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 of U.S. Provisional Patent Application No. 62 / 506,494, filed May 15, 2017; U.S. Provisional Patent Application No. 62 / 536,966, filed Jul. 25, 2017; U.S. Provisional Patent Application No. 62 / 582,186, filed Nov. 6, 2017; and U.S. Provisional Patent Application No. 62 / 648,240, filed Mar. 26, 2018, under 35 U.S.C. 119(e), the disclosures of which are hereby incorporated by reference in their entirety.

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

Background Art

[0003] Interleukin - 15 ( "IL - 15") is a pleiotropic cytokine first reported 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 pro - sequence; thus the mature protein is 114 amino acids long. IL - 15, which belongs to the four - alpha - helix - bundle cytokine family, binds to a heterotrimeric receptor where the unique alpha subunit (IL - 15Rα) confers receptor specificity to IL - 15, and the beta and gamma subunits of this receptor share features with one or more other cytokine receptors. Giri et al. (1995) EMBO J. 14:3654 - 3663.

[0004] Among cytokines, IL-15 affects 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, gives rise to the development of natural killer cells (“NK cells”) and natural killer T cells (“NK-T cells”) and maintains their survival. Consistent with its role in the innate immune system, NK cells do not specifically attack invading pathogens; rather, NK cells destroy susceptible host cells, such as tumor cells or virus-infected cells. NK-T cells produce immunomodulatory cytokines, particularly interferon-γ, which results in the general activation of the immune response.

[0005] With respect to the adaptive immune system, which defends the host from specific foreign invaders after first encountering a particular pathogen, IL-15 is required for the maintenance of immunomodulatory cytokine-producing helper T cells. Importantly, IL-15 also supports the long-term maintenance of “antigen-experienced” memory T cells, which have the ability to rapidly replicate, resulting in a more rapid and robust immune response when the host is re-exposed to a particular foreign pathogen that has invaded.

[0006] Finally, despite its specific roles within both the innate and adaptive immune systems, IL-15 has 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] IL-15 has been proposed for use in the treatment of cancer patients because it can stimulate the proliferation and maintenance of many cells within the immune system that can fight cells that appear foreign (or "non-self") to the host (Steel et al. (2012) Trends Pharmacol. Sci. 33(1):35-41). For example, an IL-15-based agonist has been proposed for the treatment of multiple myeloma (Wong et al. (2013) OncoImmunology 2(11), e26442:1-3). In addition, IL-15 drug therapy has been proposed for the treatment of patients with viral infections such as HIV infection. Despite its potential for use in the treatment of patients with several diseases, IL-15-based therapies face several challenges. For example, IL-15 is rapidly removed 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. In some approaches, attempts have been made to overcome these limitations by complexing IL-15 with the IL-15 receptor α subunit. However, such approaches can abrogate the desirable signaling that uniquely occurs via IL-15 receptor α, which is expressed in multiple cell types. Non-cleavable pegylation with a relatively low molecular weight (5 kDa) succinimidyl terminal polymer has been reported, but this resulted in a significant alteration of the biological activity of IL-15. Pettit et al. (1997) J. Biol. Chem. 272(4):2312-2318.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0009] However, despite the above-described approaches, there is still a need for new IL-15 receptor agonists having improved properties and profiles, such as a potent immune-stimulating effect, low systemic toxicity, stability and / or improved pharmacokinetics. Accordingly, among other things, the present disclosure provides long-acting IL-15 receptor agonists having a number of advantageous features as described herein and more particularly below, which are new and not fully suggested by the art, as well as compositions and kits comprising such agonists, and related methods of manufacture and use.

Means for Solving the Problems

[0010] In a first aspect, there is provided herein a long-acting IL-15 receptor agonist (including its pharmaceutically acceptable salt form). The long-acting IL-15 receptor (IL-15 R) agonist comprises at least a single straight-chain PEG (polyethylene glycol) moiety stably covalently bound to the IL-15 amino group via an amide bond. Intervening between the straight-chain PEG chain and the stable amide bond to the IL-15 amino group is a straight-chain 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 further embodiments, the unsubstituted alkylene group is (~CH2~)4; in some further embodiments, the unsubstituted alkylene group is (~CH2~)5.

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

Chemical formula

[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 one or more additional further 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 exemplary embodiments, there is provided a composition comprising a long-acting IL-15 receptor agonist according to formula (I), including each and all of its related embodiments provided herein without limitation.

[0017] In some embodiments, the long-acting IL-15 receptor agonist composition, taken together, has the following formula:

Chemical formula

[0018] For example, in some embodiments, the long-acting IL-15 receptor agonist composition contains about 10 mol percent or less of the long-acting IL-15 receptor agonist, taken together, as encompassed by formula (II).

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

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

Chemical formula

[0021] In yet another aspect, provided herein are methods for preparing long-acting interleukin-15 receptor agonists as described in formula (I), for example formula (Ia), (Ib), (Ic), (Id), and also in formula (II), for example (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 approximately 7 (e.g., 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6) is combined with an activating PEG reagent such as:

Chemical formula

Chemical formula

Chemical formula

[0022] In some preferred embodiments, the methoxy PEG-succinimidyl alkanate reagent is

Chemical formula

[0023] In some embodiments, the methoxy PEG-succinimidyl alkanate 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 alkanate reagent is added in an equimolar amount (i.e., equimolar ratio) to interleukin-15.

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

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

[0027] In some other embodiments, the methoxy PEG-succinimidyl alkanate reagent is dissolved in a suitable solvent. In certain embodiments, the methoxy PEG-succinimidyl alkanate 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., before mixing with the methoxy PEG-succinimidyl alkanate reagent. Additional exemplary concentration ranges include, for example, interleukin-15 at about 0.5 mg / mL to about 5 mg / mL, about 0.5 mg / mL to about 3 mg / mL, and about 1.0 mg / mL to about 4 mg / mL.

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

[0030] In some further embodiments, the pH of the reaction mixture is adjusted to about 8.0 after the addition of the methoxy PEG-succinimidyl alkanate reagent.

[0031] In some further embodiments, the resulting reaction mixture is stirred (or mixed) for a time sufficient for the reaction between the reaction participants. In some embodiments, the reaction participants are mixed for from about 15 minutes to about 10 hours (inclusive). In some further embodiments, the reaction participants 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, for example at room temperature, i.e., without applying 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 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. In some related embodiments, the reaction is quenched by the addition of glycine.

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

[0035] In some additional embodiments, the reaction mixture containing the methoxyPEG-alkanoate-interleukin-15 conjugate is purified.

[0036] In some particular embodiments, the reaction

Chemical formula

[0037] In some further embodiments, the reaction, considered collectively, is encompassed by the following formula:

Chemical formula

[0038] In some further 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 a decrease of about 7-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5) when compared to unmodified (i.e., non-conjugated) IL-15. For example, in one or more related embodiments, the long-acting IL-15 receptor agonist exhibits a decrease of about 6.5-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5), or a decrease of about 6-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5), or a decrease of about 5.5-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5), or a decrease of about 5-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5), or a decrease of about 4.5-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5), or a decrease of about 4-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5), or a decrease of about 3.5-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5), or even a decrease of about 3-fold or less 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 a decrease of about 50% or less in the receptor α binding (K D pM) when measured using techniques suitable for determining receptor α binding, such as surface plasmon resonance (SPR), for example, compared to non-conjugated IL-15. In some related embodiments, the long-acting IL-15 receptor agonist exhibits a decrease in the receptor α binding (KD shows a decrease of about 45% or less in (pM), or receptor α binding (K D shows a decrease of about 40% or less in (pM), or receptor α binding (K D shows a decrease of about 35% or less in (pM), or even receptor α binding (K D shows a decrease of about 30% or less in (pM).

[0041] In some further embodiments, the long-acting IL-15 receptor agonist shows a decrease of about 7-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5) compared to unmodified IL-15, and a decrease of about 50% or less in receptor α binding (K D in (pM) compared to IL-15 (including any one or more specific combinations of decreases in the EC50 value or K D value described above).

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

[0043] In some further embodiments, the long-acting IL-15 R 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 further embodiments, the long-acting IL-15 R 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, there is provided herein a method of treating a condition in a subject having a condition responsive to treatment with IL-15 by administering a therapeutically effective dose of a long-acting IL-15 R 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 straight-chain polyethylene glycol (PEG) moiety stably covalently bound to the amino group of IL-15 via an amide bond, and having a straight-chain unsubstituted alkylene group (~CH2~) having 2 to 5 carbon atoms m is interposed between the PEG moiety and the amide bond to the IL-15 amino group, a long-acting IL-15 receptor agonist, and a pharmaceutically acceptable salt form thereof. (Item 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) The long-acting IL-15 receptor agonist according to Item 1 or Item 2, wherein the unsubstituted alkylene group is selected from (~CH2~)2, (~CH2~)3, (~CH2~)4, or (~CH2~)5. (Item 4) A long-acting IL-15 receptor agonist, comprising [Chemical formula] (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 the amino group of the IL-15 moiety), a long-acting IL-15 receptor agonist. (Item 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, or about 400 to about 1300, or about 450 to about 1200. (Item 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) The long-acting IL-15 receptor agonist according to any one of Items 1 to 6, wherein m is 3. (Item 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 a long-acting IL-15 receptor agonist according to any one of items 7 to 11, wherein the composition, when considered collectively, has the formula:

Chemical formula

Chemical formula

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

[0047] Additional aspects and embodiments are provided in the following specification and claims.

Brief Description of the Drawings

[0048]

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[0049] Prior to describing one or more aspects or embodiments of the present disclosure in detail, it should be noted that the present disclosure is not intended to be limited to specific synthetic techniques, IL - 15 moieties, etc., as they can vary as would be understood by those skilled in the art of the technical field to which the present disclosure pertains.

[0050] When describing and claiming specific features of the present disclosure, the following technical terms are used according to the definitions set forth below, unless otherwise indicated.

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

[0052] In the description and claims of one or more embodiments, the following technical terms shall be used according to the following definitions.

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

[0054] An "enzymatically degradable bond" means a bond that is susceptible 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., does not undergo hydrolysis under physiological conditions to any appreciable extent over a long period of time. Examples of hydrolytically stable bonds generally include, but are not limited to: carbon-carbon bonds (e.g., in fatty chains), ethers, amides, amines, etc. Generally, a stable bond exhibits a hydrolysis rate of less than about 1-2% per day under physiological conditions. The hydrolysis rates of representative chemical bonds can be found in the most standard chemical texts.

[0056] For example, a covalent "liberating" bond in the context of a polyethylene glycol covalently bound to an active moiety, such as interleukin-15, is a bond that releases or separates the polyethylene glycol polymer from the active moiety at a clinically useful rate under physiological conditions, for example by any suitable mechanism, and includes, for example but not limited to, hydrolyzable bonds and enzymatically cleavable bonds.

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

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

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

[0060] "Pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to a component that is included in the compositions described herein and that cannot cause a significant adverse toxicological effect on the subject.

[0061] The expressions "pharmaceutically effective amount" and "pharmacologically effective amount" and "therapeutically effective amount" and "physiologically effective amount" are used interchangeably herein and refer to the amount of the long-acting IL-15 R agonist provided herein that is necessary to provide a desired level of the substance in the blood stream or target tissue to elicit a desired biological or drug response. For example, such a response can be to destroy target cancer cells in a subject or to retard or inhibit cancer progression. The term also applies to the dosage that induces a specific response in target cells. The exact amount will depend on numerous factors such as, for example, the particular condition being treated, the intended patient population, considerations of the individual patient, the components of the therapeutic composition being administered and its physical characteristics.

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

[0063] As used herein, the terms "patient" or "subject" refer to an organism that has or is susceptible to a condition that can be prevented or treated by administration of the compounds or compositions provided herein. The subject includes, but is not limited to, mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), and is preferably a human.

[0064] The molecular weight associated with a water-soluble polymer, such as PEG, can be expressed as either the number average molecular weight or the weight average molecular weight. Unless otherwise indicated, all molecular weights referred to herein refer to the weight average molecular weight. The measurement of both the number average and weight average molecular weights can be performed using gel permeation chromatography or other liquid chromatography methods (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 measurement of colligative properties (e.g., freezing point depression, boiling point elevation, or osmotic pressure), which may be used to determine the number average molecular weight, or the weight average molecular weight may be determined by using light scattering, ultracentrifugation, MALDI-TOF, or viscosity measurement methods. PEG polymers are typically polydisperse (i.e., the number average molecular weight and the weight average molecular weight of the polymer are not equal), and preferably have 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, still 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 contrasted with groups that require a strong catalyst or extremely unrealistic reaction conditions to react (i.e., "non-reactive," or "inert" groups).

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

[0067] The terms "spacer moiety", "linkage", and "linker" may be used herein to refer to a bond or an atom or collection of atoms used, for example, to optionally link the terminus of a polymeric agent to the interconnecting portion of the IL-15 moiety. The spacer moiety may be stable to hydrolysis, or may contain a linkage that is physiologically hydrolysable or enzymatically cleavable, or otherwise releasable. Unless otherwise specifically indicated in the context, the spacer moiety may optionally be present between any two elements of a compound (e.g., an IL-2 moiety and a water-soluble polymer such as PEG may be bonded either directly or indirectly through a spacer moiety).

[0068] "Alkyl" refers to a hydrocarbon chain, typically having an atomic length in the range of about 1 to 15. Such hydrocarbon chains are preferably saturated, although this is not essential, and may be branched or straight-chain, although typically straight-chain is preferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 3-methylpentyl, and the like.

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

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

[0071] The term "substituted", as in "substituted alkyl" for example, includes, but is not limited to, alkyl, C 3~8Refers 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" is 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] A "non-interfering substituent" is a group that, when present in a molecule, typically has no reactivity with other functional groups contained in that molecule.

[0073] "Aryl" means one or more aromatic rings, each having 5 or 6 central carbon atoms. Aryl includes multiple aryl rings that may be fused, as in naphthyl, or not fused, as in biphenyl. The aryl rings may also be fused or not fused to one or more cyclic hydrocarbons, heteroaryl, or heterocyclic rings. As used herein, "aryl" includes heteroaryl.

[0074] "Heteroaryl" is an aryl group containing 1 to 4 heteroatoms, preferably sulfur, oxygen, or nitrogen, or combinations thereof. The heteroaryl ring may also be fused to one or more cyclic hydrocarbons, heterocyclic rings, aryl rings, or heteroaryl rings.

[0075] "Heterocycle" or "heterocyclic" means one or more rings of 5 to 12 atoms, preferably 5 to 7 atoms, which may or may not have unsaturated or aromatic character and which have at least one ring atom other than carbon. Preferred heteroatoms include sulfur, oxygen, and nitrogen.

[0076] "Substituted heteroaryl" is heteroaryl having one or more non-interfering groups as substituents.

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

[0078] "Organic radical", as used herein, includes alkyl, substituted alkyl, aryl, and substituted aryl.

[0079] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that can optionally be included in the compositions of the present invention and that does not cause any significant toxic adverse effects to a patient.

[0080] The term "IL-15 moiety", as used herein, refers to a peptide or protein moiety having human IL-15 activity. In addition, the term "IL-15 moiety" encompasses both the IL-15 moiety before conjugate formation and the IL-15 moiety residue after conjugate formation. As will be explained in more detail below, one of ordinary skill in the art can determine whether any given moiety has IL-15 activity. A protein comprising an amino acid sequence corresponding to any one of SEQ ID NOs: 1-3, as well as any protein or polypeptide that is substantially homologous thereto, is an IL-15 moiety. 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 mutations. These terms also include analogs having 1 to 6 additional glycosylation sites, analogs having at least one additional amino acid at the carboxy-terminal end of a similar peptide or protein, said additional amino acid including at least one glycosylation site, and analogs having an amino acid sequence including at least one glycosylation site. This term includes both natural moieties and recombinantly and synthetically produced moieties.

[0081] The terms "substantially identical" or "substantially the same" mean that a particular target sequence, such as a mutant sequence, differs from a reference sequence by only one or more substitutions, deletions, or additions, but that as a net effect, there is no functionally detrimental difference between the reference sequence and the target sequence. For the purposes of the present invention, sequences having greater than 95 percent homology (identity), equivalent biological activity (not necessarily equivalent biological activity strength) to a given sequence, and equivalent expression characteristics are considered to be substantially identical (the same). For the purpose of determining homology, truncations of the native sequence need to be ignored. The exemplary IL-15 polypeptide used herein includes a sequence that is substantially identical to SEQ ID NO: 1. SEQ ID NO: 2 is substantially the same as SEQ ID NO: 1, but SEQ ID NO: 2 has a methionine required for translation initiation at the beginning of the sequence in E. coli.

[0082] The term "fragment" means any protein or polypeptide having a portion or fragment of the amino acid sequence of the IL-15 moiety and having the biological activity of IL-15, or substantially the biological activity. Fragments include proteins or polypeptides produced by proteolysis of the IL-15 moiety, 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] Summary The present disclosure relates to the provision of long-acting IL-15 receptor agonists. Such agonists would ideally have several advantages and unexpected features, 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 dosing, (ii) maintaining a high degree of binding to IL-15 receptor α (i.e., as compared to IL-15), (iii) stimulating NK cell activation and / or proliferation, and / or (iv) supporting CD8 T cell survival and / or memory formation, and (v) providing inhibition of tumor growth. Surprisingly, the applicants have arrived at long-acting IL-15 R agonists having a unique combination of advantageous properties, described in detail below.

[0085] Long-Acting IL-15 R Agonists and Related Compositions Generally, a long-acting IL-15 receptor agonist or a pharmaceutically acceptable salt form thereof comprises a single linear PEG (polyethylene glycol) moiety stably covalently bound to the IL-15 amino group via an amide bond. There is a linear unsubstituted alkylene group having 2 to 5 carbon atoms (~CH2~) m (i.e., m = 2, 3, 4, or 5) intervening between the PEG moiety and the stable amide bond to the IL-15 amino group.

[0086] When considering the IL-15 moiety, the term “IL-15 moiety” refers to the IL-15 moiety prior to conjugation and the IL-15 moiety after binding to a non-peptidic water-soluble polymer such as poly(alkylene oxide) (e.g., poly(ethylene glycol) or PEG). Hereinafter, PEG is specifically referred to hereinafter as a non-peptidic water-soluble polymer, but it will be understood that the present disclosure generally relates to non-peptidic water-soluble polymers or poly(alkylene glycols). However, if the original IL-15 moiety is bound to the polyethylene glycol moiety, it will be understood that the IL-15 moiety is slightly modified due to the presence of one or more covalent bonds related to the binding to the polymer.

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

[0088] Alternatively, the IL-15 moiety can be obtained according to the procedure described by Grabstein et al. See, for example, Grabstein et al. (1994) Science 264:965-968. The IL-15 moiety can be, for example, Immunex It can also be prepared using recombinant methods such as those described in European Patent No. 0772624 B2 to 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 can be expressed in expression systems of bacteria [e.g., Escherichia coli (E. coli), see, for example, Fischer et al. (1995) Biotechnol. Appl. Biotechnol. 21(3):295-311], mammals [see, for example, Kronman et al. (1992) Gene 121:295-304], yeast [e.g., Pichia pastoris, see, for example, Morel et al. (1997) Biochem. J. 328(1):121-129], and plants [see, for example, Mor et al. (2001) Biotechnol. Bioeng. 75(3):259-266]. Expression can occur by either exogenous expression (when the host cell naturally contains the desired genetic code) or endogenous expression.

[0090] Recombinant protein preparation methods can vary, but typically, recombinant methods involve the construction of 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, bacterium, yeast, transgenic animal cell, or mammalian cell such as Chinese hamster ovary cells or baby hamster kidney cells), and producing the desired polypeptide or fragment by expression of the nucleic acid. Methods for producing and expressing recombinant polypeptides in prokaryotic and eukaryotic host cells in vitro are known to those skilled in the art.

[0091] To facilitate the identification and purification of the recombinant polypeptide, 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 comprising the desired polypeptide and a polypeptide suitable for binding. Identification and purification of the fusion protein can be performed by first passing a mixture containing the fusion protein through an affinity column having a binding moiety (e.g., an antibody) for the epitope tag or other binding sequence in the fusion protein, thereby binding the fusion protein within the column. Subsequently, the fusion protein can be recovered by washing the column with a suitable solution (e.g., an acid) to release the bound fusion protein. The recombinant polypeptide can also be purified by lysis of the host cell, separation of the polypeptide, for example, by ion exchange chromatography, affinity binding techniques, hydrophobic interaction techniques, and then identified by MALDI or Western blot and the polypeptide recovered. These and other methods for identifying and purifying recombinant polypeptides are known to those skilled 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 for the expression of the protein having IL-15 activity, the IL-15 moiety may or may not be glycosylated, and either can be used. That is, the IL-15 moiety may not be glycosylated, or the IL-15 moiety may be glycosylated. In one or more embodiments, the IL-15 moiety is not glycosylated.

[0093] The IL-15 moiety can advantageously be modified to include and / or substitute one or more amino acid residues, such as lysine, cysteine and / or arginine, whereby the polymer is more likely to bind to atoms within the side chains of its amino acids. Examples of substitutions of the IL-15 moiety are described in U.S. Patent No. 6,177,079. In addition, 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, M.J., and Russell, R.B.

[0094] In addition, the IL-15 moiety can advantageously be modified to include a bond of a functional group (except for the addition 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 the N-terminal α-carbon. In addition, the IL-15 moiety can be modified to include one or more carbohydrate moieties. In addition, the IL-15 moiety can be modified to include an aldehyde group. In addition, the IL-15 moiety can be modified to include a ketone group. In some embodiments of the invention, it is preferred that the IL-15 moiety is not modified to include one or more of a thiol group, the N-terminal α-carbon, a carbohydrate, an aldehyde group, and a ketone group.

[0095] Exemplary IL-15 moieties are described herein, in the literature, and, for example, in 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 selected from the group consisting of SEQ ID NOs: 1-3 and sequences substantially homologous thereto (wherein, even if SEQ ID NOs: 2 and 3, and sequences substantially homologous thereto do not meet the in vitro activity criteria of the IL-15 moieties provided herein, for the purposes of the present invention, those sequences are also understood to be "IL-15 moieties"). Preferred IL-15 moieties have the amino acid sequence corresponding to SEQ ID NO: 1. In some embodiments, the IL-15 moiety is a functional homolog having at least about 85% or at least about 90% identity to any one of SEQ ID NOs: 1-3. In some embodiments, the IL-15 moiety is a functional homolog having at least about 95%, 98% or 99% identity to any one of SEQ ID NOs: 1-3.

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

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

[0098] Truncated forms, hybrid mutants, and peptidomimetics of any of the foregoing sequences can also function as the IL-15 moiety. Any biologically active fragment, deletion mutant, substitution mutant, or addition mutant of any of the foregoing that maintains at least some degree of IL-15 activity can also function as the IL-15 moiety.

[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 are 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 having IL-15 activity, as a result, a signaling cascade is induced that includes phosphorylation of STAT5 at tyrosine residue 694 (Tyr694), which can be quantitatively measured. Assay protocols and kits are known and include, for example, the MSD Phospho(Tyr694) / Total STATa,b whole cell lysate kit (Meso Scale Diagnostics, LLC, Gaithersburg, MD); using this approach, for example, pSTAT5 EC of about 300 ng / mL or less (more preferably about 150 ng / mL or less) at at least one of the time points of 5 minutes or 10 minutes 50Candidate IL-15 moieties that exhibit a value are considered to be "IL-15 moieties" relevant to the present disclosure. However, the IL-15 moieties used are more potent (e.g., less than 150 ng / mL at at least one of 5 minutes or 10 minutes, e.g., less than about 1 ng / mL at at least one of 5 minutes or 10 minutes, even more preferably less than 0.5 ng / mL pSTAT5 EC 50 value).

[0100] Other methods known in the art, including potentiometry, spectrophotometry, chromatography, and radiometry, can also be used to evaluate 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 IL-15 moieties can also be used to measure the activity of the long-acting IL-15 R agonists described herein. See, e.g., the supporting examples provided herein.

[0102] A compound is considered a long-acting IL-15 R agonist according to the present disclosure as long as, after administration to a subject, the agonist exhibits IL-15 receptor activation in vivo for a longer time than in the case of administration of IL-15. For example, conventional techniques including radiolabeling the compound, administering the compound in vivo, and determining its clearance can be used to determine long-acting IL-15 It can be evaluated whether the 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 the purposes of the present invention, the long-acting nature of the long-acting IL-15 R agonist can be determined, or typically determined, by measuring STAT5 phosphorylation in lymphocytes at various time points after administering the agonist to mice using flow cytometry. By reference, the signal is lost by IL-15 in approximately 24 hours, but in the case of a long-acting IL-15 agonist, it is persistent beyond a certain period.

[0103] As described above, preferred long-acting IL-15 R agonists will generally include a single straight-chain PEG (polyethylene glycol) moiety stably covalently attached to the IL-15 amino group via an amide bond. A straight-chain unsubstituted alkylene group (~CH2~) m having 2 to 5 carbon atoms (i.e., m = 2, 3, 4, or 5) intervenes between the PEG moiety and the stable amide bond to the IL-15 amino group.

[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:

Chemical formula

Chemical formula

Chemical formula

[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 a molecular weight corresponding to any of the aforementioned ranges of n values 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) and above.

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

[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 portion is preferably end-capped with a methoxy group as shown above in formula (I), but the PEG portion may be end-capped with any lower C 1-6 alkoxy group or may terminate with a hydroxyl group or other suitable end-capping group.

[0113] In some embodiments, the long-acting IL-15 receptor agonist composition, taken together, has the following formula:

Chemical formula

[0114] In some additional embodiments, the long-acting IL-15 receptor agonist composition, taken together, has the following formula:

Chemical formula

[0115] In some specific embodiments related to the above, the long-acting IL-15 receptor agonist composition related to formula (Ia), taken together, has the following formula:

Chemical formula

[0116] In some other preferred embodiments, the long-acting IL-15 receptor agonist compositions related to formula (Ib), taken together, have the following formula:

Chemical formula

[0117] In some other embodiments, the long-acting IL-15 receptor agonist compositions related to formula (Ic), taken together, have the following formula:

Chemical formula

[0118] In some other embodiments, the long-acting IL-15 receptor agonist compositions related to formula (Id), taken together, have the following formula:

Chemical formula

[0119] In some embodiments, the long-acting IL-15 receptor agonist composition comprises about 0.1 to 20 mol% or less of the compound of formula (II) (including the compounds of formulae (IIa), (IIb), (IIc), and (IId)). In some additional embodiments, the composition comprises about 0.1 to 15, 0.1 to 10, 0.1 to 5, 0.1 to 1, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15, or 15 to 20 mol% or less of the compound of formula (II) (including the compounds of formulae (IIa), (IIb), (IIc), and (IId)). In some embodiments, the composition comprises about 0.1, 1, 5, 10, 15, or 20 mol% or less of the compound of formula (II) (including the compounds of formulae (IIa), (IIb), (IIc), and (IId)). It will be appreciated that the composition is purified by methods known in the art with respect to the compound of formula (I) such that the compound of formula (II) is not present, is present in trace amounts, or cannot substantially be present in the composition.

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

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

[0122] In some further embodiments, the composition is of formula (I),

Chemical formula

[0123] The composition of the long-acting IL-15 R agonist may include a single species in which n' is equal to about 1 and the PEG moiety is attached at the same position for substantially all IL-15 conjugates in the composition, or alternatively, the attachment of the linear polyethylene glycol moiety occurs at different sites on the interleukin-15 moiety, i.e., the specific binding site is not the same for all of the mono-PEGylated IL-15 species included in the composition, and may include a mixture of mono-PEGylated conjugate species). Thus, such a composition is substantially uniform from the perspective of the number of PEG moieties attached to IL-15 (e.g., 1-mer), but non-uniform from the perspective of the position of the amino group attachment on the IL-15 molecule.

[0124] Additional PEG constructs and coupling chemistries can be used to arrive at long-acting IL-15 R agonists, but as will become apparent when considering in view of the supporting examples, the compounds as described above are preferred in one or more embodiments. However, additional long-acting IL-15 R agonists having the structures provided herein are also envisioned.

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

[0126] As described above, the long-acting IL-15 receptor agonist can be in the form of a pharmaceutically acceptable salt. Typically, such salts are formed by reaction with a pharmaceutically acceptable acid or acid equivalent. In this context, the term "pharmaceutically acceptable salt" generally refers to relatively non-toxic inorganic and organic acid addition salts. These salts can be prepared in situ in the dosage vehicle or dosage form manufacturing process, or by separately reacting the 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, naphthylate, oxalate, mesylate, glucoheptonate, lactobionate, and lauryl sulfonate, among others. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19). Thus, the salts described are derived from inorganic salts such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric acids; or can be prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, and isothionic acids.

[0127] In some embodiments, the long-acting IL-15 receptor agonist composition, when considered collectively, contains about 1 to 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 contains about 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, or 5 mol% or less of free (i.e., non-conjugated) IL-15.

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

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

[0130] In some further embodiments of the present method, interleukin-15 is initially present in solution at a concentration of about 0.5 mg / mL to about 10 mg / mL, i.e., before 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, about 1.5 - 10 mg / mL, 1.5 - 5 mg / mL, about 1.5 - 4 mg / mL, about 1.5 - 3 mg / mL, about 1.5 - 2 mg / mL, about 2 - 10 mg / mL, about 2 - 5 mg / mL, about 2 - 4 mg / mL, about 2 - 3 mg / mL, about 3 - 10 mg / mL, about 3 - 5 mg / mL, about 3 - 4 mg / mL, about 4 - 10 mg / mL, about 4 - 5 mg / mL or about 5 - 10 mg / mL of interleukin-15 in solution. In some specific, but non-limiting embodiments, the concentration of interleukin-15 in 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 can 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 buffer.

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

[0133] After addition of the PEG reagent, the reaction mixture may then be adjusted to a suitable pH, such as about 7.0 to 8.5, or about 8.0, if necessary. In some embodiments, the reaction mixture is adjusted to a pH of about 7.0 to 8.0 or about 7.4 to 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 can 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 pHs, while lower pH levels can result in, for example, a lower degree of conjugation at the epsilon (ε) amine, and / or several possible drawbacks such as increased and / or undesirable positional isoforms, and protein aggregation. Deamidation introduces a negative charge into the protein, which can lead to changes in the activity, structure, function, stability of the protein, and / or change the susceptibility of the protein to degradation. Thus, one of the difficulties addressed by the present agonist and related methods was to provide a long-acting interleukin-15 receptor agonist that balances, among other considerations, at least (i) the desired degree of conjugation, (ii) a low amount of deamidation of the interleukin-15 moiety both before and after conjugation to the subject PEG reagent (which can lead to, for example, a decrease in interleukin-15 activity), and (iii) protein aggregation (e.g., both before and after conjugation), while maintaining sufficient activity (i.e., for being therapeutically useful).

[0135] Based on the competing and opposing difficulties related to the reaction parameters for preparing the long-acting interleukin-15 receptor agonist described herein, 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, an optimal (lower level) of deamidation can be achieved while still promoting the conjugation of the PEG moiety to the interleukin-15 moiety (e.g., at the ε amine and N-terminus), and the long-acting IL-15 R agonist described herein is provided, as discovered by the present applicants. Without being bound by theory, based on a series of reactions in which a number of 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, is effective in still promoting the conjugation of the PEG moiety to form the product described herein while providing a lower level of deamidation in the product and also appears to maintain the desired therapeutic profile.

[0136] For example, the methods described herein are effective for producing pegylated interleukin-15 that is less than about 35% deamidated, or in some embodiments, less than about 30% deamidated, or less than about 25% deamidated, or less than about 20% deamidated. In some embodiments, the level of deamidation of 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 with a degree of deamidation less than that described above is envisioned. As shown in Experiment 2 of Example 1, adjustment of the pH within the range of about 7.0 - 8.5 resulted in deamidation levels of 21.29% (Composition 1) or 33.26% (Composition 2).

[0137] The reaction participants are generally mixed for up to about 5 - 10 hours (including both ends). In some embodiments, the reaction participants are mixed for up to about 2 - 5 hours (including both ends). In some embodiments, the reaction participants are mixed for up to about 2 hours (including about 2 hours). In some exemplary embodiments, the reaction participants 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. The mixing is generally carried out under mild conditions, such as at about 20°C to about 65°C, or about 20°C to about 40°C, or at ambient temperature or room temperature (e.g., about 22°C). Lower temperatures are used to assist in achieving a lower degree of pegylation. The reaction can be quenched, for example, by the addition of an amino acid such as glycine.

[0138] In embodiments, the pH of the composition can be further adjusted to reduce deamidation. In some embodiments, the composition is adjusted to a pH of 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 can then generally be purified by any suitable method, such as ion exchange chromatography, to obtain the desired product. For example, anion exchange chromatography can be used. The chromatography product pool is then concentrated and diafiltered into a suitable formulation buffer (e.g., sodium acetate buffer containing sucrose) using, for example, tangential flow filtration (TFF). Analysis can be performed by any suitable method, such as SDS-PAGE, reverse phase HPLC, or any other suitable analytical method.

[0140] As described previously, the amino groups on the IL-15 moiety provide the site(s) of attachment between the IL-15 moiety and the polyethylene glycol moiety, providing a long-acting IL-15 R agonist encompassed by formula (I). For example, considering the exemplary IL-15 amino acid sequences provided herein, it is apparent that there are seven lysine residues each having an ε-amino acid that may be available for conjugation. Further, the N-terminal amine of methionine can also serve as a site of attachment to the PEG moiety. It will be appreciated that the polyethylene glycol moiety can attach 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 for Lys 11 and Lys 12 ). In some embodiments, the polyethylene glycol moiety attaches at the N-terminal amine. It will be appreciated that any of the lysine sites may be suitable as the site of attachment for the PEG moiety (e.g., Lys 37 or Lys 42 of SEQ ID NO: 1). In some embodiments, the long-acting interleukin-15 receptor agonist comprises a mixture of positional isomers in which the covalent attachment of the polyethylene glycol moiety is primarily at the N-terminus (i.e., among the set of positional isomers, the isomers having the PEG moiety attached at the N-terminus are present in the greatest amount when compared to the other positional isomers).

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

[0142] Suitable gel filtration columns for performing this type of separation include Superdex™ columns and Sephadex™ columns available from GE Healthcare (Buckinghamshire, UK). The choice of a particular column may depend on the desired fractionation range desired. Elution is generally performed using a suitable buffer such as phosphate, acetate, etc. The collected fractions can be analyzed by a variety of different methods such as, 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 test for PEG content (Sims et al. (1980) Anal. Biochem, 107:60-63), (iv) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) followed by staining with barium iodide, and (v) high performance liquid chromatography (HPLC).

[0143] The long-acting IL-15 R agonists of the present invention have been found to have certain remarkable and advantageous features. The features described below are generally considered to apply to the compounds provided herein and encompassed by formula (I), although one or more of the following features may be particularly exhibited by the compounds according to formula (Ib) and the extended compounds according to formula (IIb). The long-acting IL-15 R agonists can have one or more of the following features. For example, in some embodiments, the long-acting IL-15 receptor agonist shows a decrease of about 7-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5) compared to unmodified IL-15. For example, in one or more related embodiments, the long-acting IL-15 receptor agonist shows a decrease of about 6.5-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5), or a decrease of about 6-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5), or a decrease of about 5.5-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5), or a decrease of about 5-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5), or a decrease of about 4.5-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5), or a decrease of about 4-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5), or a decrease of about 3.5-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5), or even a decrease of about 3-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5) compared to IL-15. Exemplary long-acting IL-15 R agonists according to the foregoing features are described herein and in the accompanying examples. pSTAT5) shows a decrease of about 5.5-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5), or a decrease of about 5-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5), or a decrease of about 4.5-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5), or a decrease of about 4-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5), or a decrease of about 3.5-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5), or even a decrease of about 3-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5). Exemplary long-acting IL-15 R agonists according to the foregoing features are described herein and in the accompanying examples.

[0144] As described in Example 10, the in vitro activities of exemplary conjugates (1, 3, and 5) induced IL-15 signaling in huPBMC, and conjugate 1 strongly induced such signaling. Further experiments were conducted to examine the in vitro activity of conjugate 1 against human CD8 T cells, NK cells, and CD4 T cells (Examples 16, 22, and 26-27). As shown in FIGS. 10A and 10B, at least conjugate 1 induced similar or increased signaling in CD56bright and CD56low cells compared to IL-15. Although conjugate 1 was less potent than IL-15 in its engagement with CD8 and CD56 bright NK cells (Example 22), it is important to note that conjugate 1 achieved the same maximal response as conventional IL-15 (see FIGS. 38A and 38B). As described in Example 16 for the mouse model, single injection of conjugate 1 at two different doses induced persistent pSTAT signaling in CD8 and NK cells. As described in the mouse model of Example 26, single injection of conjugate 1 resulted in an increase in %pSTAT5 compared to IL-15. In the mouse model, NK cells were the most sensitive to single-dose conjugate, followed by CD8 T cells, and CD4 T cells were the least sensitive among the cells tested.

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

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

[0147] Preferably, the long-acting IL-15 receptor agonist shows a decrease of about 7-fold or less in the EC50 value (ng / mL, CTLL-2 pSTAT5) when compared to unmodified IL-15, and shows a decrease of about 50% or less in receptor α binding (K D , pM) when compared to IL-15 (including any one or more specific combinations of decreases in the EC50 value or K D value described above).

[0148] Optionally, the long-acting IL-15 receptor agonist is included in a composition comprising one or more pharmaceutically acceptable excipients. Exemplary excipients include, but are not limited to, those selected from the group consisting of carbohydrates, inorganic salts, antibacterial 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, may be present as excipients. Specific carbohydrate excipients include, for example: monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose; disaccharides such as lactose, sucrose, trehalose, cellobiose; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myo-inositol, cyclodextrin.

[0150] Excipients may also include inorganic salts or buffers such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, monobasic sodium phosphate, dibasic sodium phosphate, and combinations thereof.

[0151] The composition may also include an antibacterial agent to prevent or inhibit the growth of microorganisms. Non-limiting examples of antibacterial 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 similarly be present in the composition. The use of antioxidants prevents oxidation, thereby preventing the degradation of conjugates 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] Surfactants may be present as excipients. Exemplary surfactants include polysorbates such as "Tween 20" and "Tween 80", and pluronics such as F68 and F88 (both available from BASF, Mount Olive, New Jersey); sorbitan esters; phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamine (preferably not in liposomal form), fatty acids, and lipids such as fatty acid esters; steroids such as cholesterol; and IL-15 chelating agents such as EDTA, zinc, and other such suitable cations.

[0154] An acid or a base may be present as an excipient in the composition. 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, 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 bases selected from the group consisting of combinations thereof.

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

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

[0157] The long-acting IL-15 R agonist is suitable for administration to patients suffering from a condition responsive to treatment with interleukin-15. The method generally comprises parenterally administering to a patient a therapeutically effective amount of a long-acting IL-15 R agonist (preferably provided as part of a pharmaceutical composition). As described above, the long-acting IL-15 R agonist can be administered parenterally (e.g., intramuscularly, subcutaneously, intravenously, or intraperitoneally). Suitable formulation types for parenteral administration include, among others, ready-to-inject solutions, dry powders to be combined with a solvent before use, ready-to-inject suspensions, dry insoluble compositions to be combined with a vehicle before use, and emulsions and liquid concentrates to be diluted before administration. In some particular 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 method of administration of a long-acting IL-15 receptor agonist (e.g., provided as part of a pharmaceutical composition) can optionally be performed so as to be localized within a particular area. For example, liquid, gel, and solid formulations containing the agonist can also be surgically implanted within the diseased area (e.g., within a tumor, near a tumor, within the area of inflammation, and near the area of inflammation, etc.). It is also convenient for organs and tissues to be able to be imaged so as to ensure that the desired location is well-exposed by the conjugate.

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

[0160] As used herein in connection with the treatment of a subject having cancer, the terms "treatment", "treating", and "being treated" are intended to include all areas of intervention in the cancer that the subject has, e.g., administration of combinations, to reduce, delay, stop, or reverse one or more symptoms of cancer, or to delay the progression of cancer even if the cancer has not actually been removed. Treatment can include, for example, a decrease in the severity of symptoms, the number of symptoms, or the frequency of recurrence, e.g., inhibition of tumor growth, suppression of tumor growth, or regression of an existing tumor.

[0161] For example, improvement of cancer or cancer-related diseases can be characterized as a complete or partial response. "Complete response" refers to the absence of clinically detectable disease, with normalization of any previous abnormal x-ray test, bone marrow, and cerebrospinal fluid (CSF) or abnormal monoclonal protein measurements. "Partial response" refers to at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% decrease 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 a physiological state in a mammal 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 precancerous and cancerous cells and tissues.

[0164] Exemplary conditions are, 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 tumor, 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 cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma (including, for example, uveal melanoma, mucosal melanoma, and leptomeningeal melanoma), neuroblastoma, retinoblastoma, and leukemia and other cancers.

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

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

[0167] In the exemplary mouse model described in Example 11, Conjugate 1 was effective in inducing an increase in NK cell proliferation and persistence of numbers, as indicated by an increase in cell number (cells / μ, as shown in FIGS. 12B and 12C) and an increase in %Ki67 (e.g., FIG. 11A). %Ki67 is used as a marker for proliferating cells. As is clear from FIGS. 12A - 12D, the increase in cell number was apparent 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 medium 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 medium dose range (e.g., 0.1 mg / kg and 0.3 mg / kg) induced an increase in %Ki67, which persisted for at least 144 hours.

[0168] The effect of Conjugate 1 is induced by a single administration and can be persistent. In the exemplary wood mouse model described in Example 11, the increase in cell number was induced by administration of a single dose of Conjugate 1 and was persistent. A single administration induced %Ki67 levels of persistence equivalent to that of repeated (e.g., Q7dx3) administrations at the same level in wood mouse CD49b cells (see FIG. 28A).

[0169] In some further embodiments, the long-acting IL-15 R agonist or composition is effective to increase NK cell activation, as demonstrated by an increase in cytotoxic protease expression by NK cells when administered at a therapeutically effective dose. In the exemplary non-human primate model described in Example 28, the expression of the NK cell lytic enzymes granzyme B and perforin was induced and enhanced by a single administration of conjugate 1. As is apparent from FIGS. 48A-48C, all dose levels increased granzyme B expression compared to pre-administration levels, and the medium / higher doses increased the expression (MFI) by at least 3-fold. As is apparent from FIGS. 49A-49C, all dose levels increased perforin expression compared to pre-administration levels, and the medium / higher doses doubled the expression (MFI). Thus, conjugate 1 is effective to increase the cytotoxicity of NK cells.

[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, single administration of conjugate 1 at all dose levels increases cell proliferation, as shown by an increase in % Ki-67 positivity in CD8 cells (Figure 30D) and in the memory and effector memory CD8 subpopulations (Figures 30E - 30F). As shown in Example 17, administration of conjugate 1 in the wood 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 wood mouse model, a single iv injection of conjugate 1 maintained an increase in cell number for at least 240 hours compared to vehicle administration (e.g., see Figure 30A). Notably, the CD8 and CD8 memory T cell numbers did not return to baseline at 240 hours post - 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. Single administration of conjugate 1 at all dose levels also increased Ki-67 positivity in all CD8 and CD8 subpopulations, indicating increased proliferation of these cells. Repeated administration with conjugate 1 further resulted in an increase in the 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 for each of the CD8, CD8 Tcm, and CD8 Tem populations in mice.

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

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

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

[0175] The actual dosage administered will vary according to the age, body 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. A therapeutically effective amount is known to those of ordinary skill in the art and / or is described in relevant reference books and literature. Generally, a therapeutically effective amount can 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 dosage may be administered periodically until, for example, a clinician determines that an appropriate endpoint (e.g., cure, regression, partial regression, etc.) has been reached.

[0176] In some embodiments, a therapeutically effective dosage ranges from about 0.25 to 25 mcg / kg. In another embodiment, a therapeutically effective dosage ranges from about 0.25 mcg / kg to about 0.1 mg / kg per day, from about 0.01 mg / kg to about 0.1 mg / kg, or from about 0.03 mg / kg to about 0.1 mg / kg per day. In another embodiment, a therapeutically effective dosage ranges from about 1 to 10 mcg / kg, from about 0.03 mg / kg to about 0.1 mg / kg. In some specific but non-limiting embodiments, a therapeutically effective dosage 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. By referring to the dosages mentioned in the examples herein, one of ordinary skill in the art can convert animal dosages (e.g., in mice) to corresponding human dosages using conversions known in the art (e.g., Nair et al., J. Basic and Clin. Pharmacy (2016) 7:27 - 31).

[0177] Any given conjugate of the unit dosage (which is also preferably provided as part of a pharmaceutical preparation) can be administered according to various dosing schedules depending on the judgment of the clinician, the needs of the patient, etc. Specific dosing schedules are well known to those skilled in the art or can be determined experimentally using routine methods. Exemplary dosing schedules include, without limitation, once daily, three times a week, twice a week, once a week, twice a month (e.g., q / 14 days), once a month (e.g., q / 30 or 31 days or q / 21 days), and administration in any combination thereof. When the desired clinical endpoint is achieved, dosing of the composition is discontinued or reduced. In some embodiments, any given conjugate of the unit dosage can be administered once to achieve a sustained effect.

[0178] Although the present invention has been described in connection with its preferred specific embodiments, 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 the present disclosure will be apparent to those skilled in the art to which the present invention pertains.

[0179] All papers, works, patents, and other publications referenced herein are hereby incorporated by reference in their entirety into this specification. In the event of any conflict between the teachings of this specification and the technology incorporated by reference, the teachings and definitions in this specification shall prevail (especially with respect to the terms used in the claims appended hereto). For example, if the present application and the publications incorporated by reference define the same term differently, the definition of the term shall be maintained within the scope of the teachings of the document in which the definition is located.

Examples

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

[0181] In the following examples, efforts were made to ensure the accuracy regarding the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations have to be taken into account. Unless otherwise indicated, the temperature is in degrees Celsius and the pressure is at or near sea level. Each of the following examples is considered to be a teaching for one or more of the embodiments described herein for those skilled in the art to practice.

[0182] Materials and Methods Recombinant IL-15 (''rIL-15'') having SEQ ID NO: 1 (provided in Figure 1) prepared using conventional techniques was used in the following examples, but any suitable IL-15 moiety can be used as well. SEQ ID NO: 1, recombinant human IL-15 derived from Escherichia coli (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 polymer reagent, linear mPEG-succinimidyl butanoate, 40 kDa (''mPEG-SBA'') has the following structure. [Chemical formula] Wherein n corresponds to the number of monomer 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 those having a weight average molecular weight of, for example, about 10 kD, 15 kD, 20 kD, 25 kD, 30 kD, 40 kD, 50 kD or 60 kD. This activated polymer reagent is effective in forming a stable amide bond between the IL-15 moiety and the polyethylene glycol moiety when reacted with the amino group (e.g., lysine or N-terminus) of IL-15.

[0184] The reactive fluorenyl-PEG reagent, PEG2-CAC-FMOC-20kD-NHS has the following structure: [Chemical formula] In the formula, mPEG is methoxy(polyethylene glycol), and the weight average molecular weight of the polymer reagent is about 20 kilodaltons (i.e., it has each mPEG moiety with a weight average molecular weight of about 10 kilodaltons). Additional PEG2-CAC-FMOC reagents having different molecular weights are accordingly named, for example, PEG2-CAC-FMOC-10kD-NHS, PEG2-CAC-FMOC-15kD-NHS, PEG2-CAC-FMOC-30kD-NHS, PEG2-CAC-FMOC-40kD-NHS, and these reagents have the structures shown above, differing only in the molecular weight of the "mPEG" moiety that binds 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. Next, the prepared samples were loaded onto a NuPAGE Novex precast gel and electrophoresed for about 30 minutes.

[0186] RP-HPLC analysis Reverse-phase chromatography (RP HPLC) analysis was performed on an Agilent 1200 HPLC system (Agilent). Samples were analyzed at 60 °C using a Poroshell 300SB-C3 column (2.1x75 mM, Agilent). The mobile phases used were 0.1% TFA / H2O (A) and 0.1% TFA / CH3CN (B). The flow rate of the column 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 after receptor binding, downstream cell signaling can then activate the 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, by a ~10-minute response to samples and reference treatments using a phosphoSTAT5 / total STAT5 multiplex assay (Meso Scale Discovery, MD).

[0188] One day prior to the assay, CTLL-2 cells were aliquoted into fresh growth medium [RPMI 1640 supplemented with 10% FBS, 10% T cell culture supplement (catalog number 354115, Corning, Inc., Tewksbury, MA), 2 mM L-glutamate, and 1 mM sodium pyruvate]. On the day of the assay, the 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. Immediately prior to the assay, dilutions of the test substance were prepared in the appropriate buffer. Stimulation of CTLL-2 cells was initiated by transferring a 25-fold dilution of the test substance solution to triplicate wells containing CTLL-2 cells. The plates were incubated at 37 °C, 5% CO2 for 10 minutes, and the reaction was stopped by cell lysis. Detection of phospho-STAT5 and total STAT5 protein levels in cell lysates was performed using the MSD Phospho(Tyr694) / Total STATa,b Whole Cell Lysate Kit (Meso Scale Discovery, MD). After 10 minutes of treatment, recombinant human IL-15 obtained from PeproTech was used as a control to demonstrate IL-15 activity by inducing STAT5 phosphorylation in CTLL-2 cells at an average EC 50 of 0.27 ng / mL.

[0189] HuPBMC-pStat5 assay The potency of IL-15 or a long-acting IL-15 R agonist against various human lymphocyte subpopulations was determined by a phospho-STAT5 (Y694) dose-response assay. Cryopreserved human PBMC from multiple donors were supplied by AllCells. 1x10 6 cells / 100ul were cultured in complete RPMI medium for 2 hours and then incubated with IL-15 or conjugate at the indicated concentrations (serial dilution from 10,000 ng / ml to 0.001 ng / ml) for 20 minutes at 37°C. The cells were then fixed (using BD Cytofix), permeabilized (using 100% pre-cooled methanol), stained with antibodies against CD3, CD4, CD8, CD4-Tregs (CD4+CD25+Foxp3+), CD56 and phosphorylated STAT5 (Y694), and analyzed by flow cytometry. The concentration-response relationship was used to calculate the EC 50 value.

[0190] Receptor affinity of a long-acting rIL-15 receptor agonist 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 active NHS esters using a 1:1 NHS:EDC mixture. It was covalently bound to the surface by injecting goat anti-human Fc antibody in 10 mM sodium acetate (pH 4) for 5 minutes. Approximately 8000 RU of antibody was bound to the surface. The remaining NHS esters were then quenched with ethanolamine.

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

[0192] The long-acting rIL-15 receptor agonist test article was in PBS (0.05% Tween It was diluted to 10 μM in (containing 20 and 0.1 mg / ml BSA). A series of three-fold dilutions were made and injected onto a sensor chip coated with IL-15Rα. k a and k d The affinity was measured by separately measuring the k d and k a The k d value was calculated using the ratio of.

[0193] Example 1 Preparation of a long-acting IL-15 receptor agonist

Chemical formula

[0194] Figure 2 shows the chromatogram after RP-HPLC analysis of the conjugation reaction mixture. The reaction provided 40% mono-conjugate (i.e., having a single PEG moiety attached to IL-15), 24% di-conjugate (having two PEGs attached to IL-15), and 6% tri-conjugate (having three PEGs attached to IL-15) species. The reaction conditions were not optimized, and approximately 30% of the 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 elution phase. The purified mono-mPEG-SBA40K-IL-15 conjugate (also referred to herein as mono-mPEG-butanamide-40K-IL-15 or mono-(methoxy PEG-N-butanamide) 40kD Interleukin-15, or mono-mPEG 40K -C4-amide-IL-15) was characterized by HPLC and SDS-PAGE. In other examples, the 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 does not contain unreacted IL-15. Figure 5 shows an RP-HPLC analysis of the purified mono-mPEG-SBA-40K-IL-15. As can be apparent from the HPLC results, the purified mono-mPEG-SBA-40K-IL-15 composition contains less than about 10% (molar amount) of di- or higher level conjugates.

[0197] Using the same approach, conjugates such as mono-mPEG-SBA-10K-IL-15, mono-mPEG-SBA-15K-IL-15, mono-mPEG-SBA-20K-IL-15, mono-mPEG-SBA-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 are prepared using mPEG SBA with different weight average molecular weights.

[0198] Experiment 2: IL-15 in a solution of approximately 2 mg / ml in buffer (50 mM sodium phosphate, 100 mM sodium chloride, 10% sucrose, pH 7.4) was transferred to each of two different reaction vessels (referred to herein as Composition 1 and Composition 2). To adjust the pH to 8.0, boric acid buffer at pH 8 (0.4 M or 0.6 M) was added. A 10-fold excess (relative to the molar amount of IL-15) of mPEG SBA-40K (mPEG SBA, 40 kDa) diluted in 2 mM HCl was added to each of the IL-15 solutions and mixed well. After the addition of mPEG SBA-40K, the pH of the reaction mixture was determined to be pH 8 or adjusted by using additional boric acid buffer if necessary. The final concentration of IL-15 in the reaction was targeted at 1 g / L and additional diluent (buffer containing 50 mM sodium phosphate, 100 mM sodium chloride, 10% sucrose, pH 7.4 was used for Composition 1 and water was used for Composition 2) was used if necessary. To couple mPEG SBA-40K to IL-15 (i.e., via the formation of amide bonds that are preferentially stable), the reaction solution was mixed for 45 or 60 minutes at room temperature for each of Composition 1 or Composition 2 to promote 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] [[ID=,21]] [Table 2]

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

[0204]

Table 3

[0205] The prepared composition predominantly contained the mPEG SBA-40K monopegylated species, contained less than 10% of the PEG dimer (i.e., two PEG moieties are bound to IL-15), and even a smaller amount of higher-order PEG species (i.e., three or more PEG moieties are bound to IL-15).

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

[0207] Example 2 Preparation of a long-acting IL-15 receptor agonist.

Chemical formula

[0208] The reaction mixture was analyzed by RP-HPLC. By SDS-PAGE, 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 mainly contained 2 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 elution phase to afford purified [mPEG2-CAC-FMOC-20kD-NHS]-IL-15 having an average degree of pegylation of about 2 (having a degree of pegylation in the range of about 1.7 - 2.5), and thus n' of the above structure for the purified composition was about 2.

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

[0210] Example 3 Preparation of a long-acting IL-15 receptor agonist, [mono-PEG2-RU-ButryALD-40K]-IL15 [Chemical formula] Branched mPEG-butylaldehyde PEG reagent, mono-PEG2-RU-ButryALD-40K [Chemical formula] Using [the above reagent], a long-acting IL-15 R agonist for the subject was prepared, and the weight average molecular weight of the PEG reagent used in the preparation of the agonist was about 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. 15-fold excess (relative to the amount of IL-15) of mPEG2-ru-ButyrALD was dissolved in MilliQ H2O to form a 10% reagent solution. The 10% reagent solution was rapidly added to the IL-15 solution and mixed well. And it was set on a RotoMixer for 15 minutes. Then, 1 / 100 volume of 1 M NaCNBH3 / H2O was added to the reaction mixture. To enable the coupling of mPEG2-ru-ButyrALD to IL-15 via secondary amine bonds, the reaction solution was placed on a RotoMixer at 4 °C for 17 hours and then quenched with glycine solution. Since the pegylation reaction was carried out at acidic pH, the binding of the PEG derivative to IL-15 was more selective for the N-terminus. To purify the conjugate, an anion exchange chromatography method using a Q Sepharose high performance column and sodium phosphate buffer was also developed. The purified mono-PEG2-ru-ButyrALD-40K-IL-15 conjugate was characterized by HPLC and SDS-PAGE.

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

[0213] Using the same procedure, conjugates were prepared using PEG2-RU-ButryALD- having 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 a long-acting IL-15 receptor agonist Mono-mPEG-ButyrALD-40K-IL-15

Chem.

Chem.

[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 H2O to form a 10% reagent solution. The 10% reagent solution was rapidly added to the IL-15 solution and mixed well. And set on a RotoMixer for 15 minutes. Then, 1 / 100 volume of 1 M NaCNBH3 / H2O was added to the reaction mixture. To enable the 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 hours and then quenched with a glycine solution. Since the pegylation reaction was carried out at acidic pH, the binding 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 Receptor bias evaluation of a long-acting IL-15 receptor agonist for 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] The test article was diluted to 10 μM in PBS (containing 0.05% Tween 20 and 0.1 mg / ml BSA). A series of 3-fold dilutions were made and injected onto a sensor chip coated with IL-15Rα. k a and k d rates were determined separately to measure affinity, and the ratio of k d to k a was used to calculate the k d value.

[0219] Preferred conjugates generally retain as much IL-15 Rα affinity as possible after pegylation compared to unmodified IL-15. Conversely, preferred conjugates generally have a minimal loss of affinity for IL-15 Rα compared to that of unmodified IL-15.

[0220] For example, in some embodiments, preferred conjugates exhibit a decrease of about 7-fold or less in EC50 value (ng / mL, CTLL-2 pSTAT5) and a decrease of about 50% or less in receptor α binding (K D , pM) compared to IL-15. For example, Conjugate 1 has a decrease in potency of about 2-fold and retains about 80% of the receptor affinity of IL-15 when compared to IL-15.

[0221] [Table 4]

[0222] As shown in the above table, conjugate 1 retains its high affinity for the IL-15 α receptor (i.e., when compared to IL-15), which is a particularly favorable characteristic for long-acting IL-15 receptor agonists. Affinity constants (K D )(in units of pM) for additional conjugates are provided below.

[0223]

Table 5

[0224] Example 6 In Vivo Study: 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 after administration (24 hours, 48 hours, 78 hours, 96 hours). The samples were pooled and evaluated by flow cytometry for pharmacodynamic analysis of the drug effect on lymphocyte cell populations and expressed as fold change relative to vehicle control (results are described in subsequent examples below). In addition to changes in cell numbers, functional markers and markers of activation were quantified. Finally, the plasma concentration of the drug was determined at each time point. See Figure 6.

[0225] As shown in Figure 6, conjugate 2 maintained measurable concentrations in plasma over an extended period, for example, exceeding 1 week (filled squares), in contrast to the rapid decline in plasma levels observed after administration of non-long-acting IL-15 (filled circles), and T 1 / 2 was approximately 20 - 30 hours.

[0226] Example 7 In Vivo Study: 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 to 7 after administration (multiple samples were collected within the first 24 hours after administration). 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 study in mice Mice were administered as described in Example 6 above, and in vivo signaling was evaluated by the degree of STAT5 phosphorylation. The degree of STAT5 phosphorylation in various lymphocytes (CD4, CD8, and NK cells) was evaluated by measuring, by flow cytometry, after staining whole blood with 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 is clear from Figure 8A, in vivo signaling activity is extremely short-lived in the case of IL-15, but exemplary conjugate 2 induces sustained STAT5 phosphorylation, most notably in NK cells (solid inverted triangle ▼) and also in CD8 cells (solid triangle ▲), and the measurable STAT5 phosphorylation activity shown in NK and CD8 cells exceeded 72 hours. STAT5 phosphorylation activity for CD4 cells is also shown (solid square ■).

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

[0231] As shown in Figures 9A - 9C, the results were similar to those observed in mice (Example 7), but in non-human primates, STAT5 phosphorylation was also observed in CD4 cells (Figure 9A). STAT5 phosphorylation in each of the three cell types substantially increased after administration, reached maximum levels at approximately 3 and 4 days after administration of the long-acting IL-15 agonist shown, and returned to approximately day -1 levels (i.e., pre-administration) by approximately days 5 - 10. Similar to Example 7, these results indicate the presence of persistent 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 (PBMC) 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 evaluated by studying signal transduction in the NK cell subset of human PBMC. STAT5 phosphorylation was evaluated as described above to assess the IL-15 signal transduction activity of the long-acting IL-15 receptor agonists.

[0233]

Table 6

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

[0235] As can be understood, each of the exemplary conjugates induces IL-15 signaling in huPBMC, and conjugate 1 induces such signaling strongly. Among the conjugates tested (not all data shown), conjugate 1 showed the greatest potency / activity against huPBMC. The data indicate that different PEG constructs and linkers can elicit very different effects on the biological activity in the resulting conjugates, even when maintaining the same degree of pegylation (i.e., the number of PEG moieties) per IL-15 protein and the same size of the PEG moieties.

[0236] A second study was conducted to examine / compare the pStat5 responses of human PBMC (CD3, CD4, CD8, CD56 (bright and dim), and CD4-Tregs (CD25+Foxp3+)) obtained from two donors to IL-15, conjugate 1, and conjugate 5. An 11-point dose response was examined with 20-minute stimulation using 10-fold dilutions in the dose range of 0.001 - 10000 ng / ml. Each of the test articles was diluted in IL-15 buffer + 0.1% BSA. The results are provided in the following table.

[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 with respect to both CD3 and CD4 induction; the potencies of conjugate 1 and conjugate 5 appear to be similar with respect to CD3 and CD4 induction.

[0239] [Table 8]

[0240] Based on the data in Table 5 above, IL-15 appears to be approximately 3 to 5 times more potent than conjugate 1 with respect to Treg and CD8 induction; the potencies of conjugate 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 than conjugate 1 with respect to CD56 induction. However, conjugate 1 appears to be more potent than conjugate 5 in the induction of CD56 bright and CD56dim.

[0243] Based on previous data, IL-15, conjugate 1, and conjugate 5 show similar pSTAT5 induction for all cell populations, and the maximum response appears to be higher for CD56bright and Treg cells.

[0244]

Table 10

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

[0246] Example 11 In vivo study: Single-dose PD study 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 (Figure 11, low dose), 0.3 mg / kg (Figure 11, medium dose), or 1 mg / kg (Figure 11, high dose). After administration, blood samples were collected at various time points after administration (24 hours, 48 hours, 78 hours, 96 hours, 120 hours). Samples from each mouse were subjected to pharmacodynamic analysis of the drug effect on lymphocyte cell populations by flow cytometry. In addition to changes in cell numbers, functional markers and markers of activation were examined.

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

[0248] Results showing the proliferation of NK cells in mice administered each of conjugate 1 at 0.03 mg / kg (Figure 11, low dose), 0.3 mg / kg (Figure 11, medium dose), and 1 mg / kg (Figure 11, high dose) are provided in Figures 11A and 11B.

[0249] NK cells and their proliferation were defined using the CD45+CD3−CD49b+ and CD45+CD3−CD49b+Ki67+ marker combinations. After administration, blood samples were obtained on a Fortessa flow cytometer running FACS DIVA software. Flowjo software was used for analysis, and NK cell absolute values and the percentage of Ki67-positive within NK cells were plotted using Prism.

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

[0251] The effects of an exemplary long-acting IL-15 receptor agonist were studied with respect to NK cells at all stages of maturity. The peripheral NK cell pool can be characterized by the expression of CD27, and CD27 lo / - NK cells are more cytotoxic, and CD27 high NK cells produce more cytokines than CD27 - NK cells (Hayakawa Y, et al., J Immunol. 2006;176:1517-1524). The mature peripheral NK cell population was further purified into four maturation stages defined by the continuous upregulation of CD11b expression followed by the downregulation of CD27, with the most immature NK cells being CD27 - CD11b - and the most mature NK cells being CD27 - D11b

[0252] In mice, the four different maturation states of NK cells are defined by CD27 and CD11b expression. Once NK marker (CD49b+), natural activating NK receptor (NKp46+), and IL-15 / IL-2RB (CD122+) triple-positive cells 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] As described above, mice were administered a single dose of conjugate 1 at 0.01, 0.03, 0.1, 0.3, 1.0, and 1.5 mg / kg, or NK cells in various maturation states were quantified in mice after the third dose on a q7dx3 schedule. Using flow cytometry, the NK population of interest was identified as positive for CD49b, NKp46, and CD122. The NK population was then further differentiated into immature (CD11b−CD27−), early NK (CD11b−CD27+), high effector (CD11b+CD27+), and terminal effector (CD11b+CD27−) subpopulations using CD11b and CD27. Peripheral blood was run through 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 the data were plotted in Prism.

[0254] The results are shown in FIGS. 12A-D. Additional results are shown in FIGS. 22A-D. The q7dx3 results are shown in FIGS. 28A-D. As can be understood from the plots, conjugate 1 was effective in increasing the number of NK cells at all maturation levels (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, and the effect persisted for at least 120 hours.

[0255] The surface expression of NKG2D was performed using flow cytometry analysis of anti-NKG2D signals and represented as the mean fluorescence intensity (MFI) in NK cells. Similarly, the level of intracellular granzyme B was determined using flow cytometry-based detection of anti-granzyme B signals, which was also represented as MFI in NK cells. After detecting NKG2D and granzyme B signals using a Fortessa flow cytometer and FACS Diva software, the analysis was carried out using Flowjo software. The 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 the ability to achieve a sustained increase in both NKG2D and granzyme B (apoptosis-promoting serine protease) by NK cells, most notably at medium and high doses, compared to the vehicle. 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 the analysis was performed using Flowjo software. The absolute CD8 cell numbers were plotted in Prism as shown in Figure 14 (blood) and Figure 24 (spleen). Figures 14 and 24 show the ability of conjugate 1 to induce the proliferation of CD8 T cells and an increase in the persistence of CD8 T cell numbers after a single i.v. administration in mice at each of the above-mentioned doses. This effect was most prominent for medium (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+CD62L− and CD45+CD3+CD4−CD8+CD44+CD62L+, respectively. Proliferation of these memory populations was determined using Ki-67 positivity. After single administration of conjugate 1 or IL-15, blood and spleens were subjected to immunophenotyping using a Fortessa flow cytometer, DIVA acquisition software, and Flowjo analysis software. Graphs were plotted in Prism. As shown in FIGS. 15A and 15B for blood, conjugate 1 induced a dose-dependent increase in both effector and central memory CD8 T cells, whereas single-dose IL-15 did not. As shown in FIGS. 25 and 26 for spleen, conjugate 1 induced a dose-dependent increase in both Ki67 and granzyme B, whereas single-dose IL-15 did not. Both effector and central memory populations proliferated in response to administration of conjugate 1, an exemplary long-acting IL-15 agonist.

[0258] Example 12 In Vivo Study: Single-Dose PD Study in Non-Human Primates In this study, cynomolgus monkeys, one female and one male, were administered 500 μg / kg of conjugate 2 intravenously. A series of blood samples were collected from each animal at multiple intervals before treatment (−6 and −1 days) and after single-dose treatment for evaluation by flow cytometry of lymphocyte cell counts (NK cells, CD8 T cells, etc.) and activation.

[0259] The number of NK cells was determined to evaluate the ability of exemplary conjugate 2 to induce persistent NK cell proliferation in non-human primates; the results are shown in FIGS. 16A and 16B. NK cells and their proliferation in blood from cynomolgus monkeys were identified by flow cytometry. Acquisition and analysis of NK cells (CD45+CD3−CD16+) and their proliferative status (CD45+CD3−CD16+Ki67+) were performed using BD FACS DIVA software. The % Ki67 positivity in the NK population was calculated using the absolute values for NK cells and proliferating NK cells. Values before and after treatment were plotted using Prism.

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

[0261] As shown in FIG. 17, the number of CD8 T cells for each animal from pre-dose to day 14 post-dose was also determined. 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 increased persistently and the effect lasted for at least 10 days. This plot further exemplifies the ability of exemplary long-acting IL-15 receptor agonist, conjugate 2, to induce proliferation and sustained increase in the number of CD8 T cells post-administration.

[0262] In monkeys, CD8 T EM cells were defined as CD45+CD3+CD4−CD8+CD45Ra−CD197−, and CD8 T CM was defined as CD45+CD3+CD4−CD8+CD45Ra−CD197+. Immunophenotyping by flow cytometry was performed, samples were acquired on DIVA software, and data were analyzed on Flowjo software. Graphs were plotted using Prism. As shown in FIGS. 18A and B, respectively, CD8 T effector memory cells (T EM cells) and CD8 T central memory cells (TCM ) The number was determined. Conjugate 2 induces a significant and persistent increase in CD8 effector and central memory T cell populations in cynomolgus monkeys. The figure shows that both CD8 effector and central memory T cell populations proliferate in response to exemplary Conjugate 2.

[0263] Example 13 Evaluation of the Antitumor Activity of a CT26-Induced Subcutaneous Lung Metastasis Tumor Model in BALB / C Mice On day 0, female Balb / c mice, 6 - 8 weeks old, were inoculated with 1×10 5 mouse CT-26 cells by 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 the administration of Conjugate 1 and Conjugate 2, but the study protocols were essentially the same for both studies). Each group was assigned one of the following interventions: 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). The animals were administered on days 1, 5, and 10.

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

[0265] The number of pulmonary tumor nodules was counted under incision for each lung, and the mean of pulmonary nodules for each group was determined. Statistical significance between the vehicle group and the intervention group could also be obtained using the independent Student's t-test.

[0266] The pulmonary metastasis results for the treatment groups corresponding to conjugate 2 and conjugate 1 respectively are shown in FIGS. 19 and 20. Both exemplary long-acting IL-15 receptor agonists were effective in promoting a decrease in pulmonary metastasis. However, conjugate 2 provided a 65% decrease in metastasis compared to the vehicle, while conjugate 1 provided an 85% decrease in metastasis.

[0267] On day 13, blood and spleen cells were analyzed for changes in immunophenotypic markers using flow cytometry and marker antibodies conjugated with various fluorescent dyes. Conjugate 2 administered at 0.3, 1, and 3 mg / kg induced a dose-dependent increase in CD8 T cells in the blood, 1.5, 2.5, and 3.3 times that of the vehicle, respectively. Similar observations were obtained in the spleen, with increases of 1.3, 1.7, and 2.2 times that of the vehicle at the 0.3, 1, and 3 mg / kg dose levels. Ki-67 immunophenotyping revealed a 1.7, 4.6, and 5.3-fold change in CD8 T cell proliferation in the blood and a significant dose-dependent increase in the spleen with 2.5, 5.7, and 6.9-fold changes at the same low, medium, and high dose levels compared to the vehicle. In addition, conjugate 2 treatment increased the survival-promoting Bcl-2+ MFI in CD8 in both blood and spleen by 1.5-fold.

[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, the isolated NK cells were stimulated overnight at 37 °C, 5% CO2 in a humidified incubator with conjugate 1 at concentrations of 3000, 1000, 300, 30, 3, or 0 (unstimulated) ng / mL before use in the cytotoxicity assay. For in vivo studies, mice were administered 0.3 mg / kg of conjugate 1, and spleen NK cells were isolated 24, 48, and 72 hours after administration and used directly in the cytotoxicity assay.

[0270] YAC-1 T cells labeled with PKH26 were used as target cells. To monitor the cytotoxicity of NK cells, 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. The cells were immediately analyzed by flow cytometry. Lysed target cells were identified as PKH26 + 7-AAD + as.

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

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

[0273] This data shows a dose-dependent increase in the cytotoxicity of NK cells in vitro and in vivo after 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 at 24 - 240 hours post - administration for immunophenotyping. After lysing red blood cells, white blood cells were labeled with a viability dye and markers specific for CD45, CD3, and CD49b to identify viable NK cells. The cells were then fixed simultaneously and permeabilized for intracellular granzyme B staining. The stained blood was run through a Fortessa flow cytometer, acquired by DIVA software, and analyzed using Flowjo software. Data are presented as the percentage of NK cells that were positive for granzyme B expression.

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

[0276] Example 16 In Vivo Study: Single - Dose IL - 15 and Conjugate 1 PK and JAK / STAT Signaling Studies in Mice For PK analysis, conjugate 1 was administered as a single intravenous dose of 0.3 mg / kg to balb / c mice (n = 3). After administration, the mice were humanely sacrificed and plasma was collected at 24, 48, 72, 96, 120, and 144 hours post-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. [The PK method is described previously herein]. For pharmacodynamic studies, balb / c mice (n = / group) received an i.v. injection of 0.03 or 0.3 mg / kg of conjugate 1 or vehicle (50 mM sodium phosphate, 100 mM sodium chloride, 10% sucrose, pH 7.4), and blood was collected before dosing and at 15 minutes, 1, 24, 48, 72, 96, and 120 hours post-treatment. Samples were analyzed individually by flow cytometry and expressed as the percent positive pSTAT5 within CD8 and NK cells.

[0277] Figure 29A is a plot of the plasma concentration of the test article (IL-15 or conjugate 1) over time up to 144 hours after administration of a single intravenous dose of 0.5 and 0.3 mg / kg of the test article in balb / c mice, respectively.

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

[0279] Figure 29B is a graph of the percent positive pSTAT5 within 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 persistent pSTAT5 signaling in CD8 T cells at both dose levels, shown over a 120-hour time course including pre-dose.

[0281] Figure 29C is a graph of the percent positive pSTAT5 within C57BL / 6 NK cells after a single injection of 0.03 and 0.3 mg / kg of conjugate 1.

[0282] Result: Conjugate 1 induces strong and persistent pSTAT5 signaling in NK cells at both dose levels.

[0283] Example 17 In Vivo Pharmacodynamic Studies of Single Dose and Q7DX3 in Mice - Cell Numbers and Proliferation Balb / c mice (n = 3 / group) were administered a single dose or three weekly doses of Conjugate 1 or vehicle at 0.01, 0.03, 0.1, 0.3, 1 or 1.5 mg / kg. Mice were sacrificed and blood was collected at various time points (24, 48, 72, 96, 120, 144, 240 hours) after administration. Samples from each mouse were subjected to flow cytometry analysis to examine the pharmacodynamic effects within the lymphocyte population and functional markers of interest (cell numbers of CD8 T cells, CD8 memory T cells and NK cells, and percent positivity of Ki-67 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 numbers, respectively, after a single dose of Conjugate 1 at 0.01, 0.03, 0.1, 0.3, 1 or 1.5 mg / kg as described in Example 17. Conjugate 1 at dose levels of 0.03 and above induces 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, the CD8 and CD8 memory T cell numbers did not return to baseline at 240 hours after injection when Conjugate 1 was administered at 0.3 - 1.5 mg / kg, indicating a persistent PD effect of Conjugate 1.

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

[0286] Figures 31A, 31B, and 31C are plots of CD8 and CD8 memory subpopulation T cell numbers after single (dotted line) or Q7dx3 (solid line) administration of conjugate 1 at 0.03 and 0.3 mg / kg, as described in Example 17. Repeated dosing further increases 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), the cell numbers did not return to baseline in the mice.

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

[0288] Figure 32C is a plot of the number of C57BL / 6 NK cells after single (solid line) or Q7dx3 (dashed line) administration of conjugate 1 at 0.03 and 0.3 mg / kg, as described in Example 17. Repeated dosing of conjugate 1 at 0.3 mg / kg induced slightly fewer NK cells compared to single dosing, but was still significant. Similar NK numbers were achieved with single versus repeated dosing at 0.03 mg / kg.

[0289] Results: Conjugate 1 at a dosage level of 0.03 or higher induces a significant increase in total CD8 T cells in the blood. The lowest dosage of 0.01 mg / kg increased CD8 Tcm (central memory) and CD8 Tem (effector memory). At 0.3 mg / kg, Conjugate 1 increased CD8, CD8 Tcm, and CD8 Tem by 6.4X, 37.9X, and 14.5X, respectively. Notably, the numbers of CD8 and CD8 memory T cells did not return to baseline at 240 hours after injection when Conjugate 1 was administered at 0.3 - 1.5 mg / kg, indicating a beneficial persistent PD effect of Conjugate 1.

[0290] Example 18 Measurement of in vitro NK cell 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 incubated at 37°C, 5% CO2 for 4 hours at NK (effector) to YAC-1 (target cell) ratios (E:T) of 12.5:1, 25:1, and 50:1. YAC-1 target cells were pre-labeled 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 points and subjected to flow cytometric measurement of granzyme B expression in NK cells. The results are shown in Figures 33A and 33B.

[0291] Figure 33A shows an in vitro NK cell cytotoxicity assay measuring changes in NK-mediated target cell lysis after treatment with test articles in mice. Shown is the time course of percent specific lysis of YAC-1 cells by spleen NK cells isolated from balb / c mice treated with conjugate 1 at 0.006, 0.03 or 0.3 mg / kg or IL-15 at 1 mg / kg, at the indicated times. Spleen NK cells from vehicle-administered mice served as controls.

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

[0293] Figure 33B is a graph of percent granzyme B positive in blood NK cells from the same mice that were sacrificed for the NK in vitro cytotoxicity assay in Figure 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 strong and sustained increase seen at 0.3 mg / kg.

[0295] Example 19 Single-dose efficacy of conjugate 1 in a CT-26 lung metastasis model Balb / c mice received an intravenous injection of 1x10 5 CT-26 colorectal cancer cells. The next day, mice (n = 9 / group) were treated twice weekly with conjugate 1 (0.03 or 0.3 mg / kg) or vehicle control. Five days after the second injection, the mice were humanely sacrificed and pulmonary nodules were counted. The results are provided in Figures 34A and 34B.

[0296] Figures 34A and 34B show percent pulmonary nodule inhibition in balb / c mice that received intravenous CT-26 tumor cell injection and were administered conjugate 1 twice at 1-week intervals at 0.03 or 0.3 mg / kg.

[0297] Results: Injections of conjugate 1 at 0.03 and 0.3 mg / kg inhibited pulmonary nodule formation by 40 and 80%, respectively. The same mice administered at 0.3 mg / kg were then given subsequent tumor cell injections for 32 days and survival was evaluated. Treatment with conjugate 1 significantly increased survival compared to tumor-injected mice that received vehicle control.

[0298] Example 20 Evaluation of NK cell dependence of conjugate 2 efficacy in a CT-26 lung metastasis model CT-26 mice (n = 7 - 11 / group) were injected with anti-asialo GM1 or two different controls (IgG or PBS) to deplete NK cells and then 1 x 10 5 CT-26 tumor cells were injected. 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 received antibody-mediated depletion of NK cells (olive green), IgG control (blue), or PBS (orange). Data are represented as percent lung nodule inhibition in 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 lost when mice were deficient in NK cells.

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

[0301] Figures 36A and 36B are plots showing the two-week time course of CD8 cell count and Ki-67 percent positive as measures of proliferation in one male (dotted line) and one female (solid line) cyno after intravenous administration of conjugate 1 at a dose of 0.1 mg / kg. As can be seen, conjugate 1 induced a significant increase in CD8 T cells in cyno, with the cell count increasing 7- to 10-fold after a single dose.

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

[0303] Figures 37A and 37B are graphs of NK cell count and Ki-67 percent positive after a single administration of 0.1 mg / kg of conjugate 1 in cyno. NK cells increased 9- to 10-fold after treatment with conjugate 1.

[0304] Example 22 Comparison of the in vitro activities of IL-15 and conjugate 1 in CD8 and CD56 BRIGHT NK cells in human PBMC The in vitro activity of conjugate 1 was evaluated by examining NK and CD8 JAK / STAT signaling after treating human PBMC with IL-15 or conjugate 1 in a dose range of 0.001-10,000 ng / ml. STAT5 phosphorylation was evaluated as described above.

[0305]

Table 12

[0306] Provide the results in FIGS. 38A and 38B; these figures are the EC50 curves for IL-15 (●) in human PBMCs, versus conjugate 1 (■) treatment, and subsequent measurements of percent positive pSTAT5 in CD8 and CD56 bright NK cells.

[0307] Results: Conjugate 1 is not 5.5 and 15X more potent than IL-15, respectively, in the involvement of CD8 and CD56 bright NK cells. However, importantly, conjugate 1 achieves the same maximum response as conventional IL-15.

[0308] Example 23 In vivo study: 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 after administration (conjugate 1: 24, 48, 72, 96, 120, 144, 240 hours; IL-15 control: 0.03, 0.08, 0.25, 0.5, 1, 2, 4, 6, 8 hours), and the plasma concentration of the drug was determined. See FIG. 39.

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

[0310] Example 24 In vivo study: 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. The plasma concentration of the drug was 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 a half-life of approximately 18 hours, persistent pharmacokinetics, and measurable plasma concentrations, with rapid elimination, compared to the plasma levels observed after administration of non-long-acting IL-15.

[0312] Example 25 In Vivo Study: Single-Dose PK Study in Non-Human Primates Cynomolgus monkeys (n = 2, 1 male and 1 female) were administered a single intravenous dose of conjugate 1 at 10, 50, and 100 μg / kg. As a control, 50 μg / kg of IL-15 was administered as a single intravenous dose. The plasma concentration of the drug was determined at the indicated time points after injection (0.03, 0.25, 1, 4, 12, 24, 48, 72, 96, 120, 144, 168 hours). See Figure 41.

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

[0314] Conjugate 1 achieved extended and persistent plasma exposure across multiple species (mice, rats, and cynomolgus monkeys) after a single dose (see Figures 39 - 41).

[0315] Example 26 In Vivo Study: Single-Dose PD Study 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 a dose of 0.3 mg / kg or 0.03 mg / kg. After administration, blood samples were collected at the indicated time points after administration (24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours, and 240 hours). Samples were subjected to immunophenotyping for CD4 T cell count (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 count, and Figure 42B is a plot of CD4 T cell proliferation measured over time by %Ki-67 positivity. CD4 T cells were the least sensitive population to conjugate 1 treatment, having a lower number and increased %Ki-67 expression compared to CD8 and NK cells (observed 72 - 144 hours after dosing) (see, for example, Example 11). In mice, NK cells were more sensitive to a single administration of conjugate 1 stimulation in the proliferative response than CD4 T cells or CD8 T cells.

[0317] Phosphorylation of STAT5 in CD4 T cells was determined using the combination of CD3+CD4+CD8- pSTAT5+ markers. Figure 43 is a plot of the percentage positive of pSTAT5 phosphorylation in CD4 T cells over time (0.25, 1, 6, 24, 48, 72, 96, and 120 hours after administration) at a dose of 0.03 mg / kg (blue, solid squares) or 0.3 mg / kg (orange, solid circles). The time-course (black) and pre-administration (white circles) levels of vehicle are also shown.

[0318] Results: CD4 T cells were the least sensitive population to conjugate 1 treatment, having an increased pSTAT5 expression lower than that of CD8 and NK cells (observed 0.25 - 72 hours after dosing). In mice, NK cells were more sensitive to a single administration of conjugate 1 stimulation in the proliferative response than CD8 T cells or CD4 T cells.

[0319] Example 27 In vivo study: Minimum effective dose study in non-human primates (NHP) Cynomolgus monkeys (n = 3 - 4 males) were administered a single intravenous injection of conjugate 1 at 0.003, 0.01, 0.1 mg / kg or vehicle control. Blood samples were collected at the indicated time points before and after administration (-5, -2, 1, 2, 3, 4, 5, 6, 7, 10, 14, 17 days) and subjected to flow cytometry analysis to examine the pharmacodynamic effects within the lymphocyte population. The cell numbers of NK, CD8 T, and CD4 T cells were examined, and the results are shown in FIGS. 44A - C. The proliferation (%Ki-67) and JAK / STAT signaling (%pSTAT5) regarding NK cells, CD8 T cells, and CD4 T cells were examined, and the results are shown in FIGS. 45A - C and FIGS. 46A - C, respectively. The proliferation (%Ki-67) of the CD8 subpopulation (T 天然 , T em , T cm and T scm ) was examined, and the results are shown in FIGS. 47A - D.

[0320] In NHP, the NK (CD45+CD3 - CD16+) cell number increased substantially and dose-dependently after a single administration of conjugate 1. At the 0.1 and 0.01 mg / kg dose levels, the maximum cell number was observed 5 days after administration and persisted until day 14. The lowest dose that resulted in a significant increase in NK cells was 0.01 mg / kg. As a confirmation of the observation of NK cell numbers, conjugate 1 also promoted a dose-dependent and strong induction of Ki-67 expression, which reached a maximum approximately 3 - 4 days after treatment and could persist until approximately day 14. A significant increase in %Ki-67 could be detected next at the 0.001 mg / kg dose level. Conjugate 1 was also strongly involved in the JAK / STAT signaling pathway in NK cells, and a dose-dependent increase in %pSTAT5 could be detected at a low dose level of 0.001 mg / kg.

[0321] FIGS. 44A, 45A, and 46A are plots of the NK cell number, %Ki-67, and %pSTAT5 over time after conjugate 1 treatment, respectively.

[0322] In NHP, conjugate 1 induced a substantial increase in total CD8 T cells (defined as CD45+CD3+CD4-CD8+), with the maximum cell number achieved approximately 5 days after treatment. This effect persisted for 7 days and returned to baseline at 10 - 14 days post - 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 a substantial amount of %Ki - 67 positivity detectable at a low dose of 0.01 mg / kg in CD8 T cells. The involvement of conjugate 1 in the JAK / STAT signaling pathway was also strong in CD8 T cells, with pSTAT5 increasing in a dose - dependent manner at 0.1 and 0.01 mg / kg dose levels.

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

[0324] Conjugate 1 had a low effect on total CD4 T cells (defined as CD45+CD3+CD4+CD8-) in NHP compared to NK and CD8 T cells. Conjugate 1 administered at the maximum dose level of 0.1 mg / kg induced a low increase in CD4 T cell numbers, %Ki - 67, and %pSTAT5.

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

[0326] NK cells were the most sensitive in the in - vivo conjugate 1 dose - response compared to CD8 T cells or CD4 T cells in NHP.

[0327] In cynomolgus monkeys, CD8 naive and memory subsets were defined by CD45Ra, CD197, and CD95. By testing the proliferation (%Ki-67) of CD8 T naive (CD45+CD3+CD4-CD8+CD45Ra+CD197+), CD8 Tscm (CD45+CD3+CD4-CD8+CD45Ra+CD197+CD95+), CD8 Tem (CD45+CD3+CD4-CD8+CD45Ra-CD197-), and CD8 Tcm (CD45+CD3+CD4-CD8+CD45Ra-CD197+), an increased sensitivity of CD8 memory subsets to conjugate 1 was revealed compared to CD8 naive T cells. Within the CD8 Tem, Tcm, and Tscm populations, conjugate 1 induced strong %Ki-67 expression in a dose-dependent manner, and a detectable increase in the proliferation marker positive started as early as day 2, reached a maximum on day 5, and returned to baseline on days 10 - 14. The Ki-67 expression and kinetics within the CD8 population support the increased persistence of CD8 T cell numbers shown in Example 27 and Figures 47A - D.

[0328] Figures 47A - D are plots of %Ki-67 over time for the CD8 T 天然 , T scm , T cm and T em populations. As is evident from the figure, the CD8 T cell memory population showed increased sensitivity to a single in vivo dose of conjugate 1 compared to naive CD8 T cells in NHP.

[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 after single administration of conjugate 1 in cynomolgus monkeys. The expression levels of granzyme B and perforin were quantified by mean fluorescence intensity (MFI) in NK cells at 0.001 mg / kg, 0.01 mg / kg or 0.1 mg / kg dose levels. Conjugate 1 increased the MFI of granzyme B by approximately 3-fold (peak, vs. pre-dose) at 0.01 and 0.1 mg / kg. Conjugate 1 also increased the MFI of perforin by approximately 2-fold (peak, vs. pre-dose) at 0.01 and 0.1 mg / kg. Overall, conjugate 1 can not only induce potent expansion of NK cells, but also improve their function.

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

[0331] Conjugate 1 increases the protein levels of cytotoxic enzymes, such as constitutively expressing granzyme B and perforin, in NHP NK cells.

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

Chem.

Chem.

Chem.

Claims

【Claim 1】 An article, method, or system described in this specification and the drawings.

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