percentage of Ki-67 positivity in NK cells.

Fig. 32C is a graph showing mouse NK cell counts following single (solid lines) or Q7dx3 (dashed lines) administration of Conjugate 1 at doses of 0.03 and 0.3 mg/kg as described in Example 17. Repeated dosing of Conjugate 1 , at 0.3 mg/kg, induced slightly lower, although still significant, numbers of NK cells compared to a single dose. Similar numbers of NK cells were achieved with a single dose compared to repeated doses of 0.03 mg/kg.

Results. Conjugate 1 at dose levels equal to or greater than 0.03 induces significant

- 39 043667 new increase in the total number of CD8 T cells in the blood. The lowest dose of 0.01 mg/kg increased the numbers of CD8 T _cm (central memory cells) and CD8 T _em (effector memory cells). At 0.3 mg/kg, conjugate 1 increased the amounts of CD8, CD8 T _cm and CD8 T _em by 6.4-fold, 37.9-fold and 14.5-fold, respectively. It is noteworthy that the numbers of CD8 T cells and memory CD8 T cells did not return to baseline levels 240 hours after injection when conjugate 1 was administered at doses of 0.3-1.5 mg/kg, demonstrating the beneficial sustained PD effects of the conjugate 1.

Example 18: In vitro measurement of NK cell cytotoxicity in mice treated with conjugate 1 and analysis of granzyme B in blood NK cells

balb/c mice (n=2/group) were treated with conjugate 1 (0.006, 0.03 or 0.3 mg/kg), IL-15 (1 mg/kg) or vehicle control. After treatment, spleens were isolated at 24, 72, and 96 h to isolate NK cells. NK cells were isolated by magnetic negative selection and incubated at ratios of NK cells (effector) to YAC-1 cells (target cells) of 12.5:1, 25:1 and 50:1 (E:T) for 4 hours at 37C, 5% CO ₂ . YAC-1 target cells were pre-labeled and then stained with 7AAD after incubation with NK cells. Detection of lysed (7AAD+) target cells (PKH26+) was performed using flow cytometry. Blood from these mice was also collected at the same time points and measured by flow cytometry for granzyme B expression in NK cells. The results are shown in FIG. 33A and 33B.

In fig. 33A illustrates an in vitro NK cell cytotoxicity assay that measures changes in the extent of NK-mediated lysis of target cells following treatment with a test article in mice. Shown is the percentage of specific lysis of YAC-1 cells by splenic NK cells isolated from balb/c mice treated with 0.006, 0.03, or 0.3 mg/kg of 1 or 1 mg/kg IL-15 conjugate at the indicated hours during the period time. Splenic NK cells from vehicle-dosed mice served as controls.

Results. Conjugate 1, administered at a dose of 0.3 mg/kg, induced an increase in NK cell cytotoxicity that was superior in magnitude and duration to NK cells from mice receiving a single injection of IL-15 at 1 mg/kg.

Fig. 33B is a graph showing the percentage of granzyme B positivity in blood NK cells from the same mice used in the in vitro NK cell cytotoxicity assay in FIG. 33A.

Results. Conjugate 1, administered at doses of 0.03 and 0.3 mg/kg, induced a significant increase in granzyme B expression levels in NK cells, with a strong and sustained increase observed at a dose of 0.3 mg/kg.

Example 19: Efficacy of Conjugate 1 as a Single Agent in a Model of CT-26 Cell-Induced Metastatic Lung Cancer

balb/c mice received a tail vein injection of 1x105 CT-26 colon carcinoma cells. The next day, mice (n=9/group) were treated twice weekly with conjugate 1 (0.03 or 0.3 mg/kg) or vehicle control. Five days after the second injection, mice were humanely sacrificed and pulmonary nodules were counted. The results are shown in Fig. 34 A and 34 B.

Fig. 34A and 34B illustrate the percentage of lung nodule suppression in balb/c mice treated with intravenous injection of CT-26 tumor cells followed by treatment with Conjugate 1 administered twice at one week intervals at 0.03 or 0.3 mg/kg.

Results. Injection of conjugate 1 in amounts of 0.03 and 0.3 mg/kg suppressed the formation of pulmonary nodules by 40 and 80%, respectively. The same mice dosed at 0.3 mg/kg were monitored for 32 days after tumor cell injection to assess survival. Treatment with conjugate 1 significantly increased survival compared to mice treated with vehicle control injected with tumor cells.

Example 20. Evaluation of NK CELL-dependent efficacy of conjugate 2 in a model of CT-26 cell-induced metastatic lung cancer

CT-26 mice (n=7-11/group) were injected with anti-asialo-GM1 antibody to deplete NK cells or two different controls (IgG or PBS), followed by an injection of 1x105 CT26 tumor cells. Mice were then treated with conjugate 2 at a dose of 0.3 mg/kg administered on days 1, 5, and 10 after injection of tumor cells or vehicle control. Mice were sacrificed and pulmonary nodules were counted three days from the last day of treatment. The results are shown in Fig. 35.

Fig. 35 is a graph showing the percentage of lung nodule suppression in CT-26 cell-injected, Conjugate 2-treated mice that received antibody-mediated NK cell depletion (olive green), IgG control (blue), or PBS (orange). Data are presented as percent suppression of lung nodule formation compared to CT-26-injected mice that were not subjected to NK cell depletion and treated with vehicle control (black). The effectiveness of conjugate 2 in this tumor model was abolished in the absence of NK cells in mice.

Example 21. In vivo pharmacodynamic study of a single dose of conjugate 1 in excellent

- 40 043667 from human primates

In this study using cyno macaques, one female and one male were intravenously administered a single dose of conjugate 1 (0.1 mg/kg). Serial blood samples were collected from each animal over a 14-day period and analyzed by flow cytometry for various lymphocytes (CD8 T cells, percentage positive for Ki-67 among total CD8, CD8 central memory T cells (Tcm) and CD8 effector memory T cells (Tem), NK cells and percentage of Ki-67 positive NK cells). The results are shown in Fig. 36A-D and 37A-B.

Fig. 36A and 36B are graphs illustrating a two-week time period for CD8 cell counts and percentage positivity for Ki-67 as a measure of proliferation in one male (dashed line) and one female (solid line) cynomolgus monkey following intravenous administration of Conjugate 1 at dose 0 .1 mg/kg. As can be seen, Conjugate 1 induces a significant increase in CD8 T cell counts in cynomolgus monkeys, with a 7- to 10-fold increase in cell counts after a single dose.

Fig. 36C and 36D illustrate increases in cynomolgus CD8 T _cm and CD8 T _em cell counts following a single injection of conjugate 1. CD8 T _cm and T _em counts increased 27-30-fold and 21-33-fold, respectively.

Fig. 37A and 37B are graphs of NK cell counts and percent positivity for Ki-67 in cynomolgus monkeys following a single dose of 0.1 mg/kg of Conjugate 1. The number of NK cells increased 9-10 times after treatment with conjugate 1.

Example 22 Comparison of in vitro activity of IL-15 and conjugate 1 on CD8 and CD56BRIGHT NK cells from human PBMCs

In vitro assessment of conjugate 1 activity was performed by examining JAK/STAT signaling in NK and CD8 following treatment of human PBMCs with IL-15 or conjugate 1 over a dose range of 0.001-10,000 ng/ml. STAT5 phosphorylation was assessed as previously described.

Table 9

Compound EC50 (mg/ml) - % pSTAT5 CD8 CD56bright IL-15 0.16 0D1 Connection 1 0.87 1.7

The results are shown in Fig. 38A and 38B; These figures represent EC50 curves for IL-15 (·) treatment compared to conjugate 1 () treatment of human PBMCs and subsequent measurement of the percentage of pSTAT5 positivity in CD8 and CD56bright NK cells.

Results. Conjugate 1 is 5.5 and 15 times less effective than IL-15 in activating CD8 and CD56bright NK cells, respectively. It is important to note, however, that conjugate 1 achieves the same maximal response as standard IL-15.

Example 23 In Vivo Study: Single Dose PK Study in Mice

balb/c mice (n=3/group) were intravenously administered a single dose of IL-15 (500 μg/kg) or conjugate 1 at 10, 30, 100, 300, and 1000 μg/kg. Blood samples were collected at the indicated time points after administration (conjugate 1: 24, 48, 72, 96, 120, 144, 240 hours; IL-15 control: 0.03, 0.08, 0.25, 0, 5, 1, 2, 4, 6, 8 hours), and the concentration of the drug in plasma was determined (See Fig. 39).

As shown in FIG. 39, conjugate 1 exhibited longer-lasting pharmacokinetic effects measurable at plasma concentrations with a half-life of approximately 14 hours compared to plasma levels observed for non-long-acting IL-15, which undergoes rapid clearance.

Example 24 In Vivo Study: Single Dose PK Study in Rat

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

As shown in FIG. 40, Conjugate 1 exhibits robust pharmacokinetic effects measurable in plasma concentrations with a half-life of approximately 18 hours compared to plasma levels observed for non-long-acting IL-15 that undergoes rapid clearance as shown in FIG. 40.

Example 25 In Vivo Study: Single Dose PK Study in Non-Human Primates

Cynomolgus monkeys (n=2, 1 male and 1 female) were intravenously administered a single dose of Conjugate 1 at 10, 50 and 100 μg/kg. IL-15 was administered intravenously as a single dose of 50 μg/kg as a control. Plasma drug concentrations were determined at the indicated time points after injection (0.03, 0.25, 1, 4, 12, 24, 48, 72, 96, 120, 144, 168 h). See fig. 41.

As shown in FIG. 41, conjugate 1 exhibits robust pharmacokinetic effects,

- 41 043667 measurable plasma concentrations with a half-life of approximately 30 hours for a dose of 100 μg/kg compared to non-long-acting IL-15, which was rapidly cleared from plasma.

Conjugate 1 achieved longer-lasting and sustained plasma exposure in several species (mice, rats and cynomolgus monkeys) after a single dose (see FIG. 3941).

Example 26 In Vivo Study: Single Dose PD Study in Mice - Cell Number, Proliferation and Activation of JAK/STAT Signaling

balb/c mice (n=3/group) were intravenously administered a single dose of vehicle (as described in Example 11) or conjugate 1 at a dose of 0.3 mg/kg or 0.03 mg/kg. After administration, blood samples were collected at specific time points after administration (24, 48, 72, 96, 120, 144, and 240 h). Samples were immunophenotyped for CD4 T cell counts (see FIG. 42A) and % Ki-67 (see FIG. 42B) at the indicated time points.

CD4 T cells and their proliferation were determined using CD45+CD3+CD4+CD8- and CD45+CD3+CD4+CD8-Ki-67+ markers, respectively. Fig. 42A is a graph of CD4 T cell counts, and FIG. 42B is a graph of CD4 T cell proliferation measured by % Ki-67 positivity over time. CD4 T cells were the least sensitive population to Conjugate 1 treatment, with relatively small increases in amounts and % Ki67 expression (observed 72-144 hours post-dose) compared to CD8 and NK cells (see, e.g., Example 11). NK cells were more sensitive to stimulation of proliferative responses by a single dose of conjugate 1 than CD4 T cells or CD8 T cells in mice.

Phosphorylation of STAT5 in CD4 T cells was determined using the CD3+CD4+CD8- pSTAT5+ marker combination. Fig. 43 is a plot of percent positivity for pSTAT5 phosphorylation in CD4 T cells over time (at 0.25, 1, 6, 24, 48, 72, 96, and 120 hours post-administration) at a dose of 0.03 mg/kg ( blue, filled squares) or 0.3 mg/kg (orange, filled circles). Levels for vehicle over time (black) and predose (open circles) are also shown.

Results. CD4 T cells were the least sensitive population to conjugate 1 treatment, with a relatively small increase in pSTAT5 expression (observed 0.25-72 hours post-dose) compared to CD8 and NK cells. NK cells are more sensitive to stimulation with a single dose of conjugate 1 in proliferative responses compared to CD8 T cells or CD4 T cells in mice.

Example 27 In Vivo Study: Minimum Effective Dose Study in Non-Human Primates (NHP)

Cynomolgus macaques (n=3-4 males) were intravenously administered a single injection of conjugate 1 in amounts of 0.003, 0.01, 0.1 mg/kg or control with vehicle medium. Blood samples were collected at the indicated pre- and post-dose time points (days -5, -2, 1, 2, 3, 4, 5, 6, 7, 10, 14, 17) and subjected to flow cytometry analysis to determine pharmacodynamic effects at lymphocyte populations. The numbers of NK cells, CD8 T cells and CD4 T cells were determined and the results are shown in FIG. 44A-C. Proliferation (%Ki-67) and JAK/STAT signaling (%pSTAT5) for NK cells, CD8 T cells and CD4 T cells were examined and the results are shown in FIG. 45A-C and figs. 46A-C respectively. The proliferation (% Ki-67) of CD8 subpopulations (T _mtive , T _em , T _c m and T _scm ) was examined, with the results shown in FIG. 47A-D.

In NHPs, NK cell counts (CD45+CD3-CD16+) were significantly increased in a dose-dependent manner following a single dose of conjugate 1. Maximum cell counts were observed five days post-dose and were sustained up to 14 days at dose levels 0.1 and 0. 01 mg/kg. The lowest dose level resulting in a significant increase in NK cells was 0.01 mg/kg. Confirming the observations regarding NK cell numbers, conjugate 1 also caused a dose-dependent and enhanced induction of Ki-67 expression, which peaked approximately 3-4 days after treatment and could be sustained for up to approximately 14 days. A significant increase in % Ki-67 can be detected after administration of a dose level of 0.001 mg/kg. Conjugate 1 also effectively activated the JAK/STAT signaling pathway in NK cells with a dose-dependent increase in pSTAT5 % that could be detected at dose levels as low as 0.001 mg/kg.

Fig. 44A, 45A and 46A are graphs of NK cell counts, % Ki-67 and % pSTAT5 over time after treatment with conjugate 1, respectively.

In NHPs, conjugate 1 induced a significant increase in total CD8 T cell numbers (defined as CD45+CD3+CD4-CD8+), with maximal cell numbers being reached around day 5 post-treatment. This effect was sustained for more than seven days, returning to baseline levels between days 10 and 14 after dosing. The effect of Conjugate 1 on total CD8 cell count can be determined at 0.003 mg/kg. In support of these results, conjugate 1 induced significant % positivity for Ki-67 in CD8 T cells, detectable at a low dose of 0.01 mg/kg. Conjugate 1 also activates the JAK/STAT signaling pathway

- 42 043667 is stable in CD8 T cells with a dose-dependent increase in pSTAT5 at dose levels of 0.1 and

0.01 mg/kg.

Fig. 44B, 45B and 46B are graphs of CD8 T cell counts, % Ki-67 and % pSTAT5, respectively, over time after treatment with conjugate 1.

Conjugate 1 had a relatively small effect on the total number of CD4 T cells (defined as CD45+ CD3+ CD4+ CD8-) in NHP compared to NK and CD8 T cells. Conjugate 1, administered at a maximum dose level of 0.1 mg/kg, induced a slight increase in CD4 T cell counts, % Ki-67 and % pSTAT5.

Fig. 44C, 45C and 46C are graphs of CD4 T cell counts, % Ki-67 and pSTAT5, respectively, over time after treatment with conjugate 1.

NK cells are the most sensitive in response to a dose of conjugate 1 compared to CD8 T cells or CD4 T cells in vivo in NHPs.

In the cynomolgus monkey, naïve and memory cell CD8 subpopulations were determined using CD45Ra, CD197, and CD95. Proliferation study (% Ki-67) of CD8 naïve T cells (CD45+CD3+CD4-CD8+CD45Ra+CD197+), CD8 T _scm (CD45+CD3+CD4-CD8+CD45Ra+CD197+CD95+), CD8 T _em ( CD45+CD3+CD4-CD8+CD45Ra-CD197-) and CD8 T _cm (CD45+CD3+CD4-CD8+CD45Ra-CD197+) revealed increased sensitivity of CD8 memory cell subpopulations to conjugate 1 compared to CD8 naïve T cells. In the CD8 T _em , T _cm , and T _scm populations, conjugate 1 induced increased expression of %Ki-67 in a dose-dependent manner with a detectable increase in proliferation marker positivity starting as early as day 2, peaking at day 5 and returning to baseline level from day 10 to day 14. Expression and kinetics of Ki-67 in CD8 populations confirm the sustained increase in CD8 T cell numbers shown in Example 27 and FIG. 47A-D.

Fig. 47A-D are plots of % Ki-67 for CD8 Tna _tive , T _scm , T _cm and T _em populations over time following treatment with conjugate 1. As can be seen from the figures, memory CD8 T cell populations showed increased sensitivity to a single dose of conjugate 1 compared with naïve CD8 T cells in vivo in NHPs.

Example 28 Induction of granzyme B or perforin by conjugate 1

Expression of the NK cytolytic enzymes granzyme B and perforin was examined in cynomolgus monkeys following a single dose of conjugate 1. Expression levels of granzyme B and perforin were quantified by mean fluorescence intensity (MFI) in NK cells at dose levels of 0.001, 0.01, or 0.1 mg/kg. Conjugate 1 increased granzyme B MFI by approximately 3-fold (peak value compared to pre-dose value) at 0.01 and 0.1 mg/kg. Conjugate 1 also increased perforin MFI by approximately 2-fold (peak value compared to predose value) at 0.01 and 0.1 mg/kg. Overall, conjugate 1 not only induced robust expansion of NK cells, but could also enhance their function.

Fig. 48A-C are MFI plots of granzyme B, and FIG. 49A-C are graphs of perforin MFI before dosing (baseline) and the time period corresponding to peak levels after treatment with conjugate 1 (0.0001-0.1 mg/kg) in NHPs.

Conjugate 1 increases protein levels of cytotoxic enzymes such as constitutively expressed granzyme B and perforin in NHP NK cells.

-

Claims (7)

Sequence Listing SEQ ID NO:1 (rhlL-15) 10 20 30 40 5060 NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDASIH 70 80 90 100110 DTVENLIILA NNSLSSSNGNVTESGCKECEE LEEKNIKEFL QSFVHIVQMF INTS SEQ ID N0:2 -1 10 20 30 40 5060 M NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDASIH 70 80 90 100110 DTVENLIILA NNSLSSSNGNVTESGCKECEE LEEKNIKEFL QSFVHIVQMF INTS SEQ ID N0:3 10 20 30 40 5060 MRISKPHLRS ISIQCYLCLL LNSHFLTEAG IHVFILGCFS AGLPKTEANW VNVISDLKKI 70 80 90 100 110120 EDLIQSMHID ATLYTESDVH PSCKVTAMKC FLLELQVISL ESGDASIHDT VENLIILANN 130 140 150160 SLSSNGNVTE SGCKECEELE EKNIKEFLQS FVHIVQMFIN TS CLAIM 1. A composition for the treatment of a condition amenable to treatment with interleukin-15 (IL-15), containing a long-acting IL-15 receptor agonist according to formula (I) О ¹¹ 111 15 CH ₃ -(OCH ₂ CH ₂ )„O-(CH ₂ ) _m C---NH-f— IL-15 where IL-15 is a fragment representing interleukin-15, n in formula (I) represents integer from 150 to 3000; m in formula (I) is an integer from 2 to 5, and -NH- in formula (I) represents the amino group of the IL-15 moiety, n' in formula (I) is 1; wherein the composition contains no more than 15 mol.% long-acting IL-15 receptor agonists covered by formula (II) ABOUT CH ₃ -(OSH ₂ CH ₂ )„O-(CH ₂ ) _t C—, ^L 2,3 or more 3 where IL-15 is a fragment representing interleukin-15, n in formula (II) is a whole number from 150 to 3000; m in formula (II) is an integer from 2 to 5 and -NH- in formula (II) represents the amino group of the fragment representing IL-15; and wherein the average number of polyethylene glycol moieties covalently attached to the amino groups of IL-15 for the long-acting receptor agonists of formula (I) and formula (II) contained in the composition ranges from 1.0 to 1.3. 2. The composition according to claim 1, wherein n in both formula (I) and formula (II) is an integer from 200 to 2000, or from 400 to 1300, or from 450 to 1200. 3. The composition according to any of the previous claims, wherein m in both formula (I) and formula (II) is 2 or 3. 4. The composition according to any of the previous claims, wherein m in both formula (I) and formula (II) is equal to 3. 5. The composition according to any of the preceding claims, wherein n in both formula (I) and formula (II) 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 , 15000, 20000, 25000, 30000, 40000, 50000 and 60000 Yes. 6. The composition according to claim 1, containing not more than 10 mol.% of the specified long-acting IL-15 receptor agonists of formula (II). 7. The composition according to claim 1, containing no more than 7 mol.% long-acting receptor agonists -