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

Abstract

To provide a long-acting IL-15 receptor agonist, related compositions and methods of preparation and use, e.g., in treatment of conditions responsive to therapy effective to provide sustained immune activation and / or anti-tumor activity.SOLUTION: In a first aspect, provided herein is a long-acting IL-15 receptor agonist including pharmaceutically acceptable salt forms thereof. The long-acting IL-15 receptor (IL-15 R) agonist comprises at least a single linear PEG (polyethylene glycol) moiety stably covalently attached to an IL-15 amino group via an amide linkage. Intervening between the linear PEG strand and the stable amide linkage to the IL-15 amino group may be a linear unsubstituted alkylene group (-CH2-)m having from 2 to 5 carbon atoms (i.e., m=2, 3, 4, or 5).SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent Application No. 62 / 506,494, filed May 15, 2017; and U.S. Provisional Patent Application No. 62 / 536,966, filed July 25, 2017; and U.S. Provisional Patent Application No. 62 / 582,186, 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 entireties.

[0002] The present disclosure relates to long-acting interleukin-15 ("IL-15") receptor agonists, related compositions, and methods of manufacture and use (among other things), for example, in the treatment of conditions responsive to effective therapy, for example, to provide sustained immune activation and anti-tumor activity. [Background technology]

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

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

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

[0006] Finally, despite its specific roles within both the innate and adaptive immune systems, IL-15 exerts important and broad effects across both categories of the immune system. Specifically, IL-15 inhibits or reduces apoptosis (or cell death) of several cell types associated with both categories of the immune system, including dendritic cells, neutrophils, eosinophils, mast cells, CD4+ T cells, and B cells.

[0007] Because IL-15 stimulates the growth and maintenance of many cells in the immune system that can fight cells that appear foreign (or "non-self") to the host, its use in treating cancer patients has been proposed (Steel et al. (2012) Trends Pharmacol. Sci. 33(1):35-41). For example, IL-15-based agonists have been proposed for the treatment of myeloma (Wong et al. (2013) OncoImmunology 2(11), e26442:1-3). In addition, IL-15 drug therapy has been proposed for the treatment of viral infections, such as HIV infection. Despite its potential for treating several diseases, IL-15-based therapies face several challenges. For example, IL-15 is rapidly cleared from plasma and is relatively unstable under physiological conditions. Furthermore, the in vivo signaling activity of IL-15 is similarly short-lived, and the molecule requires daily administration or infusion over multiple days for optimal activity, which is undesirable. Several approaches have attempted to overcome these limitations by conjugating IL-15 to the IL-15 receptor α subunit. However, such approaches may abrogate the desirable signaling that occurs uniquely through the IL-15 receptor α, which is expressed on multiple cell types. Non-releasable PEGylation with relatively low molecular weight (5 kDa) succinimidyl carbonate-terminated polymers has been reported, but this resulted in significant alterations in the biological activity of IL-15. (Pettit et al. (1997) J. Biol. Chem. 272(4):2312-2318) [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Giri et al. (1995) EMBO J.14:3654-3663. [Non-patent document 2] DiSabitino et al. (2011) Cytokine Growth Factor Rev.22:19-33 [Non-patent document 3] Steel et al.(2012)Trends Pharmacol.Sci.33(1):35-41 [Non-patent document 4] Wong et al. (2013) OncoImmunology 2(11),e26442:1-3 [Non-Patent Document 5] Pettit et al. (1997) J. Biol. Chem. 272(4):2312-2318 Summary of the Invention [Problem to be solved by the invention]

[0009] However, despite the foregoing approaches, there remains a need for new IL-15 receptor agonists with improved properties and profiles, e.g., potent immunostimulatory effects, low systemic toxicity, stability and / or improved pharmacokinetics, etc. Accordingly, among other things, the present disclosure provides long-acting IL-15 receptor agonists that are new and believed not to have been fully suggested by the art, as described herein, and that have numerous advantageous characteristics, as described in more detail below, as well as compositions and kits comprising such agonists, and related methods of making and using. [Means for solving the problem]

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

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

[0012] For example, in some embodiments, the long-acting IL-15 receptor agonist has the following structure: [ka] wherein IL-15 is an interleukin-15 moiety, n is an integer from about 150 to about 3,000; m is an integer from 2 to 5 (e.g., 2, 3, 4, or 5), and n' is 1. Formula (I) is [CHO-(CHCHO) n (CH2) m C(O)-NH-] n’ -IL15, and the two formulas can be used interchangeably. In Formula I (and similar formulas provided herein), ~NH~ in the structure represents the amino group of the IL-15 moiety.

[0013] In some further embodiments, n is an integer from about 200 to about 2000, or from about 400 to about 1300, or from about 450 to about 1200.

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

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

[0016] In one or more illustrative embodiments, compositions are provided that include a long-acting IL-15 receptor agonist according to Formula (I), including but not limited to each and every associated embodiment thereof provided herein.

[0017] In some embodiments, the long-acting IL-15 receptor agonist compositions comprise compounds having the following formula, when considered collectively: [ka] wherein the values ​​of n and m are as provided for formula (I) above. That is, in terms of the long-acting IL-15 receptor agonist component of such compositions, about 15 mol percent or less of the long-acting IL-15 receptor agonist included in the composition is of formula (II).

[0018] For example, in some embodiments, the long-acting IL-15 receptor agonist composition comprises about 10 mole percent or less of long-acting IL-15 receptor agonists encompassed by Formula (II) when considered collectively.

[0019] In some additional embodiments of the foregoing, the composition comprises about 7 mole percent or less of a long-acting IL-15 receptor agonist having an n' of 2, 3, or greater than 3 (i.e., a higher PEG-mer, also referred to as a "high-mer"). In yet some other embodiments, the composition comprises about 5 mole percent, 6 mole percent, 9 mole percent, or 10 mole percent or less of a long-acting IL-15 receptor agonist having an n' of 2, 3, or greater than 3 (i.e., 2 or greater).

[0020] In some further embodiments, the composition comprises a long-acting IL-15 receptor agonist according to Formula (I). [ka] where n and m are as defined above, n' represents the average number of polyethylene glycol moieties covalently attached to IL-15 amino groups (with respect to the composition), and n' with respect to the composition is in the range of 1.0 to about 1.3. For example, the average number of polyethylene glycol moieties per IL-15 moiety is selected from about 1.0, 1.1, 1.2, and about 1.3.

[0021] In yet another aspect, provided herein is a method for preparing a long-acting interleukin-15 receptor agonist as described in Formula (I), e.g., Formulas (Ia), (Ib), (Ic), and (Id), and Formula (II), e.g., (IIa), (IIb), (IIc), and (IId). In this method, interleukin-15 (generally dissolved in a buffer such as phosphate buffered saline or any other suitable buffer) at a pH of about 7 (e.g., 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6) is reacted with a compound having the following structure: [ka] an activated PEG reagent such as methoxy PEG-succinimidyl alkanoate (where n is an integer from about 150 to about 3,000) by [ka] (where n' is 1). Exemplary methoxy PEG-succinimidyl alkanoate reagents for reacting with interleukin-15 include: [ka]

[0022] In some preferred embodiments, the methoxy PEG-succinimidyl alkanoate reagent is [ka] mPEG-succinimidyl butanoate.

[0023] 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 about 227), about 15,000 daltons (e.g., n is about 340), about 20,000 daltons (e.g., n is about 454), about 25,000 daltons (e.g., n is about 568), about 30,000 daltons (e.g., n is about 681), about 40,000 daltons (e.g., n is about 909), about 50,000 daltons (e.g., n is about 1136), and about 60,000 daltons (e.g., n is about 1364).

[0024] In one or more embodiments of the method, the methoxy PEG-succinimidyl alkanoate reagent is added in an equimolar amount (ie, an equimolar ratio) to the interleukin-15.

[0025] In one or more alternative embodiments, the methoxy PEG-succinimidyl alkanoate reagent is added in a molar excess over interleukin-15. In some particular embodiments, the methoxy PEG-succinimidyl alkanoate reagent is present in a 2-fold molar excess, or a 5-fold molar excess, or a 7-fold molar excess, or a 10-fold molar excess, or even a 12-fold molar excess or greater. In some embodiments, the methoxy PEG-succinimidyl alkanoate reagent is added in a 5-10-fold molar excess.

[0026] In some embodiments, the methoxy PEG-succinimidyl alkanoate reagent is added as a solid.

[0027] In some other embodiments, the methoxy PEG-succinimidyl alkanoate reagent is dissolved in a suitable solvent. In certain embodiments, the methoxy PEG-succinimidyl alkanoate reagent is dissolved in an aqueous acid, such as dilute hydrochloric acid, although any suitable acid can be used.

[0028] In some further embodiments of the method, interleukin-15 is initially present in solution at a concentration of about 0.5 mg / mL to about 10 mg / mL, i.e., prior to mixing with the methoxy PEG-succinimidyl alkanoate reagent. Additional exemplary concentration ranges include, for example, about 0.5 mg / mL to about 5 mg / mL of interleukin-15, about 0.5 mg / mL to about 3 mg / mL, and about 1.0 mg / mL to about 4 mg / mL of interleukin-15.

[0029] In some further embodiments, the pH of the interleukin-15 solution is adjusted to about 8.0 prior to the addition of the methoxy PEG-succinimidyl alkanoate reagent.

[0030] 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.

[0031] In some further embodiments, the resulting reaction mixture is stirred (or mixed) for a time sufficient to allow reaction between the reactants. In some embodiments, the reactants are mixed for about 15 minutes to about 10 hours, inclusive. In some further embodiments, the reactants are mixed for about 30 minutes to about 5 hours, or about 30 minutes to about 2 hours.

[0032] In some embodiments, the reaction is carried out under ambient conditions, e.g., at room temperature, i.e., without the application of heat. Exemplary temperature ranges for carrying out the reaction include, for example, from about 5°C to about 50°C, or from about 10°C to about 40°C, or from about 15°C to about 30°C. In some further embodiments, the reaction is carried out at a temperature of from about 20°C to about 25°C.

[0033] In some embodiments of the method, the reaction is quenched by the addition of an amino acid, hi some related embodiments, the reaction is quenched by the addition of glycine.

[0034] In some further embodiments of the method, the conjugation product, ie, the methoxy PEG-alkanoate-interleukin-15 conjugate, is separated from the reaction mixture.

[0035] In some additional embodiments, the reaction mixture comprising the methoxy PEG-alkanoate-interleukin-15 conjugate is purified.

[0036] In some particular embodiments, the reaction is [ka] (where n' is 1) resulting in the formation of

[0037] In some further embodiments, the reactions, when considered collectively, are encompassed by the following equation: [ka] (wherein the values ​​of n and m are as provided for Formula (I) above) is effective to form a composition comprising about 15 mole percent (mol %) or less of a long-acting IL-15 receptor agonist (of the IL-15-containing molecules in the composition).

[0038] In still some additional embodiments, the reaction is effective to produce less than 20-35%, or less than about 25% deamidated PEGylated interleukin-15.

[0039] In some embodiments, the long-acting IL-15 receptor agonist exhibits about a 7-fold or less decrease in EC50 value (ng / mL, CTLL-2 pSTAT5) when compared to unmodified (i.e., unconjugated) IL-15. For example, in one or more related embodiments, the long-acting IL-15 receptor agonist exhibits no more than about a 6.5-fold decrease in the EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 6-fold decrease in the EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 5.5-fold decrease in the EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 5-fold decrease in the EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 4.5-fold decrease in the EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 4-fold decrease in the EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 3.5-fold decrease in the EC50 value (ng / mL, CTLL-2 pSTAT5), or even no more than about a 3-fold decrease in the EC50 value (ng / mL, CTLL-2 pSTAT5) when compared to IL-15.

[0040] In some further embodiments, the long-acting IL-15 receptor agonist exhibits increased receptor alpha binding (K) compared to unconjugated IL-15, e.g., as measured using a technique suitable for determining receptor alpha binding, such as surface plasmon resonance (SPR). D In some related embodiments, the long-acting IL-15 receptor agonist exhibits about a 50% or less decrease in receptor α binding (K , pM) when compared to unconjugated IL-15.D , pM) or exhibiting a decrease of about 45% or less in receptor α binding (K D , pM) or exhibiting a decrease of about 40% or less in receptor α binding (K D , pM) or even exhibiting a decrease of about 35% or less in receptor α binding (K D , pM) is reduced by about 30% or less.

[0041] In some still further embodiments, the long-acting IL-15 receptor agonist exhibits about a 7-fold or less decrease in EC50 value (ng / mL, CTLL-2 pSTAT5) when compared to unmodified IL-15, and about a 7-fold or less decrease in receptor alpha binding (K D , pM) (the EC50 value or K D (including any one or more specific combinations of decreases in values).

[0042] In one or more further embodiments, there is provided a composition comprising a long-acting IL-15R agonist described herein, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

[0043] In some still further embodiments, the long-acting IL-15R agonist or composition is effective to stimulate NK activation and / or proliferation when administered to a subject at a therapeutically effective dose.

[0044] In one or more still further embodiments, the long-acting IL-15R agonist or composition is effective to support CD8 T cell survival and / or memory formation when administered to a subject at a therapeutically effective dose.

[0045] In another aspect, provided herein are methods for 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-15R agonist provided herein, or a composition comprising such an agonist. In certain embodiments, for example, the following are provided: (Item 1) A long-acting IL-15 receptor agonist comprising a single linear polyethylene glycol (PEG) moiety stably and covalently attached to an amino group of IL-15 via an amide bond, wherein the PEG moiety is a linear unsubstituted alkylene group having 2 to 5 carbon atoms (~CH2~). m is interposed between the PEG moiety and the amide bond to the IL-15 amino group, and pharmaceutically acceptable salt forms thereof. (Item 2) 2. The long-acting IL-15 receptor agonist according to item 1, wherein m is an integer selected from the group consisting of 2, 3, 4 and 5. (Item 3) 3. The long-acting IL-15 receptor agonist according to item 1 or 2, wherein the unsubstituted alkylene group is selected from (-CH2-)2, (-CH2-)3, (-CH2-)4, and (-CH2-)5. (Item 4) A long-acting IL-15 receptor agonist, [ka] (wherein IL-15 is an interleukin-15 moiety, n is an integer of about 150 to about 3,000; m is an integer of 2 to 5, n' is 1, and -NH- in the structure represents an amino group of the IL-15 moiety). (Item 5) 5. The long-acting IL-15 receptor agonist according to any one of items 1 to 4, wherein n is an integer of about 200 to about 2000, about 400 to about 1300, or about 450 to about 1200. (Item 6) 6. The long-acting IL-15 receptor agonist according to any one of items 1 to 5, wherein m is 2 or 3. (Item 7) 7. The long-acting IL-15 receptor agonist according to any one of items 1 to 6, wherein m is 3. (Item 8) 8. The long-acting IL-15 receptor agonist according to any one of items 1 to 7, wherein n is an integer having a value corresponding to a polyethylene glycol polymer having a weight-average molecular weight selected from the group consisting of 10,000 daltons (about 227), 15,000 daltons (about 340), 20,000 daltons (about 454), 25,000 daltons (about 568), 30,000 daltons (about 681), 40,000 daltons (909), 50,000 daltons (about 1136), and 60,000 daltons (about 1364). (Item 9) A composition comprising the long-acting IL-15 receptor agonist according to any one of items 7 to 11, wherein the composition, when considered collectively, has the formula: [ka] A composition comprising about 15 mole percent or less of a long-acting IL-15 receptor agonist, as encompassed by: (Item 10) 10. The composition of claim 9, comprising about 10 mole percent or less of long-acting IL-15 receptor agonists encompassed by formula (II) when considered collectively. (Item 11) 11. The composition of item 9 or 10, comprising about 7 mole percent or less of long-acting IL-15 receptor agonists encompassed by formula (II) when considered collectively. (Item 12) 12. The composition of any one of items 9 to 11, comprising about 5 mole percent or less of long-acting IL-15 receptor agonists encompassed by formula (II) when considered collectively. (Item 13) Formula (I) [ka] wherein IL-15 is an interleukin-15 moiety, n is an integer from about 150 to about 3,000; m is an integer from 2 to 5, n' is 1, ~NH~ in said structure represents an amino group of the IL-15 moiety, n' represents the average number of polyethylene glycol moieties covalently bonded to IL-15 amino groups in said composition, and n' for said composition is in the range of 1.0 to about 1.3. (Item 14) 14. The composition according to any one of items 9 to 13, wherein n' for said composition is selected from 1.0, 1.1, 1.2 and about 1.3. (Item 15) 9. The long-acting IL-15 receptor agonist according to any one of items 1 to 8, wherein the long-acting IL-15 receptor agonist exhibits about a 7-fold or less decrease in EC50 value (ng / mL, CTLL-2 pSTAT5) compared to unmodified IL-15. (Item 16) The reduction in the EC50 value (ng / mL, CTLL-2 pSTAT5) of the long-acting IL-15 receptor, compared to IL-15, is about 6.5-fold or less reduction in the EC50 value (ng / mL, CTLL-2 pSTAT5), about 6-fold or less reduction in the EC50 value (ng / mL, CTLL-2 pSTAT5), about 5.5-fold or less reduction in the EC50 value (ng / mL, CTLL-2 pSTAT5), about 5-fold or less reduction in the EC50 value (ng / mL, CTLL-2 pSTAT5), about 4.5-fold or less reduction in the EC50 value (ng / mL, CTLL-2 pSTAT5), about 4-fold or less reduction in the EC50 value (ng / mL, CTLL-2 pSTAT5), about 3.5-fold or less reduction in the EC50 value (ng / mL, CTLL-2 pSTAT5), and about 4.5-fold or less reduction in the EC50 value (ng / mL, CTLL-2 pSTAT5). 16. The long-acting IL-15 receptor agonist according to any one of items 1 to 8 and 15, wherein the IL-15 receptor agonist is selected from the group consisting of about a 3-fold or less decrease in IL-15 expression level (IL-15 receptor agonist, pSTAT5), (Item 17) The long-acting IL-15 receptor agonist has a longer receptor α binding (K D17. The long-acting IL-15 receptor agonist according to any one of items 1 to 8, 15 and 16, which exhibits about 50% or less reduction in the IL-15 receptor activity (pM). (Item 18) The long-acting IL-15 receptor agonist has a longer receptor α binding (K D , pM) or exhibiting a decrease of about 45% or less in receptor α binding (K D , pM) or exhibiting a decrease of about 40% or less in receptor α binding (K D , pM) or even exhibiting a decrease of about 35% or less in receptor α binding (K D 18. The long-acting IL-15 receptor agonist according to any one of items 1 to 8 and 15 to 17, which exhibits about 30% or less decrease in the IL-15 receptor activity (pM). (Item 19) The long-acting IL-15 receptor agonist exhibits about a 7-fold or less decrease in EC50 value (ng / mL, CTLL-2 pSTAT5) when compared to unmodified IL-15, and exhibits about a 7-fold or less decrease in EC50 value (ng / mL, CTLL-2 pSTAT5) when compared to IL-15 and receptor α binding (K D 19. The long-acting IL-15 receptor agonist according to any one of items 1 to 8 and 15 to 18, which exhibits about 50% or less reduction in the IL-15 receptor activity (pM). (Item 20) A pharmaceutically acceptable composition comprising the long-acting IL-15 receptor agonist according to any one of Items 1 to 8, 15 to 19, or the composition according to any one of Items 12 to 22, and a pharmaceutically acceptable excipient. (Item 21) 21. The long-acting IL-15R agonist or composition according to any one of items 1 to 20, which is effective in stimulating NK activation and proliferation when administered to a mammalian subject at a therapeutically effective dose. (Item 22) 22. The long-acting IL-15R agonist or composition according to any one of items 1 to 21, which is effective in supporting CD8 T cell survival and memory formation when administered to a subject at a therapeutically effective dose. (Item 23) 23. A method for treating a condition that responds to treatment with IL-15 by administering to a subject having said condition a therapeutically effective dose of a long-acting IL-15R agonist or composition according to any one of items 1 to 22. (Item 24) A method for treating cancer by administering a therapeutically effective dose of the long-acting IL-15R agonist or composition according to any one of items 1 to 23 to a subject having cancer.

[0046] In yet a further aspect, there is provided a method of treating cancer by administering to a subject with cancer a therapeutically effective dose of a long-acting IL-15R agonist or composition provided herein.

[0047] Additional aspects and embodiments are provided in the following specification and claims. [Brief explanation of the drawings]

[0048] [Figure 1] FIG. 1 provides the amino acid sequence of an exemplary recombinant human IL-15 from E. coli (SEQ ID NO: 1), a single non-glycosylated polypeptide chain comprising 115 amino acids with a molecular weight of 12.9 kDa. [Figure 2] FIG. 2 is a chromatogram showing RP-HPLC analysis of an exemplary conjugation reaction mixture described in Example 1. [Figure 3] FIG. 3 is an FPLC purification profile from an anion exchange chromatography column described in Example 1. [Figure 4] 4 is an SDS-PAGE of an exemplary purified long-acting IL-15 receptor agonist, mono-mPEG-butanamide-IL-15, described in Example 1. Lane 1 provides molecular weight markers as indicated; lane 2 is the unconjugated parent molecule, IL-15, and lane 3 is mono-mPEG-butanamide-IL-15. [Figure 5] FIG. 5 is a RP-HPLC analysis of purified mono-mPEG-butanamide-IL-15 as described in Example 1. [Figure 6] FIG. 6 is a plot of the plasma concentration of the test article (IL-15, solid circles, or mPEG2-CAC-FMOC-20K-NHS-IL-15, also known as N-(2-methoxyPEG-ethyl)-7-(4-((2-methoxyPEG-ethyl)amino)-4-oxobutyl)-9-ethyl-9H-fluorene-4-carboxamidocarbamate-IL-15, i.e., conjugate 2, solid squares) over time in mice after administration of a single intravenous dose of the test article, as described in Example 6. [Figure 7] Figure 7 is a plot of the mean plasma concentration over time of mPEG2-CAC-FMOC-20K-NHS-IL-15, also known as Conjugate 2, also referred to as N-(2-methoxyPEG-ethyl)-7-(4-((2-methoxyPEG-ethyl)amino)-4-oxobutyl)-9-ethyl-9H-fluorene-4-carboxamidocarbamate-IL-15, i.e., Conjugate 2, after administration of a single intravenous dose of Conjugate 2 at doses of 0.3 (■), 0.15 (▲), and 0.075 mg / kg (●) or a single subcutaneous dose of Conjugate 2 at 0.15 mg / kg (◆) in rats, as described in Example 7. [Figure 8A-8B] Figures 8A and 8B show the degree of STAT5 phosphorylation in various lymphocytes, namely CD4 (■), CD8 (▲), and NK cells (▼), after administration of a single iv dose of either IL-15 (Figure 8A) or conjugate 2 (0.3 mg / kg, Figure 8B), as described in Example 8. [Figure 9A-9B] 9A-9C are plots showing the degree of STAT5 phosphorylation in various lymphocytes, i.e., CD4, CD8, and NK cells, following administration of a single iv dose of Conjugate 2 (0.5 mg / kg) in cynomolgus monkeys, as described in Example 9, respectively. [Figure 9C] 9A-9C are plots showing the degree of STAT5 phosphorylation in various lymphocytes, i.e., CD4, CD8, and NK cells, following administration of a single iv dose of Conjugate 2 (0.5 mg / kg) in cynomolgus monkeys, as described in Example 9, respectively. [Figure 10A] 10A and 10B are plots showing the in vitro activity of exemplary long-acting IL-15 receptor agonists (conjugates 1, 3, and 5) as measured by signaling in NK subsets of human PBMCs, CD56bright (FIG. 10A) and CD56dim (FIG. 10B) NK cells, respectively, as described in detail in Example 10. [Figure 10B] 10A and 10B are plots showing the in vitro activity of exemplary long-acting IL-15 receptor agonists (conjugates 1, 3, and 5) as measured by signaling in NK subsets of human PBMCs, CD56bright (FIG. 10A) and CD56dim (FIG. 10B) NK cells, respectively, as described in detail in Example 10. [Figure 11A] Figures 11A and 11B are plots showing NK cell proliferation in mice compared to vehicle following iv administration of mono-mPEG-SBA40K-IL-15 (also referred to herein as Conjugate 1) at doses of 0.03 mg / kg (low dose, open squares), 0.3 mg / kg (medium dose, solid circles, solid line), or 1 mg / kg (high dose, diamonds), as described in Example 11. Figure 11A shows Ki67 expression (expressed as a percentage) over time, while Figure 11B shows NK cell counts (cells / ul) versus time after administration for each of the sample groups. [Figure 11B] Figures 11A and 11B are plots showing NK cell proliferation in mice compared to vehicle following iv administration of mono-mPEG-SBA40K-IL-15 (also referred to herein as Conjugate 1) at doses of 0.03 mg / kg (low dose, open squares), 0.3 mg / kg (medium dose, solid circles, solid line), or 1 mg / kg (high dose, diamonds), as described in Example 11. Figure 11A shows Ki67 expression (expressed as a percentage) over time, while Figure 11B shows NK cell counts (cells / ul) versus time after administration for each of the sample groups. [Figure 12A]Figures 12A-D are plots showing increasing numbers of total mature-level NK cells in mice after iv administration of Conjugate 1 at doses of 0.03 mg / kg (low dose, open squares), 0.3 mg / kg (medium dose, solid circles, solid line), or 1.0 mg / kg (high dose, diamonds) compared to vehicle (solid circles, dashed line), as described in Example 11. NK cell subsets are defined by CD11b and CD27 expression. Figure 12A shows the increase in the number of terminal effector cells, expressed as cells / µL, from 24 to 120 hours post-administration; Figure 12B shows the increase in the number of pre-NK cells, expressed as cells / µL, from 24 to 120 hours post-administration; Figure 12C shows the increase in the number of high-level effector cells, expressed as cells / µL, from 24 to 120 hours post-administration; and Figure 12D shows the increase in the number of early NK cells, expressed as cells / µL, from 24 to 120 hours post-administration. [Figures 12B-12C] Figures 12A-D are plots showing increasing numbers of total mature-level NK cells in mice after iv administration of Conjugate 1 at doses of 0.03 mg / kg (low dose, open squares), 0.3 mg / kg (medium dose, solid circles, solid line), or 1.0 mg / kg (high dose, diamonds) compared to vehicle (solid circles, dashed line), as described in Example 11. NK cell subsets are defined by CD11b and CD27 expression. Figure 12A shows the increase in the number of terminal effector cells, expressed as cells / µL, from 24 to 120 hours post-administration; Figure 12B shows the increase in the number of pre-NK cells, expressed as cells / µL, from 24 to 120 hours post-administration; Figure 12C shows the increase in the number of high-level effector cells, expressed as cells / µL, from 24 to 120 hours post-administration; and Figure 12D shows the increase in the number of early NK cells, expressed as cells / µL, from 24 to 120 hours post-administration. [Figure 12D]Figures 12A-D are plots showing increasing numbers of total mature-level NK cells in mice after iv administration of Conjugate 1 at doses of 0.03 mg / kg (low dose, open squares), 0.3 mg / kg (medium dose, solid circles, solid line), or 1.0 mg / kg (high dose, diamonds) compared to vehicle (solid circles, dashed line), as described in Example 11. NK cell subsets are defined by CD11b and CD27 expression. Figure 12A shows the increase in the number of terminal effector cells, expressed as cells / µL, from 24 to 120 hours post-administration; Figure 12B shows the increase in the number of pre-NK cells, expressed as cells / µL, from 24 to 120 hours post-administration; Figure 12C shows the increase in the number of high-level effector cells, expressed as cells / µL, from 24 to 120 hours post-administration; and Figure 12D shows the increase in the number of early NK cells, expressed as cells / µL, from 24 to 120 hours post-administration. [Figure 13A] 13A and 13B are plots showing the levels of NKG2D (FIG. 13A) and granzyme B (FIG. 13B) expression by NK cells following iv administration of conjugate 1 at doses of 0.03 mg / kg (low dose, open squares), 0.3 mg / kg (medium dose, solid circles, solid line), or 1 mg / kg (high dose, diamonds) compared to vehicle (solid circles, dashed line) in mice, as described in Example 11. [Figure 13B] 13A and 13B are plots showing the levels of NKG2D (FIG. 13A) and granzyme B (FIG. 13B) expression by NK cells following iv administration of conjugate 1 at doses of 0.03 mg / kg (low dose, open squares), 0.3 mg / kg (medium dose, solid circles, solid line), or 1 mg / kg (high dose, diamonds) compared to vehicle (solid circles, dashed line) in mice, as described in Example 11. [Figure 14] FIG. 14 is a plot showing CD8 T cell counts, expressed in cells / μL, in mice after iv administration of 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, both pre-dose and 24-120 hours post-dose. [Figures 15A-15B]15A and 15B are plots showing Ki67 expression (expressed as a percentage) versus time for T effector memory cells and T central memory cells after administration in mice following iv administration of conjugate 1 at a dose of 0.3 mg / kg or 1.0 mg / kg compared to vehicle and IL-15, respectively, as described in Example 11. [Figures 16A-16B] Figures 16A and 16B are plots showing NK cell proliferation following iv administration of a 500 μg / kg dose of Conjugate 2 in cynomolgus monkeys, as described in Example 12. Figure 16A shows Ki67 expression (expressed as a percentage) on NK cells from pre-dose to day 15 after administration, while 16B shows NK cell counts from pre-dose to day 15 after administration. [Figure 17] FIG. 17 is a plot showing CD8 T cell counts (shown for each animal) from pre-dose to day 14 post-dose following iv administration of Conjugate 2 at a dose of 500 μg / kg in cynomolgus monkeys, as described in Example 12. [Figure 18A] 18A and 18B are plots showing the post-administration number of CD8 T effector memory cells (TEM cells) and CD8 T central memory cells (TCM) versus time (pre-dose to day 14 post-dose), respectively, following iv administration of a 500 μg / kg dose of Conjugate 2 in cynomolgus monkeys, as described in Example 12. [Figure 18B] 18A and 18B are plots showing the post-administration number of CD8 T effector memory cells (TEM cells) and CD8 T central memory cells (TCM) versus time (pre-dose to day 14 post-dose), respectively, following iv administration of a 500 μg / kg dose of Conjugate 2 in cynomolgus monkeys, as described in Example 12. [Figure 19]Figure 19 shows the total lesions per mouse in the lungs of female Balb / c mice inoculated with murine CT-26 colon carcinoma cells and then treated with one of the following test articles, as described in detail in Example 13: vehicle, phosphate buffered saline (Group A); native IL-15 only (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). [Figure 20] FIG. 20 shows the total lesions per mouse in the lungs of female Balb / c mice inoculated with murine CT-26 colon cancer cells and then treated with one of the following test articles: vehicle, phosphate buffered saline (Group A); Conjugate 1 at a dose of 0.03 mg / kg (Group H); and Conjugate 1 at a dose of 0.3 mg / kg, as described in detail in Example 13. [Figure 21] 21 shows percent specific lysis as a function of E:T (effector:target) ratio at 1000 ng / mL (squares), 3000 ng / mL (circles), 300 ng / mL (triangles), 30 ng / mL (upper X), 3 ng / mL (diamonds), and unstimulated (lower X), as described in Example 14. The data demonstrate a dose-dependent increase in NK cell cytotoxicity in vitro after culture with conjugate 1. [Figure 22A]Figures 22A-D are plots showing increasing numbers of all mature levels of NK cells in mice after iv administration of Conjugate 1 at doses of 0.01 mg / kg, 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, and 1.5 mg / kg, as described in Example 11. Figure 22A shows the increase in the number of terminal effector cells from 24 hours to 120 hours after administration, expressed in cells / μL; Figure 22B shows the increase in the number of pre-NK cells from 24 hours to 120 hours after administration, expressed in cells / μL; Figure 22C shows the increase in the number of high effector cells from 24 hours to 120 hours after administration, expressed in cells / μL, and Figure 22D shows the increase in the number of early NK cells from 24 hours to 120 hours after administration, expressed in cells / μL. [Figures 22B-22C] Figures 22A-D are plots showing increasing numbers of all mature levels of NK cells in mice after iv administration of Conjugate 1 at doses of 0.01 mg / kg, 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, and 1.5 mg / kg, as described in Example 11. Figure 22A shows the increase in the number of terminal effector cells from 24 hours to 120 hours after administration, expressed in cells / μL; Figure 22B shows the increase in the number of pre-NK cells from 24 hours to 120 hours after administration, expressed in cells / μL; Figure 22C shows the increase in the number of high effector cells from 24 hours to 120 hours after administration, expressed in cells / μL, and Figure 22D shows the increase in the number of early NK cells from 24 hours to 120 hours after administration, expressed in cells / μL. [Figure 22D]Figures 22A-D are plots showing increasing numbers of all mature levels of NK cells in mice after iv administration of Conjugate 1 at doses of 0.01 mg / kg, 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, and 1.5 mg / kg, as described in Example 11. Figure 22A shows the increase in the number of terminal effector cells from 24 hours to 120 hours after administration, expressed in cells / μL; Figure 22B shows the increase in the number of pre-NK cells from 24 hours to 120 hours after administration, expressed in cells / μL; Figure 22C shows the increase in the number of high effector cells from 24 hours to 120 hours after administration, expressed in cells / μL, and Figure 22D shows the increase in the number of early NK cells from 24 hours to 120 hours after administration, expressed in cells / μL. [Figure 23] 23 is a plot showing granzyme B expression as a function of treatment with 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 of conjugate 1, as described in Example 15. The data demonstrate that treatment with conjugate 1 increases NK cell granzyme B expression. [Figure 24] FIG. 24 is a plot showing the number of CD8 T cells, expressed in cells / μL, isolated from mouse spleens following iv administration of conjugate 1 at doses of 0.03 mg / kg (solid circles, dashed line) and 0.3 mg / kg (solid circles, solid line) and vehicle IL-15 buffer (open circles, dashed line) in mice, both pre-dose and 24-96 hours post-dose, as described in Example 11. [Figure 25] FIG. 25 is a plot showing the level of Ki67 expression (expressed as a percentage) on CD49b cells versus time after administration following iv administration of conjugate 1 at a dose of 0.03 mg / kg or 0.3 mg / kg in mice compared to vehicle IL-15 buffer, as described in Example 11. [Figure 26]FIG. 26 is a plot showing the level of granzyme B expression (expressed as a percentage) over time on CD49b cells versus time after administration following iv administration of conjugate 1 at a dose of 0.03 mg / kg or 0.3 mg / kg in mice compared to vehicle IL-15 buffer, as described in Example 11. [Figure 27] 27 is a plot showing in vivo cytotoxicity results following treatment with 0.3 mg / kg of Conjugate 1, as described in Example 14. Cytotoxicity was assessed 24 hours, 48 ​​hours, and 72 hours after treatment. [Figures 28A-28B] Figures 28A-D are plots showing increasing numbers of total mature-level NK cells in mice after iv administration of conjugate 1 at doses of 0.03 mg / kg and 0.3 mg / kg in a single dose or after a third dose on a q7dx3 schedule, as described in Example 11. Figure 28A shows the increase in the percentage of Ki67 CD49b cells from 24 hours to 240 hours after administration; Figure 28B shows the increase in the percentage of granzyme B CD49b cells from 24 hours to 240 hours after administration; Figure 28C shows the increase in the number of CD49b cells, expressed as cells / µL, from 24 hours to 240 hours after administration; and Figure 28D shows the increase in the percentage of granB+MFI CD49b cells from 24 hours to 240 hours after administration. [Figures 28C-28D] Figures 28A-D are plots showing increasing numbers of total mature-level NK cells in mice after iv administration of conjugate 1 at doses of 0.03 mg / kg and 0.3 mg / kg in a single dose or after a third dose on a q7dx3 schedule, as described in Example 11. Figure 28A shows the increase in the percentage of Ki67 CD49b cells from 24 hours to 240 hours after administration; Figure 28B shows the increase in the percentage of granzyme B CD49b cells from 24 hours to 240 hours after administration; Figure 28C shows the increase in the number of CD49b cells, expressed as cells / µL, from 24 hours to 240 hours after administration; and Figure 28D shows the increase in the percentage of granB+MFI CD49b cells from 24 hours to 240 hours after administration. [Figures 29A-29B]Figures 29A-C are plots related to the study described in Example 16. Figure 29A is a plot of the plasma concentration of the test article (IL-15 or Conjugate 1) over a 144-hour time course following administration of a single intravenous dose of the test article at 0.5 and 0.3 mg / kg, respectively, in BALB / c mice. Conjugate 1 exhibits a half-life of approximately 12 hours, while IL-15 is rapidly cleared from plasma with a half-life of less than 1 hour. Figure 29B is a graph of the percent pSTAT5 positivity in CD8 T cells in mice after a single injection of 0.03 and 0.3 mg / kg Conjugate 1. Conjugate 1 at both dose levels induces sustained pSTAT5 signaling in CD8 T cells. A 120-hour time course, including pre-administration, is shown. Figure 29C is a graph of the percent pSTAT5 positivity in mouse NK cells after a single injection of 0.03 and 0.3 mg / kg Conjugate 1. Conjugate 1 at both dose levels induces potent and sustained pSTAT5 signaling in NK cells. [Figure 29C] Figures 29A-C are plots related to the study described in Example 16. Figure 29A is a plot of the plasma concentration of the test article (IL-15 or Conjugate 1) over a 144-hour time course following administration of a single intravenous dose of the test article at 0.5 and 0.3 mg / kg, respectively, in BALB / c mice. Conjugate 1 exhibits a half-life of approximately 12 hours, while IL-15 is rapidly cleared from plasma with a half-life of less than 1 hour. Figure 29B is a graph of the percent pSTAT5 positivity in CD8 T cells in mice after a single injection of 0.03 and 0.3 mg / kg Conjugate 1. Conjugate 1 at both dose levels induces sustained pSTAT5 signaling in CD8 T cells. A 120-hour time course, including pre-administration, is shown. Figure 29C is a graph of the percent pSTAT5 positivity in mouse NK cells after a single injection of 0.03 and 0.3 mg / kg Conjugate 1. Conjugate 1 at both dose levels induces potent and sustained pSTAT5 signaling in NK cells. [Figure 30A]Figures 30A-C are plots of total CD8, CD8 central memory (Tcm), and CD8 effector memory (Tem) cell counts after a single dose of 0.01, 0.03, 0.1, 0.3, 1, or 1.5 mg / kg of conjugate 1, respectively, as described in Example 17. As described in Example 17, dose levels of conjugate 1 of 0.03 or higher induce a significant increase in total CD8 T cells in the blood. The lowest dose of 0.01 mg / kg increased CD8 Tcm and CD8 Tem. At 0.3 mg / kg, conjugate 1 increased CD8, CD8 Tcm, and CD8 Tem by 6.4X, 37.9X, and 14.5X, respectively. Notably, CD8 and CD8 memory T cell counts did not return to baseline at 240 hours post-injection when conjugate 1 was administered at 0.3-1.5 mg / kg, demonstrating the sustained PD effect of conjugate 1. [Figures 30B-30C] Figures 30A-C are plots of total CD8, CD8 central memory (Tcm), and CD8 effector memory (Tem) cell counts after a single dose of 0.01, 0.03, 0.1, 0.3, 1, or 1.5 mg / kg of conjugate 1, respectively, as described in Example 17. As described in Example 17, dose levels of conjugate 1 of 0.03 or higher induce a significant increase in total CD8 T cells in the blood. The lowest dose of 0.01 mg / kg increased CD8 Tcm and CD8 Tem. At 0.3 mg / kg, conjugate 1 increased CD8, CD8 Tcm, and CD8 Tem by 6.4X, 37.9X, and 14.5X, respectively. Notably, CD8 and CD8 memory T cell counts did not return to baseline at 240 hours post-injection when conjugate 1 was administered at 0.3-1.5 mg / kg, demonstrating the sustained PD effect of conjugate 1. [Fig. 30D-30E] Figures 30D, 30E, and 30F are plots of Ki-67 percent positivity within total CD8, CD8 Tcm, and CD8 Tem populations, respectively, in mice, as described in Example 17. A single dose of Conjugate 1 at all dose levels increases Ki-67 positivity in total CD8 and CD8 subpopulations. [Figure 30F]Figures 30D, 30E, and 30F are plots of Ki-67 percent positivity within total CD8, CD8 Tcm, and CD8 Tem populations, respectively, in mice, as described in Example 17. A single dose of Conjugate 1 at all dose levels increases Ki-67 positivity in total CD8 and CD8 subpopulations. [Figure 31A] Figures 31A, 31B, and 31C are plots of CD8 and CD8+ memory subpopulation T cell counts following administration of a single dose of Conjugate 1 (dotted line) or Q7dx3 (solid line) at 0.03 and 0.3 mg / kg, as described in Example 17. Repeated administration further expanded these populations, with CD8, CD8 Tcm, and CD8 Tem increasing 35.3X, 183X, and 73.8X, respectively. At the end of the time course (240 hours after the first or last dose at 0.3 mg / kg), cell counts had not returned to baseline in the mice. [Figure 31B-31C] Figures 31A, 31B, and 31C are plots of CD8 and CD8+ memory subpopulation T cell counts following administration of a single dose of Conjugate 1 (dotted line) or Q7dx3 (solid line) at 0.03 and 0.3 mg / kg, as described in Example 17. Repeated administration further expanded these populations, with CD8, CD8 Tcm, and CD8 Tem increasing 35.3X, 183X, and 73.8X, respectively. At the end of the time course (240 hours after the first or last dose at 0.3 mg / kg), cell counts had not returned to baseline in the mice. [Figure 32A-32B] Figures 32A and 32B are plots of NK cell counts and Ki-67 percent positivity in mice after a single dose of 0.01 to 1.5 mg / kg of conjugate 1, as described in Example 17. NK cell counts are significantly increased above vehicle controls at all dose levels and return to baseline by 240 hours post-dose. All dose levels induce a strong increase in Ki-67 percent positivity in NK cells. [Figure 32C]Figure 32C is a plot of NK cell counts in mice following single (solid line) or Q7dx3 (dashed line) administration of 0.03 and 0.3 mg / kg conjugate 1, as described in Example 17. Repeat administration of 0.3 mg / kg conjugate 1 induced slightly lower NK cell counts compared to single administration, but was still significant. Similar NK cell counts were achieved with single vs. repeat administration of 0.03 mg / kg. [Figure 33A] Figure 33A shows an in vitro NK cytotoxicity assay measuring changes in NK-mediated target cell lysis following test article treatment in mice, as described in Example 18. A time course of percent specific lysis of YAC-1 cells by splenic NK cells isolated from BALB / c mice treated with 0.006, 0.03, or 0.3 mg / kg of conjugate 1 or 1 mg / kg of IL-15 is shown at the indicated times. Splenic NK cells from mice administered vehicle served as a control. Conjugate 1 administered at 0.3 mg / kg induced an increase in NK cytotoxicity that was greater in magnitude and duration than NK cells from mice receiving a single injection of 1 mg / kg of IL-15. [Figure 33B] Figure 33B is a graph of percent granzyme B positivity in blood NK cells from the same mice subjected to the NK in vitro cytotoxicity assay in Figure 33A. See Example 18. Conjugate 1 administered at 0.03 and 0.3 mg / kg induced a significant increase in NK granzyme B expression, with a strong and sustained increase seen at 0.3 mg / kg. [Figure 34A-34B] Figures 34A and 34B show percent lung nodule inhibition in BALB / c mice administered two doses of conjugate 1 treatment at 0.03 or 0.3 mg / kg, one week apart, after receiving intravenous CT-26 tumor cell injections, as described in Example 19. Conjugate 1 injections at 0.03 and 0.3 mg / kg inhibited lung nodule formation by 40 and 80%, respectively. The same mice administered 0.3 mg / kg then received subsequent tumor cell injections for 32 days and survival was assessed. Treatment with conjugate 1 significantly increased survival compared to tumor-injected mice that received the vehicle control. [Figure 35] 35 is a graph of percent lung nodule inhibition in CT-26-injected mice treated with Conjugate 2 that underwent antibody-mediated depletion of NK cells (olive green), IgG control (blue), or PBS (orange), as described in Example 20. Data are expressed as percent lung nodule inhibition relative to CT-26-injected mice that were not NK cell-depleted and treated with vehicle control (black). The efficacy of Conjugate 2 in this tumor model was abolished when mice were deprived of NK cells. [Figure 36A] Figures 36A and 36B are plots showing a two-week time course of CD8 cell counts and Ki-67 percent positivity as a measure of proliferation in one male (dotted line) and one female (solid line) cyno after intravenous administration of a 0.1 mg / kg dose of Conjugate 1, as described in Example 21. Conjugate 1 induces a significant CD8 T cell expansion in the cyno, with cell counts increasing 7-10X after a single dose. [Figure 36B] Figures 36A and 36B are plots showing a two-week time course of CD8 cell counts and Ki-67 percent positivity as a measure of proliferation in one male (dotted line) and one female (solid line) cyno after intravenous administration of a 0.1 mg / kg dose of Conjugate 1, as described in Example 21. Conjugate 1 induces a significant CD8 T cell expansion in the cyno, with cell counts increasing 7-10X after a single dose. [Figure 36C] Figures 36C and 36D show the increase in cynoCD8 Tcm and CD8 Tem cell numbers after a single injection of conjugate 1, as described in Example 21. CD8 Tcm and Tem numbers increased 27-30X and 21-33X, respectively. [Figure 36D] Figures 36C and 36D show the increase in cynoCD8 Tcm and CD8 Tem cell numbers after a single injection of conjugate 1, as described in Example 21. CD8 Tcm and Tem numbers increased 27-30X and 21-33X, respectively. [Figure 37A]37A and 37B are graphs of NK cell counts and Ki-67 percent positivity in cynos after a single dose of 0.1 mg / kg of Conjugate 1. See Example 21. NK cells were expanded 9-10X after treatment with Conjugate 1. [Figure 37B] 37A and 37B are graphs of NK cell counts and Ki-67 percent positivity in cynos after a single dose of 0.1 mg / kg of Conjugate 1. See Example 21. NK cells were expanded 9-10X after treatment with Conjugate 1. [Figure 38A] Figures 38A and 38B are EC50 curves for IL-15 (red, solid circles) versus conjugate 1 (green, solid squares) treatment of human PBMCs and subsequent measurement of pSTAT5 percent positivity in CD8 and CD56 bright NK cells, as described in Example 22. Conjugate 1 is 5.5 and 15x less potent than IL-15, respectively, in engaging CD8 and CD56 bright NK cells. However, conjugate 1 achieves the same maximal response as conventional IL-15. [Figure 38B] Figures 38A and 38B are EC50 curves for IL-15 (red, solid circles) versus conjugate 1 (green, solid squares) treatment of human PBMCs and subsequent measurement of pSTAT5 percent positivity in CD8 and CD56 bright NK cells, as described in Example 22. Conjugate 1 is 5.5 and 15x less potent than IL-15, respectively, in engaging CD8 and CD56 bright NK cells. However, conjugate 1 achieves the same maximal response as conventional IL-15. [Figure 39] FIG. 39 is a plot of the plasma concentration over time of 500 μg / kg IL-15 (green, solid circles) or 10 μg / kg (pink, solid squares), 30 μg / kg (purple, solid triangles), 100 μg / kg (red, solid inverted triangles), 300 μg / kg (orange, solid diamonds), or 1000 μg / kg (dark red, open hexagons) conjugate 1 after administration of a single intravenous dose in mice, as described in Example 23. [Figure 40]FIG. 40 is a plot of the plasma concentration over time of 10 μg / kg (pink, solid squares), 75 μg / kg (purple, solid triangles), or 150 μg / kg (orange, solid inverted triangles) conjugate 1 after administration of a single intravenous dose in rats, as described in Example 24. [Figure 41] FIG. 41 is a plot of the plasma concentration of IL-15 at 50 μg / kg (green, solid circle) or at 10 μg / kg (red, solid inverted triangle), 50 μg / kg (blue, solid diamond) or 100 μg / kg (orange, solid square) Conjugate 1 over time following administration of a single intravenous dose of test article in cynomolgus monkeys, as described in Example 25. [Figure 42A-42B] Figures 42A and 42B are plots of CD4 T cell counts and Ki-67 percent positivity, respectively, after a single dose of conjugate 1 in mice at 0.03 mg / kg (blue, solid circles) or 0.3 mg / kg (orange, solid circles), as described in Example 26. Vehicle (black) and pre-dose (open circles) cell levels are also shown. All dose levels returned to baseline by 240 hours post-dose. The 0.3 mg / kg dose level induced a strong increase in Ki-67 percent positivity in CD4 T cells. [Figure 43] 43 is a graph of pSTAT5 percent positivity in CD4 T cells after a single injection of 0.03 mg / kg (orange, solid circles) or 0.3 mg / kg (blue, solid squares) of Conjugate 1 in mice, as described in Example 26. Both dose levels of Conjugate 1 induced increased pSTAT5 signaling in CD4 T cells, with 0.3 mg / kg inducing a higher increase. A 120-hour time course is shown, including vehicle (black) and pre-dose (open circles). [Figures 44A-44C]Figures 44A, 44B, and 44C are plots showing cell counts versus time post-dose for NK cells (Figure 44A), CD8 T cells (Figure 44B), and CD4 T cells (Figure 44C) following iv administration of vehicle (black) or doses of 0.003 mg / kg (blue, inverted triangles), 0.01 mg / kg (green, diamonds), or 0.1 mg / kg (orange, solid squares) of conjugate 1 in cynomolgus monkeys, as described in Example 27. [Figures 45A-45C] Figures 45A, 45B, and 45C are plots showing Ki-67 percent positivity following a single dose of conjugate 1 in cynomolgus monkeys at doses of 0.001 mg / kg (purple, solid squares), 0.003 mg / kg (blue, inverted triangles), or 0.1 mg / kg (orange, solid squares), as described in Example 27. Vehicle (black) levels are also shown. All dose levels returned to baseline by at least day 17 post-dose. The 0.1 mg / kg and 0.003 mg / kg dose levels induced a robust increase in Ki-67 percent positivity in NK cells and CD8 T cells. The 0.1 mg / kg dose level induced an increase in Ki-67 percent positivity in all cell types tested. [Figures 46A-46C] Figures 46A, 46B, and 46C show the degree of STAT5 phosphorylation in NK cell (Figure 46A), CD8 T cell (Figure 46B), and CD4 T cell (Figure 46C) lymphocytes following administration of a single iv dose of vehicle (black) or 0.001 mg / kg (purple, solid squares), 0.01 mg / kg (green, diamonds), or 0.1 mg / kg (orange, solid squares) of Conjugate 1 in cynomolgus monkeys, as described in Example 27. [Figures 47A-47D]Figures 47A-D are plots showing Ki-67 percent positivity following a single dose of conjugate 1 in cynomolgus monkeys at 0.001 mg / kg (purple, solid squares), 0.01 mg / kg (green, diamonds), or 0.1 mg / kg (orange, solid squares), as described in Example 27. Vehicle (black) levels are also shown. Figure 47A shows results for CD8 T natural cells, and Figure 47B shows results for CD8 Tscm cells. Figure 47C shows results for CD8 Tcm cells. Figure 47D shows results for CD8 Tem cells. [Figures 48A-48C] Figures 48A, 48B, and 48C are plots showing granzyme B expression as a function of time after treatment with conjugate 1 at doses of 0.001 mg / kg (Figure 48A), 0.01 mg / kg (Figure 48B), and 0.1 mg / kg (Figure 48C), as described in Example 28. The data show that treatment with conjugate 1 increases the levels of granzyme B in NK cells in non-human primates (NHPs). [Figures 49A-49C] Figures 49A, 49B, and 49C are plots showing perforin expression as a function of time following treatment with Conjugate 1 at doses of 0.001 mg / kg (Figure 49A), 0.01 mg / kg (Figure 49B), and 0.1 mg / kg (Figure 49C), as described in Example 28. The data show that treatment with Conjugate 1 increases the levels of perforin in NK cells in NHPs. DETAILED DESCRIPTION OF THE INVENTION

[0049] Before describing one or more aspects or embodiments of the present disclosure in detail, it should be noted that the present disclosure is not intended to be limited to particular synthesis techniques, IL-15 moieties, or the like, as these may vary as would be understood by one of ordinary skill in the art to which the present disclosure applies.

[0050] In describing and claiming particular features of the present disclosure, the following terminology will be used in accordance with the definitions set out below unless otherwise indicated.

[0051] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0052] In describing and claiming one or more embodiments, the following terminology will be used in accordance with the definitions set out below.

[0053] A "physiologically cleavable" or "hydrolyzable" or "degradable" bond is a relatively unstable bond that reacts with water (i.e., is hydrolyzed) under physiological conditions. The tendency of a bond to be hydrolyzed in water depends not only on the general type of bond connecting two atoms in a given molecule, but may also depend on the substituents attached to those atoms. Suitable hydrolytically unstable or weak bonds can include, but are not limited to, carboxylic acid esters, phosphate esters, anhydrides, acetals, ketals, acyloxyalkyl ethers, imines, orthoesters, peptides, oligonucleotides, thioesters, and carbonates.

[0054] "Enzymatically degradable bond" means a bond that is subject to degradation by one or more enzymes.

[0055] A "stable" linkage or bond refers to a chemical bond that is substantially stable in water, i.e., that does not undergo hydrolysis under physiological conditions to any appreciable extent over an extended period of time. Examples of hydrolytically stable linkages generally include, but are not limited to, carbon-carbon bonds (e.g., in fatty chains), ethers, amides, amines, and the like. Generally, a stable linkage is one that exhibits a rate of hydrolysis of less than about 1-2% per day under physiological conditions. Hydrolysis rates for representative chemical bonds can be found in most standard chemistry textbooks.

[0056] For example, a covalent "releasable" bond in the context of polyethylene glycol covalently attached to an active moiety such as interleukin-15 is a bond that releases or separates the polyethylene glycol polymer from the active moiety under physiological conditions, e.g., by any suitable mechanism, at a clinically useful rate, including, but not limited to, hydrolytically degradable and enzymatically degradable bond(s).

[0057] "Substantially" or "essentially" means nearly entirely or completely (eg, 95% or more of a given amount).

[0058] Similarly, as used herein, "about" or "approximately" means within ±5% of a given amount.

[0059] "Optional" or "optionally" means that the subsequently described situation may occur, but need not necessarily occur, and thus the description includes cases where the situation occurs and cases where it does not occur.

[0060] A "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to a component that can be included in the compositions described herein and that does not cause significant adverse toxicological effects in a subject.

[0061] The expressions "pharmaceutically effective amount," "pharmacologically effective amount," "therapeutically effective amount," and "physiologically effective amount" are used interchangeably herein and refer to the amount of a long-acting IL-15R agonist provided herein required to provide a desired level of a substance in the bloodstream or target tissue to elicit a desired biological or pharmaceutical response. For example, such a response can destroy targeted cancer cells or slow or arrest the progression of cancer in a subject. The term also applies to a dose that induces a particular response in target cells. The precise amount will depend on numerous factors, including, for example, the particular condition being treated, the intended patient population, individual patient considerations, the components and physical properties of the therapeutic composition being administered, and the like.

[0062] References to the long-acting IL-15R agonists described herein are intended to encompass pharmaceutically acceptable salt forms thereof.

[0063] As used herein, the term "patient" or "subject" refers to an organism suffering from or susceptible to a condition that can be prevented or treated by administration of a compound or composition provided herein. Subjects include, but are not limited to, mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), preferably humans.

[0064] Molecular weights associated with water-soluble polymers, such as PEG, can be expressed as either number-average or weight-average molecular weights. Unless otherwise indicated, all references to molecular weight herein refer to weight-average molecular weights. Both number-average and weight-average molecular weights can be measured using gel permeation chromatography or other liquid chromatography techniques (e.g., gel filtration chromatography). Gel permeation chromatography and gel filtration chromatography are most commonly used. Other methods for measuring molecular weight include end-group analysis or measuring colligative properties (e.g., freezing point depression, boiling point elevation, or osmotic pressure) to determine the number-average molecular weight, or light scattering, ultracentrifugation, MALDITOF, or viscometry to determine the weight-average molecular weight. PEG polymers are typically polydisperse (i.e., the number-average and weight-average molecular weights of the polymer are not equal), preferably with a low polydispersity value of less than about 1.2, more preferably less than about 1.15, even more preferably less than about 1.10, even more preferably less than about 1.05, and most preferably less than about 1.03.

[0065] The terms "active," "reactive," or "activated," when used in conjunction with a particular functional group, refer to a reactive functional group that readily reacts with an electrophile or nucleophile on another molecule. This is in contrast to groups that require strong catalysts or extremely impractical reaction conditions in order to react (i.e., "nonreactive" or "inert" groups).

[0066] As used herein, the term "functional group" or any synonym thereof is intended to encompass protected as well as unprotected forms thereof.

[0067] The terms "spacer moiety," "linkage," and "linker" may be used herein to refer to a bond or an atom or collection of atoms optionally used to connect, for example, the terminus of a polymeric agent and the interconnecting portion of an IL-15 moiety. A spacer moiety may be hydrolytically stable or may include a physiologically hydrolyzable, enzymatically degradable, or otherwise releasable linkage. Unless the context clearly dictates otherwise, a spacer moiety is optionally present between any two elements of a compound (e.g., an IL-2 moiety and a water-soluble polymer such as PEG can be attached directly or indirectly via a spacer moiety).

[0068] "Alkyl" refers to a hydrocarbon chain, typically ranging from about 1 to 15 atoms in length. Such hydrocarbon chains are preferably, but not necessarily, saturated and may be branched or straight-chained, although straight-chained is typically preferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 3-methylpentyl, and the like.

[0069] "Lower alkyl" refers to an alkyl group containing from 1 to 6 carbon atoms, which may be straight or branched, as exemplified by methyl, ethyl, n-butyl, i-butyl, and t-butyl.

[0070] "Alkoxy" refers to the group -OR, where R is alkyl or substituted alkyl, preferably C 1~6 It refers to the group -OR, which is alkyl (e.g., methoxy, ethoxy, propyloxy, etc.).

[0071] The term "substituted," as in "substituted alkyl," includes, but is not limited to, alkyl, C 3~8"Substituted aryl" refers to a moiety (e.g., an alkyl group) substituted with one or more non-interfering substituents, such as cycloalkyl, e.g., cyclopropyl, cyclobutyl, etc.; halo, e.g., fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl; substituted phenyl; etc. "Substituted aryl" refers to an aryl having one or more non-interfering groups as substituents. For substitution on a phenyl ring, the substituents may be in any orientation (i.e., ortho, meta, or para).

[0072] "Non-interfering substituents" are those groups that, when present in a molecule, are typically non-reactive with other functional groups contained in the molecule.

[0073] "Aryl" means one or more aromatic rings, each having five or six central carbon atoms. Aryl includes multiple aryl rings that may be fused, as in naphthyl, or unfused, as in biphenyl. Aryl rings may also be fused or unfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclic rings. As used herein, "aryl" includes heteroaryl.

[0074] "Heteroaryl" is an aryl group containing one to four heteroatoms, preferably sulfur, oxygen, or nitrogen, or a combination thereof. The heteroaryl ring may also be fused with one or more cyclic hydrocarbon, heterocyclic, aryl, or heteroaryl rings.

[0075] "Heterocycle" or "heterocyclic" means one or more rings of 5 to 12 atoms, preferably 5 to 7 atoms, with or without unsaturated or aromatic character, and having at least one ring atom other than carbon. Preferred heteroatoms include sulfur, oxygen, and nitrogen.

[0076] A "substituted heteroaryl" is a heteroaryl bearing one or more non-interfering groups as substituents.

[0077] A "substituted heterocycle" is a heterocycle having one or more side chains formed from non-interfering substituents.

[0078] "Organic radicals," as used herein, include alkyl, substituted alkyl, aryl, and substituted aryl.

[0079] 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 any significant toxic adverse effects to the patient.

[0080] The term "IL-15 moiety," as used herein, refers to a peptide or protein moiety that has human IL-15 activity. Additionally, the term "IL-15 moiety" encompasses both the IL-15 moiety prior to conjugation and the IL-15 moiety residue after conjugation. As explained in more detail below, one of skill in the art can determine whether any given moiety has IL-15 activity. Proteins containing an amino acid sequence corresponding to any one of SEQ ID NOS: 1-3, as well as any protein or polypeptide substantially homologous thereto, are IL-15 moieties. As used herein, the term "IL-15 moiety" includes such peptides and proteins that have been intentionally modified, e.g., by site-directed mutagenesis, or accidentally modified by mutation. These terms also include analogs with one to six additional glycosylation sites, analogs with at least one additional amino acid at the carboxy-terminal end of the peptide or protein, where the one or more additional amino acids comprise at least one glycosylation site, and analogs having an amino acid sequence that includes at least one glycosylation site. The term includes both naturally occurring moieties and recombinantly and synthetically produced moieties.

[0081] The terms "substantially homologous" or "substantially identical" mean that a particular subject sequence, e.g., a mutant sequence, differs from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which is no adverse functional difference between the reference and subject sequences. For purposes of the present invention, sequences having greater than 95 percent homology (identity), equivalent biological activity (equivalent to, but not necessarily equivalent strength of biological activity) to a given sequence, and equivalent expression characteristics are considered to be substantially homologous (identical). For purposes of determining homology, truncations of the native sequence should be disregarded. Exemplary IL-15 polypeptides used herein include sequences substantially homologous to SEQ ID NO:1. SEQ ID NO:2 is nearly identical to SEQ ID NO:1, except that SEQ ID NO:2 contains an initial methionine, which is required for initiation of translation in E. coli.

[0082] The term "fragment" refers to any protein or polypeptide that has the amino acid sequence of a portion or fragment of an IL-15 portion and that has the biological activity, or substantially the biological activity, of IL-15. Fragments include proteins or polypeptides produced by proteolysis of an IL-15 portion and proteins or polypeptides produced by chemical synthesis by methods routine in the art.

[0083] Amino acid residues in peptides are abbreviated as follows: phenylalanine is Phe or F; leucine is Leu or L; isoleucine is Ile or I; methionine is Met or M; valine is Val or V; serine is Ser or S; proline is Pro or P; threonine is Thr or T; alanine is Ala or A; tyrosine is Tyr or Y; histidine is His or H; glutamine is Gln or Q; asparagine is Asn or N; lysine is Lys or K; aspartic acid is Asp or D; glutamic acid is Glu or E; cysteine ​​is Cys or C; tryptophan is Trp or W; arginine is Arg or R; and glycine is Gly or G.

[0084] overview The present disclosure relates to the provision of long-acting IL-15 receptor agonists. Such agonists would ideally possess several advantageous and unexpected characteristics, such as, for example, at least one of the following: (i) the ability to deliver sustained IL-15 activity by providing a measurable pharmacodynamic effect without the need for daily administration, (ii) retain high binding to IL-15 receptor alpha (i.e., compared to IL-15), (iii) stimulate NK cell activation and / or proliferation, and / or (iv) support CD8 T cell survival and / or memory formation, and (v) provide tumor growth inhibition. Surprisingly, Applicants have arrived at long-acting IL-15R agonists that possess a unique combination of advantageous properties, described in detail below.

[0085] Long-acting IL-15 R agonists and related compositions Generally, long-acting IL-15 receptor agonists or their pharmaceutically acceptable salt forms contain a single linear PEG (polyethylene glycol) moiety stably and covalently attached to an IL-15 amino group via an amide bond. Between the PEG moiety and the stable amide bond to the IL-15 amino group is a linear unsubstituted alkylene group (~CH2~) having 2 to 5 carbon atoms. m (i.e., m=2, 3, 4, or 5).

[0086] When discussing an IL-15 moiety, the term "IL-15 moiety" refers to the IL-15 moiety prior to conjugation and to the IL-15 moiety after attachment to a non-peptidic, water-soluble polymer, such as a poly(alkylene oxide) (e.g., poly(ethylene glycol) or PEG). While specific reference will be made hereinafter to PEG as the non-peptidic, water-soluble polymer, it will be understood that the present disclosure generally relates to non-peptidic, water-soluble polymers or poly(alkylene glycol). However, it will be understood that when the original IL-15 moiety is attached to a polyethylene glycol moiety, the IL-15 moiety is slightly altered due to the presence of one or more covalent bonds associated with attachment to the polymer.

[0087] The IL-15 moiety can be obtained by non-recombinant and recombinant methods, and the disclosure is not limited in this respect. In addition, the IL-15 moiety can be derived from human, animal (including insect), fungal (including yeast), and plant sources.

[0088] Alternatively, the IL-15 moiety can be obtained according to the procedure described by Grabstein et al. (1994) Science 264:965-968. The IL-15 moiety can be obtained, for example, by Immunex It can also be prepared using recombinant methods, such as those described in European Patent No. 0772624 B2 to GenScript Corporation. The IL-15 moiety can be purchased commercially, for example, from GenScript USA Inc. (Piscataway, NJ) and Peprotech (Rockyhill, NJ).

[0089] The IL-15 moiety may be derived from bacteria [e.g., E. coli, e.g., Fischer et al. (1995) Biotechnol. Appl. Biotechnol. 21(3):295-311], mammalian [see, e.g., Kronman et al. (1992) Gene 121:295-304], yeast [e.g., Pichia pastoris, see, e.g., Morel et al. (1997) Biochem. J. 328(1):121-129], and plant [see, e.g., Mor et al. (2001) Biotechnol. Bioeng. 75(3):259-266] expression systems. Expression can occur by exogenous expression (when the host cell naturally contains the desired genetic code) or by endogenous expression.

[0090] Recombinant-based methods for preparing proteins can vary, but typically recombinant methods involve constructing a nucleic acid encoding the desired polypeptide or fragment, cloning the nucleic acid into an expression vector, transforming a host cell (e.g., a plant, bacteria, yeast, transgenic animal cell, or mammalian cell such as a Chinese hamster ovary cell or a baby hamster kidney cell), and expressing the nucleic acid to produce the desired polypeptide or fragment. Methods for producing and expressing recombinant polypeptides in vitro in prokaryotic and eukaryotic host cells are known to those of skill in the art.

[0091] To facilitate identification and purification of recombinant polypeptides, a nucleic acid sequence encoding an epitope tag or other affinity binding sequence can be inserted or added in frame with the coding sequence, thereby producing a fusion protein containing the desired polypeptide and a polypeptide suitable for binding. Fusion protein identification and purification can be achieved by first passing a mixture containing the fusion protein through an affinity column bearing a binding moiety (e.g., an antibody) for the epitope tag or other binding sequence in the fusion protein, thereby binding the fusion protein to the column. The fusion protein can then be recovered by washing the column with an appropriate solution (e.g., acid) to release the bound fusion protein. Recombinant polypeptides can also be purified by lysis of host cells, separation of the polypeptide by, for example, ion exchange chromatography, affinity binding techniques, or hydrophobic interaction techniques, followed by identification by MALDI or Western blot, and recovery of the polypeptide. These and other methods for identifying and purifying recombinant polypeptides are known to those of skill in the art. However, in one or more embodiments, the IL-15 moiety is not in the form of a fusion protein.

[0092] Depending on the system used to express the protein having IL-15 activity, the IL-15 moiety may be non-glycosylated or glycosylated, and either may be used. That is, the IL-15 moiety may be non-glycosylated, or the IL-15 moiety may be glycosylated. In one or more embodiments, the IL-15 moiety is non-glycosylated.

[0093] The IL-15 moiety can be advantageously modified to include and / or substitute one or more amino acid residues, e.g., lysine, cysteine, and / or arginine, which facilitates attachment of the polymer to atoms in the side chain of that amino acid. Examples of substitutions of the IL-15 moiety are described in U.S. Pat. No. 6,177,079. Additionally, the IL-15 moiety can be modified to include non-naturally occurring amino acid residues. Methods for adding amino acid residues and non-naturally occurring amino acid residues are known to those skilled in the art. See J. March, Advanced Organic Chemistry: Reactions Mechanisms and Structure, 4th Ed. (New York: Wiley-Interscience, 1992), and Bioinformatics for Geneticists (eds. Michael R. Barnes and Ian C Gray), 2003 John Wiley & Sons, Ltd, Chapter 14, Amino Acid Properties and Consequences of Substitutions, Betts, MJ, and Russell, RB.

[0094] In addition, the IL-15 moiety can advantageously be modified to include a functional group (other than by the attachment of an amino acid residue containing a functional group). For example, the IL-15 moiety can be modified to include a thiol group. In addition, the IL-15 moiety can be modified to include an N-terminal alpha carbon. In addition, the IL-15 moiety can be modified to include one or more carbohydrate moieties. In addition, the IL-15 moiety can be modified to include an aldehyde group. In addition, the IL-15 moiety can be modified to include a ketone group. In some embodiments of the invention, the IL-15 moiety is preferably not modified to include one or more of a thiol group, an N-terminal alpha carbon, a carbohydrate, an aldehyde group, and a ketone group.

[0095] Exemplary IL-15 moieties are described herein, in the literature, and in, for example, U.S. Patent Application Publication No. 2006 / 0104945, Pettit et al. (1997) J. Biol. Chem. 272(4):2312-2318, and Wong et al. (2013) OncoImmunology 2(11), e26442:1-3. Preferred IL-15 moieties include those having an amino acid sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 1-3, and sequences substantially homologous thereto (it will be understood that SEQ ID NOs: 2 and 3, and sequences substantially homologous thereto, are also understood to be "IL-15 moieties" for purposes of the present invention, even if they do not meet the in vitro activity criteria of an IL-15 moiety provided herein). A preferred IL-15 moiety has an amino acid sequence corresponding to SEQ ID NO: 1. In some embodiments, the IL-15 portion is a functional homolog having at least about 85% or at least about 90% identity to any one of SEQ ID NOs: 1-3. In some embodiments, the IL-15 portion is a functional homolog having at least about 95%, 98%, or 99% identity to any one of SEQ ID NOs: 1-3.

[0096] In some cases, the IL-15 moiety may be in a "monomeric" form, where a single expression of the corresponding peptide is organized as an individual unit. In other cases, the IL-15 moiety may be in a "dimeric" form, where two monomeric forms of the protein are associated with each other (e.g., a dimer of recombinant IL-15).

[0097] Additionally, the precursor form of IL-15 can be used as the IL-15 moiety. An exemplary precursor form of IL-15 has the sequence of SEQ ID NO:3.

[0098] Truncated forms, hybrid variants, and peptidomimetics of any of the foregoing sequences can also serve as IL-15 moieties. Biologically active fragments, deletion variants, substitution variants, or addition variants of any of the foregoing that maintain at least some IL-15 activity can also serve as IL-15 moieties.

[0099] For a given peptide, protein moiety, or conjugate, it is possible to determine whether the peptide, protein moiety, or conjugate has IL-15 activity. Various methods for determining in vitro IL-15 activity have been described in the art. An exemplary approach is based on the pSTAT assay. Briefly, when IL-15-dependent CTLL-2 cells are exposed to a test substance with IL-15 activity, a signaling cascade is initiated, including the phosphorylation of STAT5 at tyrosine residue 694 (Tyr694), which can be quantitatively measured. Assay protocols and kits are known, including, for example, the MSD Phospho(Tyr694) / Total STATa,b Whole Cell Lysate Kit (Meso Scal Diagnostics, LLC, Gaithersburg, MD); for example, using this approach, a pSTAT5 EC2 of about 300 ng / mL or less (more preferably about 150 ng / mL or less) at at least one of the 5 and 10 minute time points can be determined. 50Candidate IL-15 moieties exhibiting pSTAT5 EC values ​​of less than 150 ng / mL at at least one of the 5 or 10 minute time points are considered to be "IL-15 moieties" in the context of the present disclosure. However, IL-15 moieties that are used may be more potent (e.g., pSTAT5 EC values ​​of less than about 1 ng / mL at at least one of the 5 or 10 minute time points, and even more preferably less than 0.5 ng / mL). 50 It is preferred that the .alpha.-to-.alpha.

[0100] Other methods known in the art, including electrometric, spectroscopic, chromatographic, and radiometric assays, can also be used to assess IL-15 function. For one such alternative type of assay, see, e.g., Ring et al. (2012) Nat. Immunol. 13(12):1187-1195.

[0101] Assays used in connection with measuring the activity of the IL-15 moiety can also be used to measure the activity of the long-acting IL-15R agonists described herein (see, e.g., the Supporting Examples provided herein).

[0102] A compound is considered a long-acting IL-15R agonist according to the present disclosure so long as, after administration to a subject, the agonist exhibits IL-15 receptor activating activity in vivo for a longer period than administration of IL-15. Long-acting IL-15R agonists can be determined using conventional techniques, including, for example, radiolabeling the compound, administering the compound in vivo, and determining its clearance. It can be evaluated whether a compound proposed as an IL-15 R agonist is "long-acting" (i.e., has a longer clearance than IL-15 administered in the same in vivo system). For purposes of the present invention, the long-acting nature of a long-acting IL-15 R agonist can be, or typically is, determined using flow cytometry to measure STAT5 phosphorylation in lymphocytes at various time points after administration of the agonist to be evaluated to mice. For reference, the signal is lost by IL-15 in approximately 24 hours, whereas in the case of a long-acting IL-15 agonist, it persists over a period of time.

[0103] As previously mentioned, preferred long-acting IL-15R agonists will generally comprise a single linear PEG (polyethylene glycol) moiety stably and covalently attached to an IL-15 amino group via an amide bond. Between the PEG moiety and the stable amide bond to the IL-15 amino group is a linear unsubstituted alkylene group (~CH2~) having 2 to 5 carbon atoms. m (i.e., m=2, 3, 4, or 5).

[0104] For example, in some embodiments, the unsubstituted alkylene group is (-CH2-)2; or in some additional embodiments, the unsubstituted alkylene group is (-CH2-)3; in some still further embodiments, the unsubstituted alkylene group is (-CH2-)4; in some still further embodiments, the unsubstituted alkylene group is (-CH2-)5.

[0105] For example, in some embodiments, the long-acting IL-15 receptor agonist has the following structure: [ka] wherein IL-15 is an interleukin-15 moiety, n is an integer from about 150 to about 3,000; m is an integer from 2 to 5 (e.g., 2, 3, 4, or 5), and n' is 1. In Formula I (and similar formulas provided herein), ~NH~ in the structure represents the amino group of the IL-15 moiety. Formula (I) may also be represented as: [ka] where the brackets are moved to reflect the terminal PEG methoxy group, and the two formulas can be used interchangeably. Examples of exemplary compounds encompassed by formula (I) include the following: [ka]

[0106] 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).

[0107] In some further embodiments related to the structures and formulas described herein, n is an integer from about 200 to about 2000, or from about 400 to about 1300, or from about 450 to about 1200. That is, in some embodiments, n is an integer from about 200 to about 2000. In some still further embodiments, n is an integer from about 400 to about 1300. In some still further embodiments, n is an integer from about 450 to about 1200.

[0108] PEGs having molecular weights corresponding to any of the ranges of n values ​​given above are generally preferred.

[0109] In one or more embodiments, n is an integer having a value corresponding to a polyethylene glycol polymer having a weight average molecular weight selected from the group consisting of about 10,000 daltons (where n is about 227), or about 15,000 daltons (where n is about 340), or about 20,000 daltons (where n is about 454), or about 25,000 daltons (where n is about 568), or about 30,000 daltons (where n is about 681), or about 40,000 daltons (where n is about 909), or about 50,000 daltons (where n is about 1136), or even about 60,000 daltons (where n is about 1364) or greater.

[0110] Further exemplary weight average molecular weights for the polyethylene glycol portion of the compound include, in addition to those mentioned above, about 11,000 daltons, about 12,000 daltons, about 13,000 daltons, about 14,000 daltons, about 22,500 daltons, about 35,000 daltons, about 45,000 daltons, about 55,000 daltons, about 65,000 daltons, about 70,000 daltons, and about 75,000 daltons.

[0111] In some preferred embodiments, the weight average molecular weight of the polyethylene glycol polymer portion of the compound is about 40,000 daltons.

[0112] The PEG moiety is preferably end-capped with a methoxy group, as shown above in formula (I), but the PEG moiety may also terminate its terminus with any lower C 1-6 It may be capped with an alkoxy group or terminated with a hydroxyl group or other suitable end-capping group.

[0113] In some embodiments, the long-acting IL-15 receptor agonist compositions comprise compounds having the following formula, when considered collectively: [ka] wherein the values ​​of n and m are as provided for formula (I) above. That is, in terms of the long-acting IL-15 receptor agonist component of such compositions, about 20 mole percent or less of the long-acting IL-15 receptor agonists included in the compositions are of formula (II).

[0114] In some additional embodiments, the long-acting IL-15 receptor agonist compositions have the following formula, when considered collectively: [ka] where the values ​​of n and m are as provided for Formula (I) above. That is, in terms of the long-acting IL-15 receptor agonist component of such compositions, about 15 mol percent or less of the long-acting IL-15 receptor agonist included in the composition is of Formula (II). In some embodiments, the long-acting IL-15 receptor agonist composition comprises about 0.1-20 mol % or less of a compound of Formula (II). In embodiments, the composition comprises about 0.1-15, 0.1-10, 0.1-5, 0.1-1, 1-20, 1-15, 1-10, 1-5, 5-20, 5-15, 5-10, 10-20, 10-15, or 15-20 mol % or less of a compound of Formula (II).

[0115] In certain particular embodiments related to the foregoing, the long-acting IL-15 receptor agonist compositions related to Formula (Ia) have the following formula, when considered collectively: [ka] wherein the values ​​of n and m are as provided for formula (Ia) above.

[0116] In some other preferred embodiments, the long-acting IL-15 receptor agonist compositions related to Formula (Ib) have the following formula, when considered collectively: [ka] wherein the values ​​of n and m are as provided for formula (Ib) above.

[0117] In some other embodiments, the long-acting IL-15 receptor agonist compositions related to Formula (Ic) have the following formula, when considered collectively: [ka] wherein the values ​​of n and m are as provided for formula (Ic) above.

[0118] In some other embodiments, the long-acting IL-15 receptor agonist compositions related to Formula (Id) have the following formula, when considered collectively: [ka] wherein the values ​​of n and m are as provided for formula (Id) above.

[0119] In some embodiments, the long-acting IL-15 receptor agonist composition comprises about 0.1-20 mol % or less of a compound of Formula (II) (including compounds of Formulas (IIa), (IIb), (IIc), and (IId)). In some additional embodiments, the composition comprises about 0.1-15, 0.1-10, 0.1-5, 0.1-1, 1-20, 1-15, 1-10, 1-5, 5-20, 5-15, 5-10, 10-20, 10-15, or 15-20 mol % or less of a compound of Formula (II) (including compounds of Formulas (IIa), (IIb), (IIc), and (IId)). In some embodiments, the composition comprises about 0.1, 1, 5, 10, 15, or 20 mol % or less of a compound of Formula (II) (including compounds of Formulas (IIa), (IIb), (IIc), and (IId)). It will be appreciated that the composition may be purified by methods known in the art for compounds of formula (I), such that compounds of formula (II) may be absent, present in trace amounts, or substantially absent from the composition.

[0120] For example, in some embodiments, the long-acting IL-15 receptor agonist composition comprises about 12 mole percent or less, or about 10 mole percent or less, of long-acting IL-15 receptor agonists encompassed by Formula (II), which, when considered collectively, includes compounds of Formulas (IIa), (IIb), (IIc), and (IId).

[0121] In some additional embodiments of the foregoing, the composition comprises about 7 mol % or less of a long-acting IL-15 receptor agonist having an n' equal to 2, 3, or greater than 3 (i.e., a higher PEGmer). In yet some other embodiments, the composition comprises about 5 mol % or less of a long-acting IL-15 receptor agonist having an n' equal to 2, 3, or greater than 3 (i.e., 2 or greater).

[0122] In some further embodiments, the composition comprises a compound represented by Formula (I): [ka] wherein n and m are as defined above, n' represents the average number of polyethylene glycol moieties covalently attached to IL-15 amino groups (with respect to the composition), and n' with respect to the composition is in the range of 1.0 to about 1.3. For example, the average number of polyethylene glycol moieties per IL-15 moiety is selected from about 1.0, 1.1, 1.2, and about 1.3. That is, preferred long-acting IL-15 receptor agonists according to formula (I) may be referred to herein as "mono-PEGylated," although it should be understood that there is some variability in the degree of PEGylation, as described above. In some preferred embodiments related to the formulas described herein, "m" is equal to 3.

[0123] A composition of long-acting IL-15R agonists may comprise a single species in which n' is equal to about 1 and the PEG moiety is attached at the same position for substantially all of the IL-15 conjugates in the composition, or alternatively, may comprise a mixture of monoPEGylated conjugate species in which attachment of the linear polyethylene glycol moiety occurs at different sites on the interleukin-15 moiety, i.e., the specific attachment site is not the same for all of the monoPEGylated IL-15 species contained in the composition.) Thus, such compositions are substantially homogeneous in terms of the number of PEG moieties attached to IL-15 (e.g., 1-mers), but heterogeneous in terms of the position of amino group attachment on the IL-15 molecule.

[0124] Although additional PEG constructions and conjugation chemistries can be used to arrive at long-acting IL-15R agonists, as will become apparent when considered in light of the supporting examples, compounds such as those described above are preferred in one or more embodiments. However, additional long-acting IL-15R agonists having the structures provided herein are also contemplated.

[0125] In some embodiments, the long-acting IL-15 receptor agonist composition comprises at least about 80 mol % of long-acting IL-15 receptor agonists (of the IL-15-containing molecules in the composition) encompassed by Formula (I), including Formulae (Ia-d), when considered collectively. In one or more embodiments, the long-acting IL-15 receptor agonist composition comprises at least about 85 mol %, 90 mol %, 95 mol %, 98 mol %, or 99 mol % of long-acting IL-15 receptor agonists of Formula (I).

[0126] As mentioned above, long-acting IL-15R agonists can be in the form of pharmaceutically acceptable salts. Typically, such salts are formed by reaction with a pharmaceutically acceptable acid or acid equivalent. In this context, the term "pharmaceutically acceptable salts" generally refers to relatively non-toxic inorganic and organic acid addition salts. These salts can be prepared in situ during the administration vehicle or dosage form manufacturing process, or by separately reacting a long-acting interleukin-15 receptor agonist described herein with a suitable organic or inorganic acid and isolating the salt so formed. Alternative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, oxylate, mesylate, glucoheptonate, lactobionate, and lauryl sulfonate salts (see, e.g., Berge et al. (1977) "Pharmaceutical Salts," J. Pharm. Sci. 66:1-19). Thus, the salts described may be derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; or prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

[0127] In some embodiments, the long-acting IL-15 receptor agonist composition comprises, collectively, about 1-5 mol% or less of free IL-15 protein (of the IL-15-containing molecules in the composition). In some further embodiments, the long-acting IL-15 agonist composition comprises about 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, or 5 mol% or less of free (i.e., unconjugated) IL-15.

[0128] To prepare a long-acting IL-15 receptor agonist, an IL-15 moiety can be conjugated, for example, at its amino group (e.g., lysine or N-terminus) to a PEG reagent functionalized with a succinimidyl group (or other activated ester group). Using this approach, succinimidyl-activated PEG can be attached to the amino group of the IL-15 moiety in aqueous media at a pH of about 7.0-9.0, although the use of different reaction conditions (e.g., a lower pH, such as 6-7 or 7-8, or a different temperature and / or a temperature below 15°C) can result in attachment of the PEG moiety to a different position on the IL-15 moiety.

[0129] Long-acting IL-15R agonists can be prepared as described in Example 1. For example, long-acting IL-15R agonists can generally be prepared by reacting interleukin-15, e.g., purified IL-15, such as recombinant IL-15, with an activated PEG reagent, such as an activated ester, methoxy PEG-succinimidyl butanoate, or mPEG-SBA. Other suitable activated PEG reagents include methoxy PEG-succinimidyl propionate, methoxy PEG-succinimidyl pentanoate, and methoxy PEG-succinimidyl hexanoate. While a succinimidyl activating group is typically used, any suitable activated ester or activating group can be used, provided that such reactive group is suitable for forming the desired stable amide bond. Generally, interleukin-15 is dissolved in a suitable buffer, such as phosphate-buffered saline (PBS). The PEG reagent can be added to IL-15, typically in solution in a suitable buffer, in an equimolar ratio (relative to the molar amount of interleukin-15) or in a molar excess (based on the molar amount of IL-15), i.e., up to about a 15-fold molar excess, e.g., a 2-fold molar excess, or a 5-fold molar excess, or a 7-fold molar excess, or a 10-fold molar excess, or even a 12-fold molar excess or greater. In some embodiments, the PEG reagent is added in about a 5-10-fold molar excess. The PEG reagent can be added in solid form or as a solution in a suitable solvent, e.g., in an aqueous acid such as dilute hydrochloric acid.

[0130] In some further embodiments of the method, interleukin-15 is initially present in solution at a concentration of about 0.5 mg / mL to about 10 mg / mL, i.e., prior to mixing with the methoxy PEG-succinimidyl alkanoate reagent. 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, about 0.5-1 mg / mL, about 1-10 mg / mL, about 1-5 mg / mL, about 1-4 mg / mL, about 1-3 mg / mL, about 1-2 mg / mL, about 1-1.5 mg / mL, and about 1.5-10 mg / mL in solution. Examples of interleukin-15 include about 0.5 mg / mL, 1.5 to 5 mg / mL, about 1.5 to 4 mg / mL, about 1.5 to 3 mg / mL, about 1.5 to 2 mg / mL, about 2 to 10 mg / mL, about 2 to 5 mg / mL, about 2 to 4 mg / mL, about 2 to 3 mg / mL, about 3 to 10 mg / mL, about 3 to 5 mg / mL, about 3 to 4 mg / mL, about 4 to 10 mg / mL, about 4 to 5 mg / mL, or about 5 to 10 mg / mL. In some specific, but non-limiting, embodiments, the concentration of interleukin-15 in the solution is about 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, or 10 mg / mL.

[0131] It will be appreciated that any suitable buffer may be used or added to the reaction mixture, some exemplary buffers include sodium phosphate (NaPi), sodium acetate (NaAc), boric acid, bicine, citric acid, and Bis-TRIS buffers.

[0132] In some embodiments, the pH of the IL-15 solution is adjusted to approximately pH 8 prior to addition of the PEG reagent.

[0133] After addition of the PEG reagent, the reaction mixture may then be adjusted to a suitable pH, for example, about 7.0-8.5, or 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 particular embodiments, the reaction mixture is adjusted to a pH of about 8.0. It will be appreciated that the pH may be adjusted both before and after addition of the PEG reagent, as necessary to achieve the desired pH.

[0134] Interleukin-15, like many proteins, is subject to deamidation, particularly at higher pH levels; however, lower pH levels may result in several potential drawbacks, such as a lower degree of conjugation at the epsilon (ε) amine and / or increased and / or undesirable positional isoforms, as well as protein aggregation. Deamidation introduces a negative charge into the protein, which may lead to changes in the protein's activity, structure, function, stability, and / or susceptibility to degradation. Thus, one of the challenges addressed by the present agonists and related methods was to provide long-acting interleukin-15 receptor agonists that balance, among other considerations, at least (i) a desired degree of conjugation, (ii) low amounts of deamidation of the interleukin-15 moiety both before and after conjugation with a subject PEG reagent (which may lead, for example, to reduced interleukin-15 activity), and (iii) protein aggregation (e.g., both before and after conjugation), while maintaining sufficient activity (i.e., to be therapeutically useful).

[0135] Based on the competing and conflicting challenges associated with the reaction parameters for preparing the long-acting interleukin-15 receptor agonists described herein, Applicants have discovered that by adjusting the pH of the interleukin-15 solution (before or after reaction with the PEG reagent) and / or the IL-15-PEG reagent reaction mixture, optimal (lower levels of) deamidation can be reached while still facilitating conjugation of the PEG moiety to the interleukin-15 moiety (e.g., at the ε amine and N-terminus) to provide the long-acting IL-15 R agonists described herein. Without being bound by theory, based on a series of reactions in which numerous reaction parameters were varied, a pH in the range of about 7.0 to about 8.5, or about 7.5 to about 8.2, or about 7.8 to about 8.2, or about 8.0 appears to be effective in providing lower levels of deamidation in the product while still facilitating conjugation of the PEG moiety to form the products described herein and maintaining the desired therapeutic profile.

[0136] For example, the methods described herein are effective in 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 less than about 20% deamidated. In some embodiments, the level of deamidation in the product is within the range of about 20-35%, or within the range of about 20-25%, or within the range of about 25-35%, or within the range of about 25-30%. Alternatively, in some embodiments, PEGylated interleukin-15 having a degree of deamidation less than those stated above is contemplated. As shown in Experiment 2 of Example 1, adjustment of the pH to within the range of about 7.0-8.5 resulted in a deamidation level of 21.29% (Composition 1) or 33.26% (Composition 2).

[0137] The reactants are generally mixed for about 5 to 10 hours, inclusive. In some embodiments, the reactants are mixed for about 2 to 5 hours, inclusive. In some embodiments, the reactants are mixed for up to about 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 about 45 minutes to about 3.0 hours, or about 45 minutes to about 2.5 hours, or about 45 minutes to about 2.0 hours, or about 45 minutes to about 1.5 hours, or about 45 minutes to about 1.0 hour. Mixing is generally carried out under mild conditions, for example, from about 20°C to about 65°C, or from about 20°C to about 40°C, or at ambient or room temperature (e.g., about 22°C). Lower temperatures are used to favor a lower degree of PEGylation. The reaction can be quenched by the addition of an amino acid such as, for example, glycine.

[0138] In embodiments, the pH of the composition may be further adjusted to mitigate deamidation. In some embodiments, the composition is adjusted to a pH of about 6.5-7.5 or 6.5-7.0. In some embodiments, the composition is adjusted to a pH of about 6.5, 6.8, 7.0, or 7.5.

[0139] The PEGylated rIL-15 reaction product is then generally purified by any suitable method, such as ion exchange chromatography, to obtain the desired product. For example, anion exchange chromatography can be used. The chromatography product pool can then be concentrated and diafiltered into a suitable formulation buffer (e.g., sodium acetate buffer containing sucrose), for example, using tangential flow filtration (TFF). Analysis can be performed by any suitable method, such as, for example, SDS-PAGE, reverse-phase HPLC, or any other suitable analytical method.

[0140] As previously described, the amino group on the IL-15 moiety provides a site of attachment between the IL-15 moiety and the polyethylene glycol moiety to provide a long-acting IL-15R agonist encompassed by Formula (I). For example, considering the exemplary IL-15 amino acid sequence provided herein, it is apparent that there are seven lysine residues, each with an ε-amino acid that may be available for conjugation. Additionally, the N-terminal amine of methionine may also serve as a point of attachment for the PEG moiety. It will be appreciated that the polyethylene glycol moiety may be attached at any one or more of the lysine or N-terminal amine positions. In some embodiments, the polyethylene glycol moiety attachment site is at Lys 10 and Lys 11 (using the numbering shown in SEQ ID NO: 2 as an example, or using SEQ ID NO: 1, 11 and Lys 12 In some embodiments, the polyethylene glycol moiety is attached at the N-terminal amine. Any of the lysine sites may be attached at the site of attachment of the PEG moiety (e.g., Lys in SEQ ID NO: 1). 37 or Lys 42 In some embodiments, the long-acting interleukin-15 receptor agonist comprises a mixture of positional isomers in which the covalent attachment of the polyethylene glycol moiety is predominantly at the N-terminus (i.e., of the population of positional isomers, the isomer having the PEG moiety attached at the N-terminus is present in the greatest amount compared to the other positional isomers).

[0141] If desired, the product pool may be further separated into positional isomers by reversed-phase chromatography using reversed-phase high performance liquid chromatography (RP-HPLC) using a suitable column (e.g., a C18 or C3 column commercially available from companies such as Amersham Biosciences or Vydac) or by ion-exchange chromatography using an ion-exchange column, for example, a Sepharose™ ion-exchange column available from Amersham Biosciences. Either technique can be used to separate PEG-interleukin-15 positional isomers that have the same molecular weight (i.e., positional isoforms).

[0142] Suitable gel filtration columns for carrying out this type of separation include Superdex™ and Sephadex™ columns available from GE Healthcare (Buckinghamshire, UK). The choice of a particular column can depend on the desired fractionation range. Elution is generally carried out using a suitable buffer, such as phosphate or acetate. Collected fractions can be analyzed by a variety of different methods, including, for example, (i) absorbance at 280 nm for protein content, (ii) dye-based protein analysis using bovine serum albumin (BSA) as a standard, (iii) iodine testing for PEG content (Sims et al. (1980) Anal. Biochem. 107:60-63), (iv) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) followed by staining with barium iodide, and (v) high-performance liquid chromatography (HPLC).

[0143] The long-acting IL-15R agonists of the present invention have been discovered to possess certain notable and advantageous characteristics. While the characteristics described below are considered to apply generally to the compounds provided herein and encompassed by Formula (I), one or more of the following characteristics may be exhibited, in particular, by compounds according to Formula (Ib), and by extension, compounds according to Formula (IIb). A long-acting IL-15R agonist may have one or more of the following characteristics. For example, in some embodiments, the long-acting IL-15 receptor agonist exhibits no more than about a 7-fold decrease in the EC50 value (ng / mL, CTLL-2 pSTAT5) when compared to unmodified IL-15. For example, in one or more related embodiments, the long-acting IL-15 receptor agonist exhibits no more than about a 6.5-fold decrease in the EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 6-fold decrease in the EC50 value (ng / mL, CTLL-2 pSTAT5), or no more than about a 6-fold decrease in the EC50 value (ng / mL, CTLL-2 pSTAT5), when compared to IL-15. or about a 5.5-fold or less decrease in EC50 value (ng / mL, CTLL-2 pSTAT5), or about a 5-fold or less decrease in EC50 value (ng / mL, CTLL-2 pSTAT5), or about a 4.5-fold or less decrease in EC50 value (ng / mL, CTLL-2 pSTAT5), or about a 4-fold or less decrease in EC50 value (ng / mL, CTLL-2 pSTAT5), or about a 3.5-fold or less decrease in EC50 value (ng / mL, CTLL-2 pSTAT5), or even about a 3-fold or less decrease in EC50 value (ng / mL, CTLL-2 pSTAT5). Exemplary long-acting IL-15R agonists according to the foregoing characteristics are described herein and in the accompanying Examples.

[0144] As described in Example 10, the in vitro activity of illustrative conjugates (1, 3, and 5) induces IL-15 signaling in huPBMCs, with conjugate 1 potently inducing such signaling. Further experiments were conducted to examine the in vitro activity of conjugate 1 on human CD8 T cells, NK cells, and CD4 T cells (Examples 16, 22, and 26-27). As shown in Figures 10A and 10B, at least conjugate 1 induced similar or enhanced signaling compared to IL-15 in CD56 bright and CD56 low cells. It is important to note that although conjugate 1 was less potent than IL-15 in engaging CD8 and CD56 bright NK cells (Example 22), conjugate 1 achieved the same maximal response as conventional IL-15 (see Figures 38A and 38B). As described for the mouse model in Example 16, a single injection of conjugate 1 at two different doses induced sustained pSTAT signaling in CD8 and NK cells. As described for the mouse model in Example 26, a single injection of conjugate 1 resulted in an increase in %pSTAT5 relative to IL-15. In the mouse model, NK cells were 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.

[0145] Conjugate 1 also induces signaling in NK cells, CD8 T cells, and CD4 T cells in a non-human primate model (cynomolgus monkey model in Example 27). Similar to the mouse model, NK cells were most sensitive to induction by conjugate 1.

[0146] In some additional embodiments, the long-acting IL-15 receptor agonist has a reduced receptor alpha binding (K D That is, in some related embodiments, the long-acting IL-15 receptor agonist exhibits about a 50% or less decrease in receptor α binding (K , pM) when compared to IL-15. D, pM) or exhibiting a decrease of about 45% or less in receptor α binding (K D , pM) or exhibiting a decrease of about 40% or less in receptor α binding (K D , pM) or even exhibiting a decrease of about 35% or less in receptor α binding (K D , pM) is reduced by about 30% or less.

[0147] Preferably, the long-acting IL-15 receptor agonist exhibits about a 7-fold or less decrease in EC50 value (ng / mL, CTLL-2 pSTAT5) when compared to unmodified IL-15, and exhibits about a 7-fold or less decrease in receptor alpha binding (K) when compared to IL-15. D , pM) (the EC50 value or K D (including any one or more specific combinations of decreases in values).

[0148] Optionally, the long-acting IL-15 receptor agonist is included in a composition comprising one or more pharmaceutically acceptable excipients, including, without limitation, those selected from the group consisting of carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, amino acids, and combinations thereof.

[0149] Carbohydrates such as sugars, derivatized sugars, e.g., alditols, aldonic acids, esterified sugars, and / or sugar polymers can be present as excipients. Specific carbohydrate excipients include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, and sorbose; disaccharides such as lactose, sucrose, trehalose, and cellobiose; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, and starch; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosylsorbitol, myo-inositol, and cyclodextrin.

[0150] The excipient may also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.

[0151] The compositions may also include an antimicrobial agent to prevent or inhibit the growth of microorganisms. Non-limiting examples of antimicrobial agents suitable for one or more embodiments of the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.

[0152] Antioxidants may also be present in the composition. Antioxidants are used to prevent oxidation, thereby preventing deterioration of the conjugate or other components of the preparation. 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.

[0153] A surfactant may be present as an excipient. Exemplary surfactants include polysorbates such as "Tween 20" and "Tween 80," and pluronics such as F68 and F88 (both available from BASF, Mount Olive, New Jersey); sorbitan esters; lipids such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (but preferably not in liposomal form), fatty acids, and fatty acid esters; steroids such as cholesterol; and IL-15 chelators such as EDTA, zinc, and other such suitable cations.

[0154] Acids or bases may be present in the composition as excipients. Non-limiting examples of acids that can be used include acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate, and combinations thereof.

[0155] One or more amino acids can be present as excipients in the compositions described herein. Exemplary amino acids in this regard include arginine, lysine, and glycine. Suitable additional pharmaceutically acceptable excipients are described, for example, in Handbook of Pharmaceutical Excipients, 7 th ed., Rowe, R.C., Ed., Pharmaceutical Press, 2012.

[0156] The amount of long-acting IL-15R agonist contained in the composition may vary depending on various factors, but optimally, it will be a therapeutically effective dose when the composition is stored in a unit-dose container (e.g., a vial). Additionally, the pharmaceutical preparation may be contained in a syringe. A therapeutically effective dose can be determined experimentally by administering increasing amounts of the long-acting IL-15R agonist repeatedly to determine the amount that produces the clinically desired endpoint herein. The amount of any individual excipient in the composition may vary depending on the activity of the excipient and the specific needs of the composition. Typically, the optimal amount for any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing various amounts of excipient (ranging from low to high amounts) and examining stability and other parameters, and then determining at what point optimal efficacy is achieved without significant side effects.

[0157] Long-acting IL-15R agonists are suitable for administration to patients suffering from conditions responsive to treatment with interleukin-15. The methods generally involve parenterally administering a therapeutically effective amount of a long-acting IL-15R agonist (preferably provided as part of a pharmaceutical composition) to a patient. As previously described, long-acting IL-15R agonists can be administered parenterally (e.g., intramuscularly, subcutaneously, intravenously, or intraperitoneally). Formulation types suitable for parenteral administration include, among others, ready-to-inject injection solutions, dry powders that are combined with a solvent before use, ready-to-inject suspensions, dry insoluble compositions that are combined with a vehicle before use, and emulsions and liquid concentrates that are diluted before administration. In some 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.

[0158] The administration of a long-acting IL-15 receptor agonist (e.g., provided as part of a pharmaceutical composition) can optionally be performed to localize it to a specific area. For example, liquid, gel, and solid formulations containing the agonist can also be surgically implanted within a diseased area (e.g., within a tumor, near a tumor, within an inflamed area, or near an inflamed area). Organs and tissues can also be advantageously imaged to ensure that the desired location is well exposed by the conjugate.

[0159] The method of administration can be used to treat any condition that can be treated or prevented by administration of a long-acting IL-15R agonist, such as cancer, etc. For example, the long-acting agonist can be used alone or in combination with other drug therapies to treat patients with conditions that may respond to IL-15 therapy, such as cancer.

[0160] As used herein in connection with the treatment of a subject with cancer, the terms "treatment," "treat," and "treating" are intended to include the administration of a full range of interventions, e.g., combinations, for the cancer from which the subject is afflicted, to alleviate, slow, stop, or reverse one or more symptoms of the cancer, or to slow the progression of the cancer, even if the cancer is not actually eliminated. Treatment can include, for example, a reduction in the severity of symptoms, the number of symptoms, or the frequency of recurrence, e.g., inhibiting tumor growth, arresting tumor growth, or causing regression of an existing tumor.

[0161] For example, improvement in cancer or cancer-related disease can be characterized as a complete or partial response. A "complete response" refers to the absence of clinically detectable disease, accompanied by normalization of any previously abnormal radiographic tests, bone marrow, and cerebrospinal fluid (CSF) or abnormal monoclonal protein measurements. A "partial response" refers to at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in all measurable tumor burden (i.e., the number of malignant cells present in the subject, or the volume of the measured tumor mass, or the amount of abnormal monoclonal protein) in the absence of new lesions. The term "treatment" contemplates both complete and partial responses.

[0162] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.

[0163] As used herein, "tumor" and "solid tumor" refer to all lesions and neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0164] Exemplary conditions include, for example, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, colon cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, head and neck cancer, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, , medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma (including, for example, uveal melanoma, mucosal melanoma, and leptomeningeal melanoma), neuroblastoma, retinoblastoma, and leukemia.

[0165] In one particular method, the long-acting IL-15R agonist is used to treat a hematological malignancy such as leukemia or lymphoma. In yet another method, the long-acting IL-15R agonist is used to treat a solid tumor.

[0166] In some embodiments, the long-acting IL-15 R agonist or composition is effective when administered to a subject at a therapeutically effective dose to stimulate NK activation and / or proliferation.

[0167] In an exemplary mouse model described in Example 11, conjugate 1 was effective in inducing proliferation and a sustained increase in the number of NK cells, as indicated by an increase in cell number (cells / μL, as shown in Figures 12B and 12C) and an increase in %Ki67 (e.g., Figure 11A). %Ki67 is used as a marker for proliferating cells. As evident from Figures 12A-12D, an increase in cell number was evident from all mature levels of NK cells (terminal effector cells, pre-NK cells, high effector cells, and early NK cells). As shown in Example 17, the increase in NK cell number persisted for at least 96 hours at all dose levels, and for at least 144 hours at the mid- and high-dose levels. Administration of conjugate 1 in the mouse model induced an increase in %Ki67 at all dose levels compared to vehicle, which persisted for at least 120 hours. %Ki67 At least in the mid-dose range (eg, 0.1 mg / kg and 0.3 mg / kg) induced an increase in %Ki67 that lasted for at least 144 hours.

[0168] The effect of Conjugate 1 can be induced by a single administration and is long-lasting. In the exemplary mouse model described in Example 11, an increase in cell number is induced by administration of a single dose of Conjugate 1 and is long-lasting. A single administration induced a sustained %Ki67 level in mouse CD49b cells comparable to the same level of repeated administration (e.g., Q7dx3) (see Figure 28A).

[0169] In some further embodiments, the long-acting IL-15R agonist or composition, when administered at a therapeutically effective dose, is effective in increasing NK cell activation, as evidenced by increased cytotoxic protease expression by NK cells. In the exemplary non-human primate model described in Example 28, expression of the NK cytolytic enzymes granzyme B and perforin was induced and enhanced by a single administration of conjugate 1. As evident from Figures 48A-48C, all dose levels increased granzyme B expression compared to pre-dose levels, with mid / higher doses at least tripling expression (MFI). As evident from Figures 49A-49C, all dose levels increased perforin expression compared to pre-dose levels, with mid / higher doses doubling expression (MFI). Thus, conjugate 1 is effective in increasing NK cell cytotoxicity.

[0170] In some still further embodiments, the long-acting IL-15 R agonist or composition is effective when administered to a subject at a therapeutically effective dose to support CD8 T cell survival and memory formation.

[0171] In an exemplary mouse model, a single administration of conjugate 1 at all dose levels increases cell proliferation, as indicated by an increase in % Ki-67 positivity in CD8 cells (Figure 30D) and memory and effector memory CD8 subpopulations (Figures 30E-30F). As shown in Example 17, administration of conjugate 1 in a mouse model induced a significant increase in total CD8 T cells in the blood (see Figure 30A). The lowest dose increased CD8 Tcm and CD8 Tem (see Figures 30B-30C). In an exemplary mouse model, a single iv injection of conjugate 1 maintained an increase in cell numbers for at least 240 hours compared to administration of vehicle (see, e.g., Figure 30A). Notably, CD8 and CD8 memory T cell numbers did not return to baseline 240 hours after injection when conjugate 1 was administered at several dose levels. Thus, conjugate 1 maintains the population of CD8+ memory T cells for an extended period of time. A single administration of conjugate 1 at all dose levels also increased Ki-67 positivity in total CD8 and CD8 subpopulations, indicating increased proliferation of these cells. Repeated administration with conjugate 1 further increased CD8, CD8 Tcm, and CD8 Tem populations (see Figures 31A-31C). Repeated administration also resulted in a long-term increase in cell proliferation for at least 240 hours in mice for each of the CD8, CD8 Tcm, and CD8 Tem populations.

[0172] Conjugate 1 also induced a sustained increase in the proliferation and number of NK cells and CD8 T cells in a non-human primate model (the cynomolgus monkey model in Example 27) compared to vehicle administration. Conjugate 1 at each dose level induced and sustained increases in NK cell numbers for at least 14 days (see Figure 44A). Conjugate 1 at each dose level induced and sustained increases in CD8 T cell numbers for at least 10 days.

[0173] In one or more still further embodiments, the IL-15R agonist is administered intravenously. In still further embodiments, the IL-15R agonist is administered subcutaneously.

[0174] In some still further embodiments, after administration, the IL-15R agonist is effective to induce sustained signaling in lymphocytes, resulting in proliferation of CD8 T cells and / or preferential expansion of CD8 central memory populations.

[0175] The actual dose administered may 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 medical professional, and the conjugate being administered. Therapeutically effective amounts are known to those skilled in the art and / or are described in relevant reference books and literature. Generally, a therapeutically effective amount may range from about 0.001 mg to 100 mg, preferably from 0.01 mg / day to 75 mg / day, and more preferably from 0.10 mg / day to 50 mg / day. A given dose may be administered periodically, for example, until a clinician determines that an appropriate endpoint (e.g., cure, regression, partial regression, etc.) has been reached.

[0176] In some embodiments, the therapeutically effective dose is in the range of about 0.25-25 mcg / kg. In other embodiments, the therapeutically effective dose is in the range of about 0.25 mcg / kg to about 0.1 mg / kg per day, about 0.01 mg / kg to about 0.1 mg / kg per day, or about 0.03 mg / kg to about 0.1 mg / kg per day. In other embodiments, the therapeutically effective dose is in the range of about 1-10 mcg / kg, or about 0.03 mg / kg to about 0.1 mg / kg per day. In some specific, but non-limiting embodiments, the therapeutically effective dose is about 0.25 mcg / kg, 0.3 mcg / kg, 0.5 mcg / kg, 1 mcg / kg, 2 mcg / kg, 3 mcg / 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 mentioned in the Examples herein, one of skill in the art can convert animal doses (e.g., mice) to corresponding human doses using conversions known in the art (e.g., Nair et al., J. Basic and Clin. Pharmacy (2016) 7:27-31).

[0177] The unit dosage of any given conjugate (again, preferably provided as part of a pharmaceutical preparation) can be administered in a variety of dosing schedules depending on the clinician's judgment, the patient's needs, etc. Specific dosing schedules will be known to those of skill in the art or can be determined empirically using routine methods. Exemplary dosing schedules include, without limitation, administration once daily, three times per week, twice weekly, once weekly, twice monthly (e.g., 14 days), once monthly (e.g., 30 or 31 days or 21 days), and any combination thereof. Once the desired clinical endpoint is achieved, dosing of the composition is discontinued or reduced. In some embodiments, the unit dose of any given conjugate can be administered once to achieve a sustained effect.

[0178] While the present invention has been described in connection with preferred specific embodiments thereof, it should be understood that the foregoing description and the following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Other aspects, advantages, and modifications within the scope of this disclosure will be apparent to those skilled in the art to which the invention pertains.

[0179] All articles, books, patents, and other publications referenced herein are incorporated herein by reference in their entirety. In the event of a conflict between the teachings of this specification and the art incorporated by reference, the meaning of the teachings and definitions in this specification shall prevail (particularly with respect to the terms used in the claims appended hereto). For example, if this application and a publication incorporated by reference define the same term differently, the definition of the term shall be maintained within the teachings of the document in which the definition is located. [Example]

[0180] It should be understood that the foregoing description and the following examples are illustrative of the invention provided herein and are not intended to be limiting. Other aspects, advantages, and modifications will be apparent to those skilled in the art to which this disclosure pertains.

[0181] In the following examples, efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, temperatures are in degrees Celsius and pressures are at or near marine pressure. Each of the following examples is considered instructional for one of ordinary skill in the art to practice one or more of the embodiments described herein.

[0182] Materials and Methods Recombinant IL-15 ("rIL-15") SEQ ID NO: 1 (provided in Figure 1), prepared using conventional techniques, was used in the following examples, although any suitable IL-15 portion can similarly be used. SEQ ID NO: 1, recombinant human IL-15 from E. coli, is a single, non-glycosylated polypeptide chain containing 115 amino acids and has a molecular weight of 12.9 kDa.

[0183] The reactive polymeric reagent, linear mPEG-succinimidyl butanoate, 40 kDa (“mPEG-SBA”), has the following structure: [ka] where n corresponds to the number of monomeric subunits providing a polymer having a weight-average molecular weight of about 40 kilodaltons, i.e., n is about 909. Additional mPEG-succinimidyl butanoate reagents suitable for use include, for example, those having a weight-average molecular weight of about 10 kD, 15 kD, 20 kD, 25 kD, 30 kD, 40 kD, 50 kD, or 60 kD. This activated polymer reagent, when reacted with an amino group (e.g., lysine or N-terminus) of IL-15, is effective to form a stable amide bond between the IL-15 moiety and the polyethylene glycol moiety.

[0184] The reactive fluorenyl-PEG reagent, PEG2-CAC-FMOC-20kD-NHS, has the following structure: [ka] where mPEG is methoxy(polyethylene glycol) and the weight-average molecular weight of the polymeric reagent is about 20 kilodaltons (i.e., with each mPEG moiety having a weight-average molecular weight of about 10 kilodaltons). Additional PEG2-CAC-FMOC reagents having different molecular weights are designated accordingly, e.g., PEG2-CAC-FMOC-10 kD-NHS, PEG2-CAC-FMOC-15 kD-NHS, PEG2-CAC-FMOC-30 kD-NHS, PEG2-CAC-FMOC-40 kD-NHS, which reagents have the structure shown above and differ only in the molecular weight of the "mPEG" moiety attached to the FMOC core.

[0185] 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 prepared samples were then loaded onto a NuPAGE Novex precast gel and run for approximately 30 minutes.

[0186] 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 / HO (A) and 0.1% TFA / CHCN (B). The column flow rate was 0.5 ml / min. Eluted proteins and PEG-protein conjugates were detected using UV at 280 nm.

[0187] Bioassay Potency assay based on STAT5 phosphorylation in CTLL-2 cells (mouse T cells) In the phospho-STAT5 assay, downstream cell signaling following receptor binding can then activate signal transducer and activator of transcription 5 (STAT5) via phosphorylation to promote gene expression and induce cell proliferation. Activation of phospho-STAT5 is measured in CTLL-2 cells, a murine T lymphocyte cell line, using a phospho-STAT5 / total STAT5 multiplex assay (Meso Scale Discovery, MD) in response to sample and reference treatments over a 10-minute period.

[0188] The day before the assay, CTLL-2 cells were split into fresh growth medium [RPMI 1640 supplemented with 10% FBS, 10% T cell culture supplement (catalog no. 354115, Corning, Inc., Tewksbury, MA), 2 mM L-glutamate, and 1 mM sodium pyruvate]. On the day of the assay, cells were preincubated in assay medium (RPMI 1640 supplemented with 1% FBS, 2 mM L-glutamate, and 1 mM sodium pyruvate) for at least 4 hours and then plated at 50,000 cells / well in assay medium in a 96-well plate. Test article dilutions were prepared in the appropriate buffer immediately before the assay. Stimulation of CTLL-2 cells was initiated by transferring 25x test article solution to triplicate wells containing CTLL-2 cells. Plates were incubated at 37°C and 5% CO2 for 10 minutes, and the reaction was terminated 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, MD). After 10 min of treatment, recombinant human IL-15 obtained from PeproTech had a mean EC 50 IL-15 activity was demonstrated by inducing STAT5 phosphorylation in CTLL-2 cells, which served as a control.

[0189] HuPBMC-pStat5 assay The efficacy of IL-15 or long-acting IL-15R agonists on various human lymphocyte subpopulations was determined by a phospho-STAT5 (Y694) dose-response assay. Frozen human PBMCs from multiple donors were provided by AllCells. 1x10 6 Cells / 100 μl were cultured in complete RPMI medium for 2 hours and then incubated with the indicated concentrations of IL-15 or conjugates (serial dilutions from 10,000 ng / ml to 0.001 ng / ml) for 20 minutes at 37°C. Cells were then fixed (using BD Cytofix), permeabilized (using 100% prechilled methanol), stained with antibodies against CD3, CD4, CD8, CD4- Tregs (CD4+CD25+Foxp3+), CD56, and phosphorylated STAT5 (Y694), and analyzed by flow cytometry. EC values ​​were calculated using concentration-response relationships. 50 values ​​were calculated.

[0190] Receptor affinity of long-acting rIL-15 receptor agonists for IL-15Rα Surface plasmon resonance ("SPR") was used to measure the affinity of IL-15 and an exemplary long-acting rIL-15 receptor agonist using a BIAcore™ SPR system. Briefly, the surface of a Biacore CM5 sensor chip was activated by generating an active NHS ester using a 1:1 NHS:EDC mixture. A goat anti-human Fc antibody was covalently bound to the surface by injecting it in 10 mM sodium acetate (pH 4) for 5 minutes. Approximately 8000 RU of antibody bound to the surface. The remaining NHS ester was then quenched with ethanolamine.

[0191] At the start of each injection cycle, IL-15-Rα-Fc was captured onto the sensor chip channel by a 5-min injection step in PBSP, typically resulting in 150-200 RU of receptor bound to the surface.

[0192] Long-acting rIL-15 receptor agonist test articles were administered in PBS (0.05% Tween The antibody was diluted to 10 μM in 100 μL of IL-15Rα (containing 0.20 and 0.1 mg / ml BSA). A series of 3-fold dilutions was made and injected over a sensor chip coated with IL-15Rα. a and k d The affinity is measured by separately measuring the rate, k d and k a Using the ratio of k d values ​​were calculated.

[0193] Example 1 Preparation of long-acting IL-15 receptor agonists [ka] Experiment 1: A 2.7 ml solution of IL-15 (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 under nitrogen at -20°C, was warmed to ambient temperature. A 10-fold excess (relative to the molar amount of IL-15) of mPEG SBA-40K was dissolved in 2 mM HCl to form a 10% PEG reagent solution. The 10% PEG reagent solution was rapidly added to the IL-15 solution and mixed well. After the addition of mPEG SBA-40K, the pH of the reaction mixture was measured and adjusted to pH 8 using conventional techniques. To couple mPEG SBA-40K to IL-15 (i.e., via the formation of a stable amide bond), the reaction solution was placed on a Slow Speed ​​Lab Rotator for 1.5 hours to facilitate conjugation at room temperature. The reaction was quenched by the addition of a glycine solution.

[0194] Figure 2 shows a chromatogram after RP-HPLC analysis of the conjugation reaction mixture. The reaction yielded 40% mono-conjugate (i.e., with a single PEG moiety attached to IL-15), 24% di-conjugate (with two PEGs attached to IL-15), and 6% tri-conjugate (with three PEGs attached to IL-15) species. Although the reaction conditions were not optimized, approximately 30% unreacted IL-15 remained in the reaction mixture.

[0195] The desired mono-conjugate was separated / isolated by anion exchange chromatography using a Q Sepharose high performance column and sodium phosphate buffer as the eluent. The purified mono-mPEG-SBA40K-IL-15 conjugate (also referred to herein as mono-mPEG-butanamide-40K-IL-15 or mono(methoxyPEG-N-butanamide) 40kD Interleukin-15, or mono-mPEG 40K -C4-amide-IL-15) was characterized by HPLC and SDS-PAGE. In other examples, purified mono-mPEG-SBA40K-IL-15 is referred to as conjugate 1.

[0196] Figure 3 provides an FPLC purification profile from an anion exchange chromatography column. Figure 4 shows an SDS gel of the purified mono-mPEG-SBA-40K-IL-15 conjugate. As shown by the gel, the purified conjugate has a high level of purity and is free of unreacted IL-15. Figure 5 shows RP-HPLC analysis of purified mono-mPEG-SBA-40K-IL-15. As can be seen from the HPLC results, the purified mono-mPEG-SBA-40K-IL-15 composition contains less than about 10% (molar amount) of di- or higher levels of conjugates.

[0197] Using this same approach, mPEG SBAs with different weight average molecular weights are used to prepare conjugates such as mono-mPEG-SBA-10K-IL-15, mono-mPEG-SBA-15K-IL-15, mono-mPEG-SBA-20K-IL-15, mono-mPEG-SBA-25K-IL-15, mono-mPEG-SBA-30K-IL-15; mono-mPEG-SBA-40K-IL-15; mono-mPEG-SBA-50K-IL-15; and mono-mPEG-SBA-60K-IL-15.

[0198] Experiment 2: IL-15 in an approximately 2 mg / ml solution 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). pH 8 borate buffer (0.4 M or 0.6 M) was added to adjust the pH to 8.0. A 10-fold excess (relative to the molar amount of IL-15) of mPEG SBA-40K (mPEG 1000) diluted in 2 mM HCl was added. mPEG SBA-40K was added to each of the IL-15 solutions and mixed thoroughly. After the addition of mPEG SBA-40K, the pH of the reaction mixture was determined to be pH 8, or adjusted by using additional borate buffer if necessary. The final concentration of IL-15 in the reaction was targeted to 1 g / L, and additional diluent was used if necessary (a buffer containing 50 mM sodium phosphate, 100 mM sodium chloride, and 10% sucrose, pH 7.4, was used for Composition 1, and water was used for Composition 2). To couple mPEG SBA-40K to IL-15 (i.e., via the formation of a predominantly stable amide bond), the reaction solutions were mixed at room temperature for 45 or 60 minutes for Composition 1 or Composition 2, respectively, to facilitate conjugation. The reaction was quenched by adding a 71-fold excess of glycine (relative to the molar amount of PEG initially added to the reaction) at pH 8.0 for 30 minutes. For Composition 1, the pH was adjusted by titrating to pH 7.0 using 0.2 M phosphoric acid.

[0199] The resulting compositions were characterized by reverse phase HPLC (RP-HPLC), SDS-PAGE, and ion exchange HPLC (IEX-HPLC). The results of the RP-HPLC analysis are provided in Table 1A below.

[0200] [Table 1]

[0201] The results of the SEC-HPLC analysis are provided in Table 1B below.

[0202] [Table 2]

[0203] The results of the IEX-HPLC analysis are provided in Table 1B below.

[0204] [Table 3]

[0205] The prepared compositions contained predominantly mPEG SBA-40K monoPEGylated species, with less than 10% PEG dimers (i.e., two PEG moieties attached to IL-15), and even smaller amounts of higher PEG species (i.e., three or more PEG moieties attached to IL-15).

[0206] Additionally, the compositions had a relatively low degree of deamidation (identified as "acidic region" in Table 1C): Composition 1 was 21.29% deamidated, and Composition 2 was 33.26% deamidated.

[0207] Example 2 Preparation of long-acting IL-15 receptor agonists. [ka] mPEG2-CAC-fmoc-20K-NHS, stored under nitrogen at -80 °C, was warmed to ambient temperature under a nitrogen purge. A stock solution of mPEG2-CAC-fmoc-20K-NHS (200 mg / mL) was prepared in 2 mM HCl, and mPEG2-CAC-fmoc-20K-NHS was added to rIL-15 at molar ratios 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 reach a final concentration of 100 mM, and conjugation was allowed to proceed for 30 minutes to provide the [mPEG2-CAC-fmoc-20K-NHS]-[rIL-15] conjugate (the informal name of the conjugate reflects the polymeric reagent used in its preparation; it is understood that, with respect to the resulting product, the reactive moiety of the polymeric reagent has, in this case, been replaced by a bond to IL-15). After 30 minutes, quenching was achieved by adding 1 M glycine (pH 6.0) to the reaction mixture to reach a final concentration of 100 mM. The pH of the quenched reaction mixture was then adjusted to 4.0 using glacial acetic acid prior to column chromatographic purification and characterization.

[0208] The reaction mixture was analyzed by RP-HPLC analysis. SDS-PAGE revealed that the reaction mixture contained approximately 10-20% mono-conjugate, approximately 50-70% di-conjugate, and approximately 20-30% tri-conjugate. That is, the reaction mixture contained primarily di-conjugated species. The conjugate mixture was separated / isolated by anion exchange chromatography using a Q Sepharose high-performance column and sodium phosphate buffer as the eluent to provide purified [mPEG2-CAC-FMOC-20kD-NHS]-IL-15 with an average PEGylation degree of approximately 2 (with a PEGylation degree ranging from approximately 1.7 to 2.5), and thus n' in the above structure for the purified composition is approximately 2.

[0209] In the following examples, purified [mPEG2-CAC-FMOC-20kD-NHS]-IL-15 is referred to as conjugate 2.

[0210] Example 3 Preparation of long-acting IL-15 receptor agonist, [mono-PEG2-RU-ButryALD-40K]-IL15 [ka] Branched mPEG-Butryaldehyde PEG Reagent, Mono-PEG2-RU-ButryALD-40K [ka] was used to prepare the subject long-acting IL-15 R agonists, and the weight average molecular weight of the PEG reagent used in preparing the agonists was approximately 40,000 daltons.

[0211] 2.7 ml of IL-15 (1.23 mg / ml in PBS buffer, pH 7.4) was transferred to a small reaction vial, and 0.3 ml of 1 M sodium acetate buffer (pH 5) was added to adjust the pH to pH 6. mPEG2-ru-ButyrALD, 40 kDa, stored at -20 °C under nitrogen, was warmed to ambient temperature. A 15-fold excess (relative to the amount of IL-15) of mPEG2-ru-ButyrALD was dissolved in MilliQ HO to form a 10% reagent solution. The 10% reagent solution was rapidly added to the IL-15 solution, mixed well, and placed on a RotoMixer for 15 minutes. Next, 1 / 100th volume of 1 M NaCNBH3 / HO was added to the reaction mixture. To allow coupling of mPEG2-ru-ButyrALD to IL-15 via a secondary amine bond, the reaction solution was placed on a RotoMixer at 4 °C for 17 hours and then quenched with glycine solution. Because the PEGylation reaction was performed at acidic pH, conjugation of the PEG derivative to IL-15 was more selective for the N-terminus. An anion-exchange chromatography method using a Q Sepharose high-performance column and sodium phosphate buffer was also developed to purify the conjugate. The purified mono-PEG2-ru-ButyrALD-40K-IL-15 conjugate was characterized by HPLC and SDS-PAGE.

[0212] In the following examples and accompanying disclosure, purified [mono-PEG2-RU-ButryALD-40K]-IL-15, i.e., having a single PEG moiety having the structure shown above covalently attached to IL-15 via an amine bond, is referred to as conjugate 3.

[0213] This same approach was used to prepare conjugates using PEG2-RU-ButryALD- with different weight-average molecular weights. For example, mono-PEG2-RU-ButryALD-20K]-IL-15 was prepared as described above using a 20 kD polymer reagent (referred to herein as conjugate 4).

[0214] Example 4 Preparation of long-acting IL-15 receptor agonists, Mono-mPEG-ButyrALD-40K-IL-15 [ka] structure [ka] The subject long-acting IL-15 R agonists were prepared using a PEG reagent, linear mPEG-Butyraldehyde, 40 kDa ("mPEG-ButyrALD"), having a weight average molecular weight of approximately 40,000 daltons.

[0215] 2.7 ml of IL-15 (1.23 mg / ml in PBS buffer, pH 7.4) was transferred to a small reaction vial, and 0.3 ml of 1 M sodium acetate buffer (pH 5) was added to adjust the pH to pH 6. mPEG-ButyrALD, 40 kDa, stored at -20 °C under nitrogen, was warmed to ambient temperature. A 10-fold excess (relative to the amount of IL-15) of mPEG-ButyrALD was dissolved in MilliQ HO to form a 10% reagent solution. The 10% reagent solution was rapidly added to the IL-15 solution, mixed well, and placed on a RotoMixer for 15 min. Next, 1 / 100th volume of 1 M NaCNBH3 / HO was added to the reaction mixture. To allow coupling of mPEG-ButyrALD to IL-15 via a secondary amine bond, the reaction solution was placed on a RotoMixer at 4 °C for 17 h and then quenched with glycine solution. Because the PEGylation reaction was performed at acidic pH, conjugation of the PEG derivative to IL-15 was more selective for the N-terminus. An anion-exchange chromatography method using a Q Sepharose high-performance column and sodium phosphate buffer was also developed to purify the conjugate. The purified mono-mPEG-ButyrALD-40K-IL-15 conjugate was characterized by HPLC and SDS-PAGE.

[0216] In the following examples, mono-mPEG-ButryALD-40k-IL-15 is referred to as conjugate 5.

[0217] Example 5 Evaluation of receptor bias of long-acting IL-15 receptor agonists targeting IL-15Rα The affinity of an exemplary long-acting IL-15 receptor agonist (test article) for the IL-15 α receptor was measured and compared to IL-15. Affinity was measured by BIAcore using IL-15Rα:Fc captured by immobilized anti-Fc.

[0218] Test articles were diluted to 10 μM in PBS (containing 0.05% Tween 20 and 0.1 mg / ml BSA). Serial 3-fold dilutions were made and injected over a sensor chip coated with IL-15Rα. a and k d The affinity is measured by separately determining the ratio, k d and k a Using the ratio of k d values ​​were calculated.

[0219] Preferred conjugates generally retain as much IL-15Rα affinity as possible after pegylation compared to unmodified IL-15, or conversely, preferred conjugates generally exhibit minimal loss of affinity for IL-15Rα compared to unmodified IL-15.

[0220] For example, in some embodiments, preferred conjugates exhibit about a 7-fold or less reduction in EC50 values ​​(ng / mL, CTLL-2 pSTAT5) and receptor alpha binding (K D , pM). For example, conjugate 1 has about a two-fold reduction in potency when compared to IL-15, and retains about 80% of the receptor affinity of IL-15.

[0221] [Table 4]

[0222] As shown in the table above, conjugate 1 retains its high affinity for the IL-15 α receptor (i.e., compared to IL-15), a particularly favorable characteristic for a long-acting IL-15 receptor agonist. The affinity constants (K D ) (units: pM) are provided below.

[0223] [Table 5]

[0224] Example 6 In vivo studies: Single-dose PK study in mice C57BL / 6 mice (n=3 / group) were administered a single intravenous 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 various time points (24, 48, 78, and 96 hours) post-administration. Samples were pooled and evaluated by flow cytometry for pharmacodynamic analysis of drug effects on lymphocyte cell populations, expressed as fold changes relative to vehicle control (results are described in subsequent Examples below). In addition to changes in cell number, functional and activity markers were quantified. Finally, plasma concentrations of the drug were determined at each time point. See Figure 6.

[0225] As shown in Figure 6, conjugate 2 maintained measurable concentrations in plasma for extended periods, e.g., greater than 1 week (solid squares), in contrast to the rapid decline in plasma levels observed after administration of non-long-acting IL-15 (solid circles). 1 / 2 was about 20 to 30 hours.

[0226] Example 7 In vivo studies: Single-dose PK study in rats Rats (n=3 / group) were administered a single intravenous dose of Conjugate 2 at doses of 0.3, 0.15, and 0.075 mg / kg, or a single subcutaneous dose of Conjugate 2 at 0.15 mg / kg. After administration, blood was collected on days 1-7 post-dose (multiple samples were collected in the first 24 hours post-dose). At each time point, the plasma concentration of the drug was determined. See Figure 7.

[0227] As shown in Figure 7, similar to the results shown in Figure 6 for mice, administration of conjugate 2 resulted in sustained and dose-proportional exposure to the drug.

[0228] Example 8 In vivo IL-15 signaling studies in mice Mice were dosed as described in Example 6 above to evaluate in vivo signaling, as assessed by the degree of STAT5 phosphorylation. The degree of STAT5 phosphorylation in various lymphocytes (CD4, CD8, and NK cells) was assessed by measuring by flow cytometry after staining whole blood for leukocyte surface markers and pSTAT5. The results are shown in Figures 8A and 8B for IL-15 and Conjugate 2, respectively.

[0229] STAT5 phosphorylation is early and transient in IL-15 / IL-2 receptor signaling. As can be seen in Figure 8A, while in vivo signaling activity is extremely short-lived in the case of IL-15, exemplary conjugate 2 induced sustained STAT5 phosphorylation, most notably in NK cells (solid inverted triangles ▼) and also in CD8 cells (solid triangles ▲), with measurable STAT5 phosphorylation activity displayed in NK and CD8 cells for over 72 hours. STAT5 phosphorylation activity for CD4 cells is also shown (solid squares ■).

[0230] Example 9 In vivo IL-15 signaling studies in non-human primates In this study, one female and one male cynomolgus monkey (cyno) were each administered a single dose of conjugate 2 (0.5 mg / kg intravenously. Serial blood samples were collected from each animal before treatment (days -6 and -1) and at multiple intervals after treatment for flow cytometric assessment of STAT5 phosphorylation in various types of lymphocytes (CD4, CD8, and NK cells). The results are presented in Figures 9A (CD4), 9B (CD8), and 9C (NK).

[0231] As shown in Figures 9A-9C, the results are similar to those observed in mice (Example 7), except that in non-human primates, STAT5 phosphorylation was also observed in CD4 cells (Figure 9A). STAT5 phosphorylation in each of the three cell types increased substantially following administration, reaching maximum levels at approximately days 3 and 4 after administration of the long-acting IL-15 agonist shown, and returning to near day -1 levels (i.e., pre-administration) by approximately days 5-10. As in Example 7, these results demonstrate the persistent presence of active IL-15 species.

[0232] Example 10 In vitro IL-15 activity of exemplary long-acting IL-15 receptor agonists in the NK subset of human peripheral blood mononuclear cells (PBMCs) As shown in Figures 10A (CD56bright cells) and 10B (CD56dim cells), the in vitro activity of exemplary long-acting IL-15 receptor agonists (e.g., conjugates 1, 3, and 5) was assessed by studying signaling in NK cell subsets of human PBMCs. To assess the IL-15 signaling activity of long-acting IL-15 receptor agonists, STAT5 phosphorylation was assessed as previously described.

[0233] [Table 6]

[0234] The results are shown in Figures 10A (CD56bright) and 10B (CD56dim).

[0235] As can be seen, each of the illustrative conjugates induces IL-15 signaling in huPBMCs, with conjugate 1 potently inducing such signaling. Among the conjugates tested (full data not shown), conjugate 1 demonstrated the greatest potency / activity on huPBMCs. The data indicate that even when maintaining the same degree of PEGylation (i.e., number of PEG moieties) and the same size of PEG moieties per IL-15 protein, different PEG constructs and linkers can elicit very different effects on bioactivity in the resulting conjugates.

[0236] A second study was performed to examine / compare the pStat5 response of human PBMCs (CD3, CD4, CD8, CD56 (bright and dim) and CD4- Tregs (CD25+Foxp3+)) obtained from two donors to IL-15, Conjugate 1, and Conjugate 5. An 11-point dose response was performed with 10-fold dilutions ranging in dose from 0.001 to 10,000 ng / ml, with a 20-minute stimulation. Each of the test articles was diluted in IL-15 buffer + 0.1% BSA. The results are provided in the table below.

[0237] [Table 7]

[0238] Based on the data in Table 3 above, IL-15 appears to be approximately 4-6 times more potent than conjugate 1 for both CD3 and CD4 induction; the potencies of conjugate 1 and conjugate 5 appear to be similar for CD3 and CD4 induction.

[0239] [Table 8]

[0240] Based on the data in Table 5 above, IL-15 appears to be approximately 3-5 times more potent than conjugate 1 with respect to Treg and CD8 induction; the potencies of conjugates 1 and 5 appear to be substantially similar with respect to CD4 and CD8 induction.

[0241] [Table 9]

[0242] Based on Table 6, IL-15 appears to be ∼10 times more potent at inducing CD56 than conjugate 1. However, conjugate 1 appears to be more potent than conjugate 5 in inducing CD56 bright and CD56 dim.

[0243] Based on previous data, it appears that IL-15, conjugate 1 and conjugate 5 show similar pSTAT5 induction for all cell populations, with maximal responses being higher for CD56bright and Treg cells.

[0244] [Table 10]

[0245] Based on the foregoing data, IL-15 is approximately 3-4 times more potent than conjugate 1 and approximately 5-8 times more potent than conjugate 5 in inducing CD3, CD4, CD8, and Treg cells, and approximately 12 times more potent than conjugate 1 and approximately 40-60 times more potent than conjugate 5 in inducing CD56bright and CD56dim cells, suggesting certain unexpected and particularly advantageous characteristics of conjugate 1.

[0246] Example 11 In vivo studies: Single-dose PD studies in mice - cell proliferation and activation Balb / c mice (n=3 / group) were administered a single intravenous dose of vehicle (50 mM sodium phosphate, 100 mM sodium chloride, 10% sucrose, pH 7.4) or Conjugate 1 at doses 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 various time points (24, 48, 78, 96, and 120 hours) post-dose. Samples from each mouse were subjected to pharmacodynamic analysis of drug effects on lymphocyte cell populations by flow cytometry. In addition to changes in cell numbers, functional and activity markers were examined.

[0247] Additional administrations of Conjugate 1 were given at doses of 0.01 mg / kg, 0.1 mg / kg, and 1.5 mg / kg.

[0248] Results showing NK cell proliferation in mice administered 0.03 mg / kg ( FIG. 11 , low dose), 0.3 mg / kg ( FIG. 11 , medium dose), and 1 mg / kg ( FIG. 11 , high dose) of each of Conjugate 1 are provided in FIGS. 11A and 11B.

[0249] The CD45+CD3-CD49b+ and CD45+CD3-CD49b+Ki67+ marker combinations were used to define NK cells and their proliferation. After administration, blood samples were acquired on a Fortessa flow cytometer running FACS DIVA software. Flowjo software was used for analysis, and absolute NK cell counts and % Ki67 positivity within NK cells were plotted using Prism.

[0250] Figure 11A is a plot of Ki67 expression (as a percentage) over time; Figure 11B provides NK cell counts over time. The plots demonstrate the ability of conjugate 1 to induce sustained NK cell proliferation in mice.

[0251] The effects of exemplary long-acting IL-15 receptor agonists were studied on all levels of mature NK cells. The peripheral NK cell pool can be delineated by the expression of CD27, lo / - NK cells are more cytotoxic and CD27 high They produce larger amounts of cytokines than NK cells (Hayakawa Y, et al., J Immunol. 2006;176:1517-1524). Mature peripheral NK cell populations have been further refined into four maturation stages defined by the sequential upregulation of CD11b expression and the subsequent downregulation of CD27, with the most immature NK cells expressing CD27. - CD11b - and the most mature NK cells express CD27 - D11b - (Chiossone L., et al., Blood, 2009;113:5488-5496).

[0252] In mice, four distinct maturation states of NK cells are defined by CD27 and CD11b expression. Once triple-positive cells for NK markers (CD49b+), natural activating NK receptor (NKp46+), and IL-15 / IL-2RB (CD122+) were identified, immature (CD11b-CD27-), early (CD11b-CD27+), high effector (CD11b+CD27+), and terminal effector (CD11b+CD27-) NK cells were quantified by flow cytometry.

[0253] NK cells at various maturation states were quantified in mice receiving a single dose of 0.01, 0.03, 0.1, 0.3, 1.0, and 1.5 mg / kg of conjugate 1 or after the third dose of the q7dx3 schedule, as described above. Using flow cytometry, NK populations of interest were identified by CD49b, NKp46, and CD122 positivity. CD11b and CD27 were then used to further differentiate NK populations into immature (CD11b-CD27-), early NK (CD11b-CD27+), high effector (CD11b+CD27+), and terminal effector (CD11b+CD27-) subpopulations. Peripheral blood was run on a Fortessa flow cytometer, and absolute values ​​for each population were determined using counting beads during sample acquisition using BD FACS DIVA software. Flow cytometry analysis was performed using Flowjo software, and data were plotted in Prism.

[0254] The results are shown in Figures 12A-D. Additional results are shown in Figures 22A-D. The q7dx3 results are shown in Figures 28A-D. As can be seen from the plots, conjugate 1 was effective in increasing the numbers of all mature levels of NK cells (terminal effector cells, pre-NK cells, high effector cells, and early NK cells). A dose-dependent increase in NK cells was observed in all mature subpopulations, with the effect lasting for at least 120 hours.

[0255] Surface expression of NKG2D was measured using flow cytometry analysis of anti-NKG2D signals and expressed as mean fluorescence intensity (MFI) in NK cells. Similarly, intracellular granzyme B levels were measured using flow cytometry-based detection of anti-granzyme B signals and also expressed as MFI in NK cells. NKG2D and granzyme B signals were detected using a Fortessa flow cytometer and FACS Diva software, followed by analysis using Flowjo software. MFI values ​​were plotted using Prism. The results are shown in Figures 13A and 13B. These figures further demonstrate the ability of conjugate 1 to increase NK cell activation, as evidenced by its ability to achieve sustained increases in both NKG2D and granzyme B (a proapoptotic serine protease) by NK cells compared to vehicle, most notably for the mid- and high-dose regimens. A dose-dependent increase in both NK activation markers was observed after a single administration of conjugate 1.

[0256] In mice, CD8 T cells were defined as CD45+CD3+CD4-CD8+. Blood and spleens from mice were subjected to immunophenotyping using a Fortessa flow cytometer, and analysis was performed using Flowjo software. Absolute CD8 cell counts were plotted in Prism as shown in Figure 14 (blood) and Figure 24 (spleen). Figures 14 and 24 demonstrate the ability of conjugate 1 to induce CD8 T cell proliferation and a sustained increase in CD8 T cell numbers after a single iv administration in mice at each of the doses mentioned above. This effect was most pronounced for the mid (0.1 mg / kg, 0.3 mg / kg) and high (1.0 mg / kg, 1.5 mg / kg) doses.

[0257] In mice, CD8 effector memory (Tem) and CD8 central memory (Tcm) T cells were identified as CD45+CD3+CD4-CD8+CD44+CD62- and CD45+CD3+CD4-CD8+CD44+CD62L+. Expansion of these memory populations was monitored 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 plotted in Prism. As shown in Figures 15A and 15B for blood, conjugate 1, but not a single dose of IL-15, induced a dose-dependent increase in both effector and central memory CD8 T cells. As shown in Figures 25 and 26 for spleen, conjugate 1, but not a single dose of IL-15, induced a dose-dependent increase in both Ki67 and granzyme B. Both effector and central memory populations expand in response to administration of Conjugate 1, an exemplary long-acting IL-15 agonist.

[0258] Example 12 In vivo studies: Single-dose PD studies in non-human primates In this study, cynomolgus monkeys, one female and one male, were administered 500 μg / kg intravenously with Conjugate 2. Serial blood samples were taken from each animal before treatment (days −6 and −1) and at multiple intervals after single-dose treatment for flow cytometric assessment of lymphocyte cell counts (NK cells, CD8 T cells, etc.) and activation.

[0259] NK cell numbers were determined to assess the ability of exemplary conjugate 2 to induce sustained NK cell proliferation in non-human primates; the results are shown in Figures 16A and 16B. NK cells and their proliferation in the blood from cynomolgus monkeys were identified by flow cytometry. Acquisition and analysis of NK cells (CD45+CD3-CD16+) and their proliferation status (CD45+CD3-CD16+Ki67+) was 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. Pre- and post-treatment values ​​were plotted using Prism.

[0260] As shown in these figures, administration of a single dose of conjugate 2 was effective in inducing sustained NK cell proliferation in non-human primates.

[0261] As shown in Figure 17, CD8 T cell counts were also determined for each animal from pre-dose through 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 described above. In the monkeys, CD8 T cells were sustainedly expanded, an effect that lasted for at least 10 days. This plot further illustrates the ability of an exemplary long-acting IL-15 receptor agonist, Conjugate 2, to induce a sustained increase in the proliferation and number of CD8 T cells following administration.

[0262] In monkeys, CD8 T EM Cells were defined as CD45+CD3+CD4-CD8+CD45Ra-CD197- and CD8 T CM The CD8 T effector memory cell (T) count was defined as CD45+CD3+CD4-CD8+CD45Ra-CD197+. Immunophenotyping was performed by flow cytometry, samples were acquired using DIVA software, and data were analyzed using Flowjo software. Graphs were plotted using Prism. The CD8 T effector memory cell (T) count for each animal was calculated from pre-dose to day 14 post-dose, as shown in Figures 18A and B, respectively. EM cells) and CD8 T central memory cells (TCM ) numbers were determined. Conjugate 2 induces a significant and sustained expansion of CD8 effector and central memory T cell populations in cynomolgus monkeys. The figure shows that both CD8 effector and central memory T cell populations proliferate in response to exemplary conjugate 2.

[0263] Example 13 Evaluation of antitumor activity in a CT26-induced subcutaneous lung metastasis tumor model in BALB / C mice On day 0, 6- to 8-week-old female Balb / c mice were treated with 1 × 10 5 Mice were inoculated with CT-26 cells via tail vein injection. On day 1, 24 hours after administration of CT-26 cells, the mice were divided into 10 groups. Each group consisted of 6-9 animals (for conjugate 2) or 9-12 animals (for conjugate 1). (Two separate studies were conducted for administration of conjugate 1 and conjugate 2, but the study protocol was essentially the same for both studies.) Each group was assigned to one intervention as follows: 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); for conjugate 1: vehicle, phosphate-buffered saline (Group H); conjugate 1 at a dose of 0.03 mg / kg (Group I) and conjugate 1 at a dose of 0.3 mg / kg (Group J). Animals were dosed on days 1, 5, and 10.

[0264] Thirteen days after CT-26 tumor cell administration, mice were anesthetized, and blood and spleen cells were collected for further analysis of immunophenotypic markers, while lungs were fixed in Bouin's solution containing picric acid and formaldehyde for 24–48 h.

[0265] The number of lung tumor nodules was counted under dissection for each lung, and the mean lung nodule count for each group was determined. Statistical significance of the vehicle group versus the intervention group was also obtained using an unpaired Student's t-test.

[0266] Lung metastasis results for the treatment groups corresponding to Conjugate 2 and Conjugate 1, respectively, are shown in Figures 19 and 20. Both illustrative long-acting IL-15 receptor agonists were effective in promoting the reduction of lung metastases, with Conjugate 2 providing a 65% reduction in metastases compared to vehicle, while Conjugate 1 provided an 85% reduction in metastases.

[0267] Blood and spleen cells were analyzed for changes in immunophenotypic markers on day 13 using flow cytometry and marker antibodies conjugated to various fluorochromes. Conjugate 2 administered at 0.3, 1, and 3 mg / kg each significantly increased CD8 in the blood. Conjugate 2 induced a dose-dependent increase in CD8 T cells by 1.5, 2.5, and 3.3-fold compared to vehicle. Similar observations were observed in the spleen, with 1.3, 1.7, and 2.2-fold increases compared to vehicle at the 0.3, 1, and 3 mg / kg dose levels. 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 compared to vehicle at the same low, mid, and high dose levels. In addition, Conjugate 2 treatment increased the pro-survival Bcl-2+ MFI in CD8 T cells by 1.5-fold in both the blood and spleen.

[0268] [Table 11]

[0269] Example 14 In vitro and in vivo cytotoxicity of NK cells after treatment with conjugate 1 NK cell-mediated cytotoxicity against target tumor cells was evaluated in vitro using a flow cytometry-based assay. NK cells were isolated from the spleens of Balb / c mice using negative selection magnetic cell isolation (Mouse NK Cell Enrichment Kit, Stemcell Technologies) and used as effector cells. For in vitro studies, isolated NK cells were stimulated overnight with conjugate 1 at concentrations of 3000, 1000, 300, 30, 3, or 0 (unstimulated) ng / mL in a humidified incubator at 37°C and 5% CO2 before use in cytotoxicity assays. For in vivo studies, mice were administered 0.3 mg / kg conjugate 1, and splenic NK cells were isolated 24, 48, and 72 hours after administration and used directly in cytotoxicity assays.

[0270] YAC-1 T cells labeled with PKH26 were used as target cells. To monitor NK cell cytotoxicity, NK and YAC-1 cells were co-cultured at various effector:target ratios (50:1, 25:1, and 12.5:1) at 37°C, 5% CO2 for 4 hours, and then stained with 7-AAD for 10 minutes to label dead cells. Cells were immediately analyzed by flow cytometry. Lysed target cells were then labeled with PKH26. + 7-AAD + was identified as.

[0271] In vitro results: After 4 hours of co-culture, cytotoxicity was assessed by flow cytometry. The results are presented in Figure 21.

[0272] In vivo results: Cytotoxicity after treatment with 0.3 mg / kg was assessed after 24, 48 and 72 hours. The results are presented in Figure 27.

[0273] The data demonstrate a dose-dependent increased cytotoxicity of NK cells in vitro and in vivo following treatment with conjugate 1.

[0274] Example 15 Induction of granzyme B by conjugate 1 NK cell granzyme B expression 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 24 to 240 hours post-dose for immunophenotyping. After lysis of red blood cells, white blood cells were labeled with a viability dye and markers specific for CD45, CD3, and CD49b to identify live NK cells. Cells were then simultaneously fixed and permeabilized for intracellular granzyme B staining. Stained blood was run on a Fortessa flow cytometer, acquired using DIVA software, and analyzed using Flowjo software. Data are expressed as the percentage of NK cells positive for granzyme B expression.

[0275] The results are provided in Figure 23. The data show that treatment with conjugate 1 increases granzyme B expression in NK cells.

[0276] Example 16 In vivo studies: Single-dose IL-15 and conjugate 1 PK and JAK / STAT signaling studies in mice For PK analysis, Conjugate 1 was administered intravenously at a single 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 hours after treatment. From a separate study, mice were administered a single intravenous dose of IL-15 (0.5 mg / kg). Samples from these mice were collected at the indicated time points within 6 hours of treatment. [PK methods are described previously herein]. For pharmacodynamic testing, BALB / c mice (n = 1 / group) received an intravenous injection of 0.03 or 0.3 mg / kg Conjugate 1 or vehicle (50 mM sodium phosphate, 100 mM sodium chloride, 10% sucrose, pH 7.4), and blood was collected pre-dose and at 15 minutes, 1, 24, 48, 72, 96, and 120 hours after treatment. Samples were analyzed individually by flow cytometry and expressed as percent pSTAT5 positivity in CD8 and NK cells.

[0277] FIG. 29A is a plot of the plasma concentrations of the test article (IL-15 or Conjugate 1) over a 144 hour time course following administration of a single intravenous dose of the test article at 0.5 and 0.3 mg / kg, respectively, in balb / c mice.

[0278] Results: Conjugate 1 exhibits a half-life of approximately 12 hours, while IL-15 is rapidly cleared from plasma with a half-life of less than 1 hour.

[0279] FIG. 29B is a graph of pSTAT5 percent positivity in CD8 T cells after a single injection of 0.03 and 0.3 mg / kg of Conjugate 1 in mice.

[0280] Results: Conjugate 1 induces sustained pSTAT5 signaling in CD8 T cells at both dose levels. A 120 hour time course is shown, including pre-dose.

[0281] FIG. 29C is a graph of pSTAT5 percent positivity in mouse NK cells after a single injection of 0.03 and 0.3 mg / kg of Conjugate 1.

[0282] Results: Conjugate 1 induces potent and sustained pSTAT5 signaling in NK cells at both dose levels.

[0283] Example 17 In vivo single-dose and Q7DX3 pharmacodynamic studies in mice - cell number and proliferation Balb / c mice (n=3 / group) were administered 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 (24, 48, 72, 96, 120, 144, and 240 hours) post-dose. Samples from each mouse were subjected to flow cytometry analysis to examine pharmacodynamic effects within lymphocyte populations and functional markers of interest (CD8 T cell, CD8 memory T cell, and NK cell counts, as well as percent Ki-67 positivity within each population). The results are shown in Figures 30A-F, 31A-C, and 32A-C.

[0284] Figures 30A-C are plots of total CD8, CD8 central memory (Tcm), and CD8 effector memory (Tem) cell counts after a single administration of 0.01, 0.03, 0.1, 0.3, 1, or 1.5 mg / kg of conjugate 1, respectively, as described in Example 17. Dose levels of conjugate 1 of 0.03 and above induce a significant increase in total CD8 T cells in the blood, as described in Example 17. The lowest dose of 0.01 mg / kg increased CD8 Tcm and CD8 Tem. At 0.3 mg / kg, conjugate 1 increased CD8, CD8 Tcm, and CD8 Tem by 6.4X, 37.9X, and 14.5X, respectively. Notably, CD8 and CD8 memory T cell counts did not return to baseline at 240 hours post-injection when conjugate 1 was administered at 0.3-1.5 mg / kg, demonstrating the sustained PD effect of conjugate 1.

[0285] Figures 30D, 30E, and 30F are plots of Ki-67 percent positivity within total CD8, CD8 Tcm, and CD8 Tem populations, respectively, in mice, as described in Example 17. A single dose of Conjugate 1 increases Ki-67 positivity in total CD8 and CD8 subpopulations at all dose levels.

[0286] Figures 31A, 31B, and 31C are plots of CD8 and CD8+ memory subpopulation T cell counts following administration of a single dose of Conjugate 1 (dotted line) or Q7dx3 (solid line) at 0.03 and 0.3 mg / kg, as described in Example 17. Repeated administration further expanded these populations, with CD8, CD8 Tcm, and CD8 Tem increasing 35.3X, 183X, and 73.8X, respectively. At the end of the time course (240 hours after the first or last dose at 0.3 mg / kg), cell counts had not returned to baseline in the mice.

[0287] Figures 32A and 32B are plots of NK cell counts and Ki-67 percent positivity in mice after a single dose of 0.01 to 1.5 mg / kg of conjugate 1, as described in Example 17. NK cell counts are significantly increased above vehicle controls at all dose levels and return to baseline by 240 hours post-dose. All dose levels induce a strong increase in Ki-67 percent positivity in NK cells.

[0288] Figure 32C is a plot of NK cell counts in mice following single (solid line) or Q7dx3 (dashed line) administration of 0.03 and 0.3 mg / kg conjugate 1, as described in Example 17. Repeat administration of 0.3 mg / kg conjugate 1 induced slightly lower NK cell counts compared to single administration, but was still significant. Similar NK cell counts were achieved with single vs. repeat administration of 0.03 mg / kg.

[0289] Results: Conjugate 1 at dose levels of 0.03 and above induced a significant increase in total CD8 T cells in the blood. The lowest dose, 0.01 mg / kg, increased CD8 Tcm (central memory) and CD8 Tem (effector memory). At 0.3 mg / kg, conjugate 1 increased CD8, CD8 Tcm, and CD8 Tem by 6.4X, 37.9X, and 14.5X, respectively. Notably, CD8 and CD8 memory T cell counts did not return to baseline 240 hours post-injection when conjugate 1 was administered at 0.3-1.5 mg / kg, demonstrating the beneficial and sustained PD effect of conjugate 1.

[0290] Example 18 Measurement of in vitro NK cytotoxicity and blood NK cell granzyme B analysis in mice treated with conjugate 1 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 hours for NK cell isolation. NK cells were isolated by a magnet-based negative selection method and were analyzed at 12.5:1, 25:1, and 50:1 ratios. The NK (effector) to YAC-1 (target cell) ratio (E:T) was incubated at 37°C, 5% CO for 4 hours. YAC-1 target cells were prelabeled and then stained with 7AAD after NK cell incubation. Detection of lysed (7AAD+) target cells (PKH26+) was performed by flow cytometry. Blood from these mice was also collected at the same time point and subjected to flow cytometric measurement of granzyme B expression on NK cells. The results are shown in Figures 33A and 33B.

[0291] Figure 33A shows an in vitro NK cytotoxicity assay measuring changes in NK-mediated target cell lysis following test article treatment in mice. The time course of percent specific lysis of YAC-1 cells by splenic NK cells isolated from BALB / c mice treated with 0.006, 0.03, or 0.3 mg / kg of Conjugate 1 or 1 mg / kg of IL-15 is shown at the indicated times. Splenic NK cells from mice administered vehicle served as a control.

[0292] Results: Conjugate 1 administered at 0.3 mg / kg induced an increase in NK cytotoxicity that was greater in magnitude and duration compared to NK cells from mice receiving a single injection of 1 mg / kg IL-15.

[0293] FIG. 33B is a graph of percent granzyme B positivity in blood NK cells from the same mice dedicated to the NK in vitro cytotoxicity assay in FIG. 33A.

[0294] Results: Conjugate 1 administered at 0.03 and 0.3 mg / kg induced a significant increase in NK granzyme B expression, with a potent and sustained increase seen at 0.3 mg / kg.

[0295] Example 19 Conjugate 1 single-drug efficacy in the CT-26 lung metastasis model Balb / c mice: 1x10 5 Mice received tail vein injections of CT-26 colorectal cancer cells. The following day, mice (n=9 / group) were treated twice weekly with Conjugate 1 (0.03 or 0.3 mg / kg) or vehicle control. Five days after the second injection, mice were humanely sacrificed and lung nodules were counted. The results are presented in Figures 34A and 34B.

[0296] Figures 34A and 34B show percent lung nodule inhibition in balb / c mice that received intravenous CT-26 tumor cell injection followed by two doses of Conjugate 1 treatment at 0.03 or 0.3 mg / kg, one week apart.

[0297] Results: Conjugate 1 injections at 0.03 and 0.3 mg / kg inhibited lung nodule formation by 40 and 80%, respectively. The same mice dosed at 0.3 mg / kg then received subsequent tumor cell injections for 32 days and survival was assessed. Treatment with Conjugate 1 significantly increased survival compared to tumor-injected mice that received the vehicle control.

[0298] Example 20 Evaluation of NK cell dependency of Conjugate 2 efficacy in the CT-26 lung metastasis model CT-26 mice (n=7-11 / group) were injected with anti-asialoGM1 or two different controls (IgG or PBS) to deplete NK cells, and then 1x10 5 CT-26 tumor cells were injected into the mice. Mice were then treated with 0.3 mg / kg of Conjugate 2 administered on days 1, 5, and 10 after tumor cell injection or vehicle control. Mice were sacrificed and lung nodules were counted 3 days after the last day of treatment. The results are shown in Figure 35.

[0299] Figure 35 is a graph showing percent lung nodule inhibition in CT-26-injected mice treated with Conjugate 2 that underwent antibody-mediated depletion of NK cells (olive green), IgG control (blue), or PBS (orange). Data are expressed as percent lung nodule inhibition relative to CT-26-injected mice that were not NK cell-depleted and treated with vehicle control (black). The efficacy of Conjugate 2 in this tumor model was abolished when mice were depleted of NK cells.

[0300] Example 21 In vivo pharmacodynamic study of a single dose of conjugate 1 in non-human primates In this study, one female and one male cynomolgus monkey (cyno) were each administered a single dose of Conjugate 1 (0.1 mg / kg) intravenously. Serial blood samples were collected from each animal over a 14-day time course and subjected to flow cytometry analysis of various lymphocytes (CD8 T cells, total CD8, CD8 central memory T cells (Tcm), and CD8 effector memory T cells (Tem) Ki-67 percent positive, NK cells, and NK cell Ki-67 percent positive). The results are presented in Figures 36A-D and 37A-B.

[0301] Figures 36A and 36B are plots showing a two-week time course of CD8 cell counts and Ki-67 percent positivity as a measure of proliferation in one male (dotted line) and one female (solid line) cyno after intravenous administration of a 0.1 mg / kg dose of Conjugate 1. As can be seen, Conjugate 1 induces a significant CD8 T cell expansion in the cyno, with cell counts increasing 7-10X after a single dose.

[0302] Figures 36C and 36D show the increase in cynoCD8 Tcm and CD8 Tem cell numbers after a single injection of conjugate 1. CD8 Tcm and Tem numbers increased 27-30X and 21-33X, respectively.

[0303] 37A and 37B are graphs of NK cell counts and Ki-67 percent positivity in cynos after a single dose of 0.1 mg / kg of Conjugate 1. NK cells were expanded 9-10X after treatment with Conjugate 1.

[0304] Example 22 Comparison of the in vitro activity of IL-15 and conjugate 1 in CD8 and CD56 BRIGHT NK cells in human PBMCs The in vitro activity of Conjugate 1 was assessed by examining NK and CD8 JAK / STAT signaling after treatment of human PBMCs with doses ranging from 0.001 to 10,000 ng / ml of IL-15 or Conjugate 1. STAT5 phosphorylation was assessed as previously described.

[0305] [Table 12]

[0306] The results are presented in Figures 38A and 38B, which show the EC50 curves for IL-15 (●) versus conjugate 1 (■) treatment of human PBMCs and subsequent measurement of pSTAT5 percent positivity in CD8 and CD56 bright NK cells.

[0307] Results: Conjugate 1 is 5.5 and 15x less potent than IL-15 in engaging CD8 and CD56 bright NK cells, respectively. Importantly, however, conjugate 1 achieves the same maximal response as conventional IL-15.

[0308] Example 23 In vivo studies: Single-dose PK study in mice Balb / c mice (n=3 / group) were administered a single intravenous 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 post-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) to determine plasma concentrations of drug. See Figure 39.

[0309] As shown in Figure 39, conjugate 1 exhibited extended pharmacokinetics, measurable plasma concentrations, and was rapidly cleared with a half-life of approximately 14 hours compared to plasma levels observed with non-long-acting IL-15.

[0310] Example 24 In vivo studies: Single-dose PK study in rats Sprague Dawley rats (n=3) were administered a single intravenous dose of Conjugate 1 at 10, 75, and 150 μg / kg. Plasma concentrations of the drug were determined at the indicated time points after injection (0.03, 0.08, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 72, 96, 120, 144 hours). See Figure 40.

[0311] As shown in Figure 40, conjugate 1 exhibited sustained pharmacokinetics, measurable plasma concentrations, and was rapidly cleared with a half-life of approximately 18 hours compared to plasma levels observed after administration with non-long-acting IL-15.

[0312] Example 25 In vivo studies: Single-dose PK studies in non-human primates Cynomolgus monkeys (n=2, 1 male and 1 female) were administered a single intravenous dose of 10, 50, and 100 μg / kg of Conjugate 1. As a control, a single intravenous dose of 50 μg / kg of IL-15 was administered. Plasma concentrations of the drug were determined at the indicated time points (0.03, 0.25, 1, 4, 12, 24, 48, 72, 96, 120, 144, 168 hours) after injection. See Figure 41.

[0313] As shown in Figure 41, conjugate 1 exhibited sustained pharmacokinetics, measurable plasma concentrations, and was rapidly cleared from plasma with a half-life of approximately 30 hours compared to non-long-acting IL-15 for the 100 μg / kg dose.

[0314] Conjugate 1 achieved prolonged and sustained plasma exposure across multiple species (mouse, rat, and cynomolgus monkey) after a single dose (see Figures 39-41).

[0315] Example 26 In vivo studies: Single-dose PD studies in mice - involvement of cell number, proliferation and JAK / STAT signaling Balb / c mice (n=3 / group) were administered a single intravenous dose of vehicle (described in Example 11) or Conjugate 1 at doses of 0.3 mg / kg or 0.03 mg / kg. After administration, blood samples were collected at post-dose time points (24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, 144 hours, and 240 hours). Samples were subjected to immunophenotyping for CD4 T cell counts (see Figure 42A) and % Ki-67 (see Figure 42B) at the indicated time points.

[0316] CD4 T cells and their proliferation were identified by CD45+CD3+CD4+CD8- and CD45+CD3+CD4+CD8-Ki-67+ markers, respectively. Figure 42A is a plot of CD4 T cell counts, and Figure 42B is a plot of CD4 T cell proliferation over time as measured by % Ki-67 positivity. CD4 T cells were the least sensitive population to Conjugate 1 treatment, with lower numbers and increased % Ki-67 expression compared with CD8 and NK cells (observed 72-144 hours post-dose) (see, e.g., Example 11). In mice, NK cells were more sensitive than CD4 or CD8 T cells to stimulation with a single dose of Conjugate 1 in their proliferative responses.

[0317] STAT5 phosphorylation in CD4 T cells was determined using a combination of CD3+CD4+CD8- pSTAT5+ markers. Figure 43 shows plots of percent positive pSTAT5 phosphorylation in CD4 T cells over time (0.25, 1, 6, 24, 48, 72, 96, and 120 hours post-dose) at doses of 0.03 mg / kg (blue, solid squares) or 0.3 mg / kg (orange, solid circles). Vehicle levels over time (black) and pre-dose (open circles) are also shown.

[0318] Results: CD4 T cells were the least sensitive population to Conjugate 1 treatment and had a lower increase in pSTAT5 expression compared to CD8 and NK cells (observed 0.25-72 hours post-dose). In mice, NK cells were more sensitive than CD8 or CD4 T cells to a single dose of Conjugate 1 stimulation in their proliferative responses.

[0319] Example 27 In vivo studies: Minimum effective dose studies in non-human primates (NHPs) Cynomolgus monkeys (n=3-4 males) were administered a single intravenous injection of 0.003, 0.01, or 0.1 mg / kg of Conjugate 1 or vehicle control. Blood samples were collected pre-dose and at the indicated time points post-dose (days -5, -2, 1, 2, 3, 4, 5, 6, 7, 10, 14, and 17) and subjected to flow cytometry analysis to examine the pharmacodynamic effects within lymphocyte populations. NK, CD8 T, and CD4 T cell counts were examined, and the results are shown in Figures 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 Figures 45A-C and 46A-C, respectively. The CD8 subpopulation (T 天然 , T em , T cm and T scm The proliferation (% Ki-67) of these cells was examined, and the results are shown in Figures 47A to 47D.

[0320] In NHPs, NK (CD45+CD3-CD16+) cell counts were substantially and dose-dependently increased after a single administration of Conjugate 1. At the 0.1 and 0.01 mg / kg dose levels, maximum cell counts were observed 5 days after administration and persisted through day 14. The lowest dose resulting in a significant increase in NK cells was 0.01 mg / kg. In support of the NK cell count observations, Conjugate 1 also promoted a dose-dependent and robust induction of Ki-67 expression, which reached a maximum approximately 3-4 days after treatment and could persist through approximately day 14. A significant increase in %Ki-67 could be detected at dose levels as low as 0.001 mg / kg. Conjugate 1 also strongly engaged the JAK / STAT signaling pathway in NK cells, and a dose-dependent increase in %pSTAT5 could be detected at doses as low as 0.001 mg / kg.

[0321] Figures 44A, 45A and 46A are plots of NK cell counts, % Ki-67 and % pSTAT5, respectively, over time following Conjugate 1 treatment.

[0322] In NHPs, conjugate 1 induced a significant expansion of total CD8 T cells (defined as CD45+CD3+CD4-CD8+), with maximum cell numbers achieved approximately 5 days after treatment. This effect persisted for 7 days, returning to baseline 10-14 days after administration. The effect of conjugate 1 on total CD8 cell numbers was detectable at 0.003 mg / kg. In support of these findings, conjugate 1 induced significant %Ki-67 positivity in CD8 T cells, detectable at doses as low as 0.01 mg / kg. Conjugate 1 engagement of the JAK / STAT signaling pathway was also potent in CD8 T cells, with a dose-dependent increase in pSTAT5 at the 0.1 and 0.01 mg / kg dose levels.

[0323] Figures 44B, 45B and 46B are plots of CD8 cell counts, % Ki-67 and % pSTAT5, respectively, over time following Conjugate 1 treatment.

[0324] Conjugate 1 significantly increased the number of total CD4 T cells compared to NK and CD8 T cells in NHPs. It has a low effect on T cells (defined as CD45+CD3+CD4+CD8-). Conjugate 1 administered at the highest dose level of 0.1 mg / kg induced a low increase in CD4 T cell counts, % Ki-67 and % pSTAT5.

[0325] Figures 44C, 45C and 46C are plots of CD4 T cell counts, % Ki-67 and pSTAT5 over time, respectively, following Conjugate 1 treatment.

[0326] NK cells are the most sensitive to the Conjugate 1 dose response in vivo compared to CD8 or CD4 T cells in NHPs.

[0327] In cynomolgus monkeys, CD8 naive and memory subpopulations were defined by CD45Ra, CD197, and CD95. Examination of proliferation (% Ki-67) of CD8 T naive (CD45+CD3+CD4-CD8+CD45Ra+CD197+), CD8 Tscm (CD45+CD3+CD4-CD8+CD45Ra+CD197+CD95+), CD8 Tem (CD45+CD3+CD4-CD8+CD45Ra-CD197-), and CD8 Tcm (CD45+CD3+CD4-CD8+CD45Ra-CD197+) revealed increased sensitivity of the CD8 memory subpopulation to conjugate 1 compared to CD8 naive T cells. Within the CD8 Tem, Tcm, and Tscm populations, conjugate 1 induced robust % Ki-67 expression in a dose-dependent manner, with detectable increases in proliferation marker positivity beginning as early as day 2, reaching a maximum at day 5, and returning to baseline between days 10 and 14. Ki-67 expression and kinetics within the CD8 population support the sustained increases in CD8 T cell numbers shown in Example 27 and Figures 47A-D.

[0328] Figures 47A-D show CD8 T cells after treatment with Conjugate 1. 天然 , T scm , T cm and T em 1 is a plot of population %Ki-67 over time. As can be seen, CD8 T cell memory populations showed increased sensitivity to a single dose of Conjugate 1 in vivo compared to naive CD8 T cells in NHPs.

[0329] Example 28 Induction of Granzyme B or Perforin by Conjugate 1 The expression of NK cell lytic enzymes, granzyme B and perforin, was examined in cynomolgus monkeys after a single administration of conjugate 1. Granzyme B and perforin expression levels were quantified by mean fluorescence intensity (MFI) in NK cells at dose levels of 0.001 mg / kg, 0.01 mg / kg, or 0.1 mg / kg. Conjugate 1 increased the MFI of granzyme B approximately three-fold (peak vs. pre-dose) at 0.01 and 0.1 mg / kg. Conjugate 1 also increased the MFI of perforin approximately two-fold (peak vs. pre-dose) at 0.01 and 0.1 mg / kg. Overall, conjugate 1 not only induces potent expansion of NK cells but can also improve their function.

[0330] Figures 48A-C are plots of granzyme B at pre-dose (baseline) and peak levels and Figures 49A-C are plots of perforin MFI after conjugate 1 treatment (0.0001-0.1 mg / kg) in NHPs.

[0331] Conjugate 1 increases the protein levels of cytotoxic enzymes, including constitutive expression of granzyme B and perforin, in NHP NK cells.

[0332] Sequence Listing SEQ ID NO: 1 (rhIL-15) [ka] SEQ ID NO: 2 [ka] SEQ ID NO: 3 [ka]

Claims

1. 1. A pharmaceutical composition for use in the treatment of cancer, comprising:

1. A long-acting interleukin-15 (IL-15) receptor agonist having the formula: 【Chemistry 1】 wherein IL-15 is an interleukin-15 moiety, n is an integer from about 150 to about 3,000, m is an integer from 2 to 5, n' is 1, and ~NH~ in the structure represents an amino group of the IL-15 moiety. or a pharmaceutically acceptable salt form thereof, The cancer is selected from hematological malignancies, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, colon cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, head and neck cancer, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial carcinoma, melanoma, neuroblastoma, and retinoblastoma; composition.

2. The composition of claim 1 , wherein m is 2 or 3.

3. The composition of claim 1 , wherein m is 3.

4. 2. The composition of claim 1, wherein n is an integer from about 200 to about 2000, or from about 400 to about 1300, or from about 450 to about 1200.

5. 10. The composition of claim 1, wherein n is about 909.

6. 2. The composition of claim 1, wherein n is an integer having a value corresponding to a polyethylene glycol polymer having a weight average molecular weight selected from the group consisting of 10,000 daltons, 15,000 daltons, 20,000 daltons, 25,000 daltons, 30,000 daltons, 40,000 daltons, 50,000 daltons and 60,000 daltons.

7. 7. The composition of any one of claims 1-6, wherein about 15 mole percent or less of the long-acting IL-15 receptor agonist has the formula: 【Chemistry 32】 wherein for formula (II), the variables n and m are as defined for formula (I) above.

8. 8. The composition of claim 7, wherein about 10 mole percent or less of the long-acting IL-15 receptor agonists has the formula (II).

9. 8. The composition of claim 7, wherein about 7 mole percent or less of the long-acting IL-15 receptor agonists has the formula (II).

10. 8. The composition of claim 7, wherein about 5 mole percent or less of the long-acting IL-15 receptor agonists has the formula (II).

11. The composition of any one of claims 1 to 10, wherein the average number n' in the composition is in the range of 1.0 to about 1.

3.

12. The composition of any one of claims 1 to 10, wherein n' for said composition is selected from 1.0, 1.1, 1.2 and about 1.

3.

13. The composition of any one of claims 1 to 12, comprising a mixture of positional isomers of the long-acting IL-15 receptor agonist having the structure of formula (I).

14. 14. The composition of claim 13, wherein for the mixture of positional isomers of formula (I), the amino group is predominantly at the N-terminus of the IL-15 moiety.

15. The composition of any one of claims 1 to 14, wherein the IL-15 has an amino acid sequence having at least about 95% identity to any one of SEQ ID NOs: 1 to 3.

16. The composition of any one of claims 1 to 15, comprising a pharmaceutically acceptable excipient.

17. The composition of any one of claims 1 to 16, wherein the hematological malignancy is myeloma.

18. The composition of any one of claims 1 to 16, wherein the hematological malignancy is selected from leukemia or lymphoma.

19. The composition of any one of claims 1 to 18, having an EC50 value (ng / mL, CTLL-2 pSTAT5) that is reduced by about 7-fold or less compared to the EC50 value (ng / mL, CTLL-2 pSTAT5) of unmodified IL-15.

20. 19. The composition of any one of claims 1 to 18, having an EC50 value (ng / mL, CTLL-2 pSTAT5) that is reduced by about 6.5-fold or less, about 6-fold or less, about 5.5-fold or less, about 5-fold or less, about 4.5-fold or less, about 4-fold or less, about 3.5-fold or less, or about 3-fold or less, when compared to the EC50 value (ng / mL, CTLL-2 pSTAT5) of unmodified IL-15.

21. Receptor α binding of unmodified IL-15 (K Ｄ , pM) values, the receptor α binding (K Ｄ 19. The composition of claim 1, having a pH of 1., pM.

22. Receptor α binding of unmodified IL-15 (K Ｄ α receptor binding (K) values ​​that are reduced by about 45% or less, about 40% or less, about 35% or less, or about 30% or less compared to the K values. Ｄ 19. The composition of claim 1, having a pH of 1., pM.

23. When compared with the EC50 value (ng / mL, CTLL-2 pSTAT5) of unmodified IL-15, it had an EC50 value (ng / mL, CTLL-2 pSTAT5) that was reduced by about 7-fold or less, and the receptor α binding value (K Ｄ , pM) was reduced by about 50% or less when compared to the receptor α binding value (K Ｄ 19. The composition of claim 1, wherein the hydroxyl group is hydroxypropyl methylcellulose, ...

24. 19. The composition of any one of claims 1 to 18, which is effective in stimulating NK activation and proliferation when administered to a mammalian subject at a therapeutically effective dose.

25. 19. The composition of any one of claims 1 to 18, which is effective in supporting CD8 T cell survival and memory formation when administered to a subject at a therapeutically effective dose.

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