Il-2 fusion polypeptide compositions and methods of making and using the same

Abstract

Provided herein are compositions comprising polypeptides comprising a circularly permuted interleukin-2 (IL-2) fused to the extracellular portion of an IL-2Rα chain, and methods of making and using such compositions.

Description

[Technical Field]

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 022,853, filed on May 11, 2020, the entire disclosure of which is incorporated herein by reference.

[0003] This disclosure relates to a composition comprising a polypeptide and a method for manufacturing and using such a composition, wherein the polypeptide comprises a circular sequence recombinant interleukin-2 (IL-2) fused to the extracellular portion of an IL-2Rα chain. [Previous Technology]

[0004] Polypeptides comprising a circular sequence recombinant interleukin-2 (IL-2) [interleukin-2 receptor α (IL-2Rα)] fused to the extracellular portion of the IL-2Rα chain show great promise as anticancer agents. These peptides retain the full signaling capability via the intermediate-affinity IL-2R complex expressed on memory CD8+ T cells and natural killer (NK) cells, but spatially block binding to the high-affinity IL-2R complex predominantly expressed on CD4+ FOXP3+ regulatory T cells (CD4+ Tregs) and endothelial cells. Due to this selective IL-2R binding, these peptides selectively activate CD8+ T cells and NK cells, thereby enhancing tumor cell killing. Loss of the ability to activate high-affinity IL-2R on endothelial cells also reduces the risk of toxicity due to capillary leakage syndrome (a known risk of IL-2 therapy).

[0005] When used to treat human subjects, the aforementioned polypeptides must be stored and transported to the point of administration before use. To repeatedly achieve the desired amount of polypeptide in a subject, the polypeptide needs to be stored in a formulation that maintains its biological activity. Therefore, stable polypeptide compositions are required in this art. Preferably, such compositions will have a long shelf life and be stable during storage and transport. [Summary of the Invention]

[0006] This disclosure provides compositions comprising polypeptides and methods for manufacturing and using such compositions, wherein the polypeptides comprise a circular sequence recombinant interleukin-2 (IL-2) fused to the extracellular portion of an IL-2Rα chain. These compositions are specially formulated to improve the stability and shelf life of the polypeptides contained therein.

[0007] In one aspect, this disclosure provides a composition comprising: a) a polypeptide of about 1 mg to about 50 mg, including a circular sequence recombinant IL-2 fused to the extracellular portion of an IL-2Rα chain; b) sucrose; c) mannitol; d) a citrate buffer; and e) an emulsifier.

[0008] In a specific embodiment, the polypeptide system comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 1. In a specific embodiment, the polypeptide system comprises the amino acid sequence of SEQ ID NO: 1. In a specific embodiment, the polypeptide system is composed of the amino acid sequence of SEQ ID NO: 1.

[0009] In a specific example, the constituent system comprises about 1 mg to about 15 mg of a polypeptide. In a specific example, the constituent system comprises about 1 mg of a polypeptide. In a specific example, the constituent system comprises about 5 mg of a polypeptide. In a specific example, the constituent system comprises about 15 mg of a polypeptide. In a specific example, the constituent system comprises about 20 mg of a polypeptide. In a specific example, the constituent system comprises about 30 mg of a polypeptide.

[0010] In a particular specific instance, the composition system comprises about 60 mg to about 72 mg of sucrose. In a particular specific instance, the composition system comprises about 66 mg of sucrose.

[0011] In a specific instance, the composition system comprises about 60 mg to about 72 mg of mannitol. In a specific instance, the composition system comprises about 66 mg of mannitol.

[0012] In a specific instance, the composition system comprises about 4.0 mg to about 6.0 mg of citrate anion. In a specific instance, the composition system comprises about 5.0 mg of citrate anion.

[0013] In a particular specific instance, the composition system comprises citric acid and trisodium citrate dihydrate in a mass ratio of about 1:10 to about 1:2 (i.e., about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3 and about 1:2).

[0014] In a specific embodiment, the composition system comprises citric acid and trisodium citrate dihydrate in a mass ratio of approximately 1:9. In a specific embodiment, the composition system comprises citric acid and trisodium citrate dihydrate in a mass ratio of approximately 1:2.

[0015] In a specific example, the emulsifier system includes polysorbate 20. In a specific example, the composition system includes about 0.20 mg to about 0.24 mg of polysorbate 20. In a specific example, the composition system includes about 0.22 mg of polysorbate 20.

[0016] In a specific instance, the composition system comprises about 0.10 mg to about 0.12 mg of polysorbate 20. In a specific instance, the composition system comprises about 0.11 mg of polysorbate 20.

[0017] In a specific instance, the component is a freeze-dried cake block.

[0018] In a specific embodiment, the freeze-dried cake is dissolved in water to produce an aqueous solution having a pH of about 5.5 to about 6.5. In a specific embodiment, the freeze-dried cake is dissolved in water to produce an aqueous solution having a pH of about 6.1.

[0019] In a specific example, the freeze-dried cake is dissolved in water to produce an aqueous solution with an isotonic osmotic pressure. In a specific example, the freeze-dried cake is dissolved in water to produce an aqueous solution with an osmotic pressure of about 240 to about 340 mOsm / kg. In a specific example, the freeze-dried cake is dissolved in water to produce an aqueous solution with an osmotic pressure of about 280 to about 320 mOsm / kg. In a specific example, the freeze-dried cake is dissolved in water to produce an aqueous solution with an osmotic pressure of about 285 mOsm / kg. In a specific example, the freeze-dried cake is dissolved in water to produce an aqueous solution with an osmotic pressure of about 300 mOsm / kg.

[0020] In a specific instance, the composition is an aqueous solution.

[0021] In a particular specific example, the aqueous solution comprises about 0.03 mg / mL of polypeptide to about 0.2 mg / mL of polypeptide.

[0022] In a particular specific instance, the composition system comprises a polypeptide of about 0.5 mg / mL to about 30 mg / mL.

[0023] In a specific instance, the composition system comprises about 1 mg / mL of polypeptide.

[0024] In a specific instance, the composition system comprises about 5 mg / mL of polypeptide.

[0025] In a specific instance, the composition system comprises about 15 mg / mL of polypeptide.

[0026] In a specific instance, the constituent system comprises about 20 mg / mL of a polypeptide. In a specific instance, the constituent system comprises about 30 mg / mL of a polypeptide.

[0027] In a specific instance, the compositional system comprises about 25 mg / mL to about 35 mg / mL of sucrose. In a specific instance, the compositional system comprises about 30 mg / mL of sucrose.

[0028] In a specific instance, the composition system comprises about 25 mg / mL to about 35 mg / mL mannitol. In a specific instance, the composition system comprises about 30 mg / mL mannitol.

[0029] In a specific example, the compositional system comprises about 10 mM to about 20 mM of citrate buffer. In a specific example, the compositional system comprises about 12 mM of citrate buffer. In a specific example, the citrate buffer is formed by mixing 2.03 mg / mL trisodium citrate dihydrate and 0.97 mg / mL citrate monohydrate in an aqueous solution. In a specific example, the citrate buffer is formed by mixing 2.91 mg / mL trisodium citrate dihydrate and 0.34 mg / mL citrate monohydrate in an aqueous solution. In a specific example, the citrate buffer is formed by mixing 2.96 mg / mL trisodium citrate dihydrate and 0.30 mg / mL citrate monohydrate in an aqueous solution.

[0030] In a specific instance, the composition system comprises about 0.09 mg / mL to about 0.11 mg / mL of polysorbate 20. In a specific instance, the composition system comprises about 0.1 mg / mL of polysorbate 20.

[0031] In a particular specific instance, the pH of the composition is about 5.5 to about 6.5. In a particular specific instance, the pH of the composition is about 6.1.

[0032] In a specific instance, the osmotic pressure of the composition is about 240 to about 340 mOsm / kg. In a specific instance, the osmotic pressure of the composition is about 280 to about 320 mOsm / kg. In a specific instance, the osmotic pressure of the composition is about 285 mOsm / kg. In a specific instance, the osmotic pressure of the composition is about 300 mOsm / kg.

[0033] In a specific instance, the composition is a single unit dose of polypeptide.

[0034] In one aspect, this disclosure provides a composition comprising: a) a polypeptide of about 1 mg to about 30 mg, including a circular recombinant IL-2 fused to the extracellular portion of an IL-2Rα chain; b) sucrose of about 25 mg / mL to about 35 mg / mL; c) mannitol of about 25 mg / mL to about 35 mg / mL; d) citrate buffer of about 10 mM to about 20 mM; and e) polysorbate 20 of about 0.09 mg / mL to about 0.11 mg / mL, wherein the pH of the solution is about 5.5 to about 6.5.

[0035] In a specific instance, the polypeptide system comprises an amino acid having at least 95% identity with SEQ ID NO: 1. In a specific instance, the polypeptide system comprises the amino acid sequence of SEQ ID NO: 1.

[0036] In one aspect, this disclosure provides a composition comprising: a) a polypeptide comprising about 1 mg / mL to about 30 mg / mL of the amino acid sequence of SEQ ID NO: 1; b) sucrose comprising about 25 mg / mL to about 35 mg / mL; c) mannitol comprising about 25 mg / mL to about 35 mg / mL; d) citrate buffer comprising about 10 mM to about 20 mM; and e) polysorbate 20 comprising about 0.09 mg / mL to about 0.11 mg / mL, wherein the pH of the solution is about 5.5 to about 6.5.

[0037] In a specific instance, the composition system comprises about 30 mg / mL of sucrose.

[0038] In a specific instance, the composition system comprises about 30 mg / mL mannitol.

[0039] In a particular specific instance, the composition system includes about 12 mM of citrate buffer.

[0040] In a particular specific instance, the composition system comprises about 0.11 mg / mL of polysorbate 20.

[0041] In a specific instance, the pH of the solution is approximately 6.1.

[0042] In a specific example, the constituent system comprises about 1 mg / mL of a polypeptide. In a specific example, the constituent system comprises about 5 mg / mL of a polypeptide. In a specific example, the constituent system comprises about 15 mg / mL of a polypeptide. In a specific example, the constituent system comprises about 20 mg / mL of a polypeptide. In a specific example, the constituent system comprises about 30 mg / mL of a polypeptide.

[0043] In another aspect, this disclosure provides an aqueous composition comprising: a) a polypeptide comprising about 1, 5, 15 or 30 mg / mL of the amino acid sequence of SEQ ID NO: 1; b) about 25 mg / mL to about 35 mg / mL of sucrose; c) about 25 mg / mL to about 35 mg / mL of sucrose; d) about 8 mM of citrate buffer to about 14 mM of citrate buffer (e.g., 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM or 14 mM); and e) about 0.1 mg / mL of polysorbate 20, wherein the pH of the composition is about 6.1.

[0044] In another aspect, this disclosure provides an aqueous composition comprising: a) a polypeptide comprising about 1, 5, 15 or 30 mg / mL of the amino acid sequence of SEQ ID NO: 1; b) about 25 mg / mL to about 35 mg / mL of sucrose; c) about 25 mg / mL to about 35 mg / mL of sucrose; d) about 12 mM of citrate buffer; and e) about 0.1 mg / mL of polysorbate 20, wherein the pH of the composition is about 6.1.

[0045] In another aspect, this disclosure provides an aqueous composition comprising: a) about 1, 5, 15 or 30 mg / mL of a polypeptide including the amino acid sequence of SEQ ID NO: 1; b) about 25 mg / mL to about 35 mg / mL of sucrose; c) about 25 mg / mL to about 35 mg / mL of sucrose; d) about 2 mg / mL of trisodium citrate dihydrate; e) about 1 mg / mL of citrate monohydrate; and f) about 0.1 mg / mL of polysorbate 20, wherein the pH of the composition is about 6.1.

[0046] In another aspect, this disclosure provides an aqueous composition comprising: a) a polypeptide comprising about 1, 5, 15 or 30 mg / mL of the amino acid sequence of SEQ ID NO: 1; b) about 25 mg / mL to about 35 mg / mL of sucrose; c) about 25 mg / mL to about 35 mg / mL of sucrose; d) about 2.03 mg / mL of trisodium citrate dihydrate; e) about 0.97 mg / mL of citrate monohydrate; and f) about 0.1 mg / mL of polysorbate 20, wherein the pH of the composition is about 6.1.

[0047] In another aspect, this disclosure provides an aqueous composition comprising: a) about 1, 5, 15 or 30 mg / mL of a polypeptide including the amino acid sequence of SEQ ID NO: 1; b) about 25 mg / mL to about 35 mg / mL of sucrose; c) about 25 mg / mL to about 35 mg / mL of sucrose; d) about 3 mg / mL of trisodium citrate dihydrate; e) about 0.3 mg / mL of citrate monohydrate; and f) about 0.1 mg / mL of polysorbate 20, wherein the pH of the composition is about 6.1.

[0048] In another aspect, this disclosure provides an aqueous composition comprising: a) a polypeptide comprising about 1, 5, 15 or 30 mg / mL of the amino acid sequence of SEQ ID NO: 1; b) about 25 mg / mL to about 35 mg / mL of sucrose; c) about 25 mg / mL to about 35 mg / mL of sucrose; d) about 2.91 mg / mL of trisodium citrate dihydrate; e) about 0.34 mg / mL of citrate monohydrate; and f) about 0.1 mg / mL of polysorbate 20, wherein the pH of the composition is about 6.1.

[0049] In another aspect, this disclosure provides an article comprising any of the foregoing components. In a particular specific example, the article is a glass vial.

[0050] In another aspect, this disclosure provides a freeze-dried composition made by freeze-drying any of the aforementioned aqueous solutions.

[0051] In another aspect, this disclosure provides a method for manufacturing a lyophilized composition, the method comprising lyophilizing any of the aforementioned aqueous solutions.

[0052] In another aspect, this disclosure provides a method for manufacturing an aqueous composition, the method comprising dissolving any of the aforementioned lyophilized composition in an aqueous solvent. In a particular specific example, the aqueous solvent is water for injection. In a particular specific example, the aqueous solvent is an aqueous solution of sodium chloride.

[0053] In a specific instance, the pH of the aqueous component is adjusted to approximately 6.1. In a specific instance, the pH of the aqueous component is adjusted to approximately 6.1 using an alkali. In a specific instance, this alkali is sodium hydroxide.

[0054] In a specific example, the aqueous composition is further diluted with an aqueous solution comprising about 1% (w / w) of a surfactant. In a specific example, the surfactant is polysorbate 20. In a specific example, the aqueous composition further comprises about 0.1% (w / w) citrate monohydrate, 0.2% (w / w) trisodium citrate dihydrate, and 98.7% (w / w) water for injection.

[0055] In a particular specific instance, the composition system includes a pharmaceutical composition.

[0056] In another aspect, this disclosure provides a method for activating natural killer (NK) cells in a subject, the method comprising administering to the subject an effective amount of any of the aforementioned components.

[0057] In another aspect, this disclosure provides a method for treating cancer in a subject with this need, the method comprising administering to the subject an effective amount of any of the aforementioned components. In specific instances, the cancer is renal cell carcinoma, melanoma, ovarian cancer, or lung cancer. In specific instances, the cancer includes refractory solid tumors.

Implementation Method

[0058] This document provides compositions including polypeptides and methods for manufacturing and using such compositions, wherein the polypeptides include a circular sequence recombinant IL-2 fused to the extracellular portion of an IL-2Rα chain.

[0059] The formulations disclosed herein provide enhanced stability and shelf life for the peptides contained therein. Specifically, the peptide product retains its biological activity, including after lyophilization in the formulation and recombination with water for injection (WFI) or a similar acceptable diluent. Importantly, the formulations described herein are designed to allow the lyophilized product to be recombinated in WFI and are readily available to patients or healthcare professionals. When recombinated with WFI, the formulations described herein have a physiologically acceptable osmotic pressure, allowing the recombined product to be administered subcutaneously. This eliminates the need for specialized diluents to recombine the lyophilized product with appropriate osmotic pressure, making the medication easier for patients or healthcare professionals to use and thus improving medication adherence.

[0060] Subcutaneous administration is also advantageous for drug delivery. When delivered via the subcutaneous route, the drug can be delivered more quickly compared to other delivery routes (e.g., intravenous). Subcutaneous delivery can also be performed by the patient at home, rather than by healthcare professionals in a medical facility. This patient-oriented delivery also improves drug use compliance.

[0061] The formulations provided herein also produce freeze-dried cakes with a superior appearance. Specifically, the cakes are whole (not broken into fragments), exhibit minimal shrinkage in containers (e.g., glass vials), and have a smooth, concave surface. (Choose a definition)

[0062] Unless otherwise defined herein, the scientific and technical terms used herein shall have the meaning as normally understood by one of ordinary skill in the art. In the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definition. Unless required by the context, singular terms shall include plural terms and plural terms shall include singular terms. Unless otherwise indicated, the use of "or" means "and / or". The use of the term "including" and other forms such as "comprising" and "containing" is without limitation.

[0063] As used herein, the terms “comprising,” “including,” and “having,” and their grammatical variations, are used to specify the contents, integers, steps, or components described herein, but do not exclude the inclusion of one or more of their additional contents, integers, steps, components, or groups. These terms cover the terms “consisting of” and “substantially consisting of.”

[0064] As used herein, the term "circular recombination" refers to the process of taking a straight-chain protein or its homologous nucleic acid sequence and fusing it with the native N- and C-termini (using protein or recombinant DNA methods, directly or via a linker) to form a circular molecule, and then cleaving (opening) the circular molecule at different positions to form new straight-chain protein or homologous nucleic acid molecules whose endpoints are different from those of the original molecule. Circular recombination thus preserves the sequence, structure, and function of the protein while generating new C- and N-termini at different positions, thus enhancing the orientation towards a desired fusion polypeptide partner compared to the original molecule.

[0065] As used herein, those skilled in the art should understand that the term "about" will vary to some extent in its application. As used herein, when referring to a measurable value, such as quantity, duration of a short period of time, etc., the term "about" refers to a variation of up to ±5%, including ±5%, ±1%, and ±0.1% compared to the value indicated, and thus such variations apply to the methods disclosed.

[0066] As used herein, the term “treat, treated, treating, or treatment” includes reducing or alleviating at least one symptom related to or caused by the state, condition, or disease for which treatment is desired.

[0067] As used herein, the term "effective dose" in the context of treatment of a subject refers to the therapeutic dose that achieves the desired preventive or therapeutic effect.

[0068] As used herein, the terms "patient," "individual," or "object" refer to human or non-human mammals. Non-human mammals include, for example, livestock and pets, such as sheep, cattle, pigs, dogs, cats, and rodents. In a specific instance, the object is a human. IL-2 fusion peptide

[0069] In one aspect, this disclosure provides a composition of a polypeptide comprising a circular recombinant interleukin-2 (IL-2) fused to the extracellular portion of an IL-2Rα chain. The polypeptide used in the composition disclosed herein exhibits preferential binding to intermediate-affinity IL-2R complexes (including IL-2Rβ and a shared γ chain, IL-2Rγ) relative to high-affinity IL-2R complexes (including IL-2Rα, IL-2Rβ, and IL-2Rγ) and acts as a selective agonist for intermediate-affinity IL-2R complexes. The design and production of these polypeptides are described in U.S. Patent No. 9,359,415, which is incorporated herein by reference in its entirety.

[0070] Example peptide lines that can be included in the compositions disclosed herein are as described in the following SEQ ID NO: 1: SKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTG (SEQ ID NO: 1)

[0071] Therefore, in a specific instance, the amino acid sequence of the polypeptide includes the amino acid sequence of SEQ ID. NO: 1. In a specific instance, the amino acid sequence of the polypeptide is composed of the amino acid sequence of SEQ ID. NO: 1.

[0072] Those skilled in the art will understand that amino acid sequence variants of SEQ ID. NO: 1 may also be used in the compositions described herein. For example, in a particular instance, the amino acid sequence of the polypeptide comprises or is composed of amino acid sequences having at least 80% (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identity with the amino acid sequence of SEQ ID. NO: 1. In a particular instance, the amino acid sequence of the polypeptide comprises or is composed of amino acid sequences having at least 95% identity with the amino acid sequence of SEQ ID. NO: 1.

[0073] Those skilled in the art should also understand that the amino acid sequences of the polypeptides used in the composition described herein may be derivatized or modified, such as by pegification, acetylation, etc.

[0074] In specific examples, the amount of peptide in the formulation is from about 1 mg to about 50 mg (e.g., about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 44 mg, about 45 mg, or about 50 mg). In specific examples, the amount of peptide is from about 1 mg to about 30 mg. In specific examples, the amount of peptide is from about 1 mg to about 15 mg. In specific examples, the amount of peptide is about 1 mg. In specific examples, the amount of peptide is about 2.2 mg. In specific examples, the amount of peptide is about 5 mg. In specific examples, the amount of peptide is about 11 mg. In a specific instance, the amount of peptide was approximately 15 mg. In a specific instance, the amount of peptide was approximately 20 mg. In a specific instance, the amount of peptide was approximately 30 mg. In a specific instance, the amount of peptide was approximately 44 mg.

[0075] In a specific instance, the concentration of the peptide in the aqueous formulation is from about 0.5 mg / mL to about 50 mg / mL. In a specific instance, the concentration of the peptide is from about 0.5 mg / mL to about 20 mg / mL (e.g., about 0.5 mg / mL, about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, or about 50 mg / mL). In specific instances, the peptide concentration is approximately 1 mg / mL. In specific instances, the peptide concentration is approximately 5 mg / mL. In specific instances, the peptide concentration is approximately 15 mg / mL. In specific instances, the peptide concentration is approximately 20 mg / mL. In specific instances, the peptide concentration is approximately 30 mg / mL. Excipients & Buffers

[0076] In specific instances, the constituent systems disclosed herein include one or more excipients and / or buffers.

[0077] As used herein, the term "excipient" means any non-therapeutic agent added to a composition or formulation to provide a desired consistency, viscosity, or stabilizing effect. Suitable excipients for use in the compositions disclosed herein may be, for example, thickeners, stabilizers, solubilizers, etc. Excipients may be ionic or nonionic. Suitable ionic excipients include salts, such as NaCl, or amino acid components, such as arginine-HCl. Suitable nonionic excipients include sugars, such as monosaccharides (e.g., fructose, maltose, galactose, glucose, D-mannose, sorbitol, etc.); disaccharides (e.g., lactose, sucrose, trehalose, cellobiose, etc.); polysaccharides (e.g., raffinose, melitriose, maltodextrin, dextran, starch, etc.); and sugar alcohols (e.g., mannitol, xylitol, maltitol, lactitol, sorbitol (glucosyl alcohol), etc.). For example, this sugar can be sucrose, trehalose, raffinose, maltose, sorbitol, or mannitol. Alternatively, this sugar can be a sugar alcohol or an amino sugar. In a specific instance, this sugar is sucrose and mannitol.

[0078] In specific examples, the amount of excipients (e.g., sucrose and mannitol) in the formulation is from about 1 mg to about 150 mg (e.g., about 1 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, or about 150 mg). In specific examples, the amount of excipients (e.g., sucrose and mannitol) in the formulation is from about 30 mg to about 90 mg. In specific examples, the amount of excipients (e.g., sucrose and mannitol) in the formulation is from about 60 mg to about 72 mg. In specific examples, the amount of excipients (e.g., sucrose and mannitol) in the formulation is about 66 mg.

[0079] In specific examples, the concentration of excipients (e.g., sucrose and mannitol) in the aqueous formulation is from about 1 mg / mL to about 100 mg / mL (e.g., about 1 mg / mL, about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, or about 100 mg / mL). In specific examples, the concentration of excipients (e.g., sucrose and mannitol) is from about 10 mg / mL to about 50 mg / mL. In specific examples, the concentration of excipients (e.g., sucrose and mannitol) is from about 25 mg / mL to about 35 mg / mL. In specific examples, the concentration of excipients (e.g., sucrose and mannitol) is about 30 mg / mL.

[0080] Suitable buffers for use with the compositions disclosed herein include organic acids and salts, such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tris(hydroxymethyl)aminomethane hydrochloride, or phosphate buffers. In addition, amino acid components may also be used as buffers. These amino acid components include glycine, histidine, and methionine. In a particular specific example, the buffer is a citrate buffer. As used herein, the term "citrate buffer" refers to a pH buffering system (in aqueous or lyophilized form) utilizing citrate ions. Citrate buffers can be manufactured using any known method, including by mixing: (i) citric acid, trisodium citrate dihydrate, and citrate monohydrate; or (ii) citrate monohydrate, disodium hydrogen phosphate, and citric acid. In a particular specific example, the citrate buffer is prepared using sodium citrate dihydrate and citric acid.

[0081] In specific examples, the amount of buffer (e.g., citrate) in the formulation is from about 1 mg to about 10 mg (e.g., about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg). In specific examples, the amount of buffer (e.g., sodium citrate) is from about 5.9 mg to about 7.2 mg (e.g., about 5.9 mg, about 6.0 mg, about 6.1 mg, about 6.2 mg, about 6.3 mg, about 6.4 mg, about 6.5 mg, about 6.6 mg, about 6.7 mg, about 6.8 mg, about 6.9 mg, about 7.0 mg, about 7.1 mg, or about 7.2 mg). In specific examples, the amount of buffer (e.g., citrate) is about 6.6 mg. In specific instances, the amount of citrate anions in the buffer (e.g., citrate) is from about 4.0 mg to about 6.0 mg. In specific instances, the amount of citrate anions in the buffer (e.g., citrate) is about 5.0 mg.

[0082] In a specific instance, the concentration of the buffer (e.g., citrate) in the aqueous formulation disclosed herein is from about 1 mM to about 50 mM (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM or about 50 mM). In specific examples, the concentration of the buffer (e.g., sodium citrate) is from about 11 mM to about 13 mM (e.g., about 11.1 mM, 11.2 mM, 11.3 mM, 11.4 mM, 11.5 mM, 11.6 mM, 11.7 mM, 11.8 mM, 11.9 mM, 12.1 mM, 12.2 mM, 12.3 mM, 12.4 mM, 12.5 mM, 12.6 mM, 12.7 mM, 12.8 mM, or 12.9 mM). In specific examples, the concentration of the buffer (e.g., citrate) is about 12 mM. In specific examples, the concentration of the buffer (e.g., citrate) is about 11.95 mM. In specific examples, the concentration of the buffer (e.g., citrate) is about 11.67 mM. In a specific instance, the citrate buffer contains 2.03 mg / mL (6.90 mM) trisodium citrate dihydrate and 0.97 mg / mL (5.05 mM) citric acid. In another specific instance, the citrate buffer contains 2.91 mg / mL (9.90 mM) trisodium citrate dihydrate and 0.34 mg / mL (1.77 mM) citric acid.

[0083] In specific examples, the composition system disclosed herein has a pH of about 5.0 to about 8.0, about 5.5 to about 7.5, about 5.0 to about 7.0, about 6.0 to about 8.0, or about 6.0 to about 7.0. In specific examples, the composition system has a pH of about 5.4 to about 6.5. In specific examples, the composition system has a pH of about 5.8 to about 6.4. In specific examples, the composition system has a pH of about 6.1. In specific examples, the pH of the composition is adjusted to about pH 6.1. In specific examples, the pH is adjusted with a base. In specific examples, the base is a hydroxide salt, such as sodium hydroxide (NaOH) or potassium hydroxide (KOH). In specific examples, the composition is an aqueous composition and the pH of the aqueous composition is adjusted to about pH 6.1.

[0084] In specific examples, the composition system disclosed herein has isotonic osmotic pressure. In specific examples, the osmotic pressure of the composition is about 240 to about 340 mOsm / kg. In specific examples, the osmotic pressure of the composition is about 280 to about 320 mOsm / kg. In specific examples, the osmotic pressure of the composition is about 285 mOsm / kg. In specific examples, the osmotic pressure of the composition is about 300 mOsm / kg.

[0085] As used herein, the term "surfactant" refers to an organic substance with an amphiphilic structure; that is, it is composed of groups with opposite solubility tendencies, typically an oil-soluble hydrocarbon chain and a water-soluble ionic group. Based on the charge of the surface-active groups, surfactants can be classified as anionic, cationic, and dispersants used in the preparation of various pharmaceutical compositions and biological substances. Surfactants suitable for use in the compositions disclosed herein include nonionic surfactants, ionic surfactants, and zwitterionic surfactants. Typical surfactants used in this invention include sorbitan fatty acid esters (e.g., sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate), sorbitan trioleate, glyceryl fatty acid esters (e.g., glyceryl monocaprylate, glyceryl monomyristate, glyceryl monostearate), polyglycerol fatty acid esters (e.g., decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl monolinoleate), and polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate, polyoxyethylene...). Sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate, polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan tetrastearate, polyoxyethylene sorbitan tetraoleate), polyoxyethylene glycerol fatty acid esters (e.g., polyoxyethylene glycerol monostearate), polyethylene glycol fatty acid esters (e.g., polyethylene glycol distearate), polyoxyethylene alkyl ethers (e.g., polyoxyethylene lauryl ether). Polyoxyethylene polyoxypropylene alkyl ethers (e.g., polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether), polyoxyethylene alkylphenyl ethers (e.g., polyoxyethylene nonylphenyl ether), polyoxyethylene hydrogenated castor oil (e.g., polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil), polyoxyethylene beeswax derivatives (e.g., polyoxyethylene sorbitan beeswax), polyoxyethylene lanolin derivatives (e.g., polyoxyethylene lanolin), and polyoxyethylene fatty acid amides (e.g., polyoxyethylene stearate amide). The composition may include: C10-C18 alkyl sulfates (e.g., sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate), polyoxyethylene C10-C18 alkyl ether sulfates (e.g., sodium polyoxyethylene lauryl sulfate) having an average of 2 to 4 moles of ethylene oxide units added, and C1-C18 alkyl sulfosuccinate salts (e.g., sodium lauryl sulfosuccinate); and natural surfactants such as lecithin, glycerophospholipids, sphingomyelins (e.g., myelin), and sucrose esters of C12-C18 fatty acids. The composition may include one or more of these surfactants. In specific examples, the composition system disclosed herein includes polyoxyethylene sorbitan fatty acid esters, such as polysorbate 20, 40, 60, or 80. In specific examples, the composition system disclosed herein includes polysorbate 20.

[0086] In a specific instance, the amount of surfactant (e.g., polysorbate 20) in the formulation is from about 0.1 mg to about 1 mg (e.g., about 0.1 mg, about 0.15 mg, about 0.2 mg, about 0.25 mg, about 0.3 mg, about 0.35 mg, about 0.4 mg, about 0.45 mg, about 0.5 mg, about 0.55 mg, about 0.6 mg, about 0.65 mg, about 0.7 mg, about 0.75 mg, about 0.8 mg, about 0.85 mg, about 0.9 mg, about 0.95 mg, or about 1 mg). In specific examples, the amount of surfactant (e.g., polysorbate 20) is from about 0.15 mg to about 0.3 mg (e.g., about 0.16 mg, about 0.17 mg, about 0.18 mg, about 0.19 mg, about 0.21 mg, about 0.22 mg, about 0.23 mg, about 0.24 mg, about 0.26 mg, about 0.27 mg, about 0.28 mg, or about 0.29 mg). In specific examples, the amount of surfactant (e.g., polysorbate 20) is from about 0.20 mg to about 0.24 mg. In specific examples, the amount of surfactant (e.g., polysorbate 20) in the aqueous formulation is about 0.22 mg. In specific examples, the composition system comprises about 0.10 mg to about 0.12 mg of polysorbate 20. In a specific instance, the composition system comprises approximately 0.11 mg of polysorbate 20.

[0087] In a specific instance, the concentration of the surfactant (e.g., polysorbate 20) in the formulation is from about 0.01 mg / mL to about 1 mg / mL (e.g., about 0.01 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL or about 1 mg / mL). In specific examples, the concentration of the surfactant (e.g., polysorbate 20) is from about 0.05 mg / mL to about 0.15 mg / mL (e.g., about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, or about 0.08 mg / mL, about 0.09 mg / mL, about 0.1 mg / mL, about 0.11 mg / mL, about 0.12 mg / mL, about 0.13 mg / mL, about 0.14 mg / mL, or about 0.15 mg / mL). In specific examples, the concentration of the surfactant (e.g., polysorbate 20) is from about 0.09 mg / mL to about 0.11 mg / mL. In specific examples, the concentration of the surfactant (e.g., polysorbate 20) in the aqueous formulation is about 0.1 mg / mL.

[0088] Those skilled in the art will understand that the components of the composition and the components of the present invention can be described in units other than mg / mL. For example, the components of the composition and the components of the present invention can be described in units of volume molar concentration. The components of the composition and the components of the present invention can be further described in units of weight or mass percentage. Lyophilization

[0089] In one aspect, this disclosure provides lyophilized components of the polypeptides described herein (e.g., lyophilized cakes), and methods for manufacturing them.

[0090] Freeze-drying generally includes three main stages: freezing, primary drying, and secondary drying. Freezing involves converting water into ice or certain amorphous components into crystalline form. Primary drying is a process that removes ice from the frozen product through direct sublimation at low pressure and low temperature. Secondary drying is a process that removes bound water from the product matrix by diffusing residual water to an evaporation surface. During secondary drying, the product temperature is generally higher than during primary drying. See, Tang 281-353; Wang et al. (2000) "Lyophilization and development of solid protein pharmaceuticals," MJ Pharm., 203: 1-60; Williams NA et al. (1984) "The lyophilization of pharmaceuticals; A literature review." J. Parenteral Sci. Technol, 38: 48-59; and WO 2010 / 148337 A1.

[0091] Due to the temperature and pressure variations throughout the freeze-drying process, appropriate excipients or other components, such as stabilizers, buffers, extenders, and surfactants, are required to prevent the peptides described herein from degrading (e.g., protein aggregation, deamination, and / or oxidation) during freeze-drying and storage. The freeze-dried compositions disclosed herein contain a specific combination of components that enables the stable long-term storage of the peptides disclosed herein (which include a circular sequence recombinant interleukin-2 (IL-2) fused to the extracellular portion of the IL-2Rα chain).

[0092] In another aspect, this disclosure provides a lyophilized composition manufactured by lyophilizing any of the aqueous compositions disclosed herein, wherein the aqueous compositions disclosed herein include a circular sequence recombinant interleukin-2 (IL-2) fused to the extracellular portion of an IL-2Rα chain. In a particular specific example, the lyophilized composition is a lyophilized cake. In a particular specific example, the lyophilized composition is prepared by lyophilizing any of the aqueous compositions disclosed herein according to the lyophilization method described in Table 11A or Table 11B.

[0093] In another aspect, this disclosure provides a method for manufacturing a lyophilized composition, the method comprising lyophilizing any of the aqueous compositions disclosed herein, wherein the aqueous compositions disclosed herein comprise a circular sequence recombinant interleukin-2 (IL-2) fused to the extracellular portion of an IL-2Rα chain. In a particular specific example, the method for manufacturing the lyophilized composition comprises following the lyophilization method described in Table 11A or Table 11B.

[0094] In another aspect, this disclosure provides a method for manufacturing an aqueous composition, the method comprising dissolving any of the lyophilized compositions disclosed herein in an aqueous solvent, wherein the lyophilized compositions disclosed herein comprise a circular sequence recombinant interleukin-2 (IL-2) fused to the extracellular portion of an IL-2Rα chain. In a particular example, the lyophilized composition is a lyophilized cake. In a particular example, the lyophilized composition is dissolved in 1.1 ml of water. In a particular example, the lyophilized composition is dissolved in 2.2 ml of water. Use of the polypeptide composition

[0095] The components disclosed herein may be used specifically to treat, prevent or improve any disease or condition related to interleukin-2 receptor signaling.

[0096] In one aspect, a method for activating natural killer (NK) cells in a subject is provided, the method comprising administering to the subject an effective amount of any of the components disclosed herein, wherein the components disclosed herein comprise a circular sequence recombinant IL-2 fused to the extracellular portion of an IL-2Rα chain.

[0097] In another aspect, a method for treating cancer in a subject with this need is provided, the method comprising administering to the subject an effective amount of any of the compositions disclosed herein, wherein the compositions disclosed herein comprise a circular sequence recombinant IL-2 fused to the extracellular portion of an IL-2Rα chain. Cancers suitable for treatment using the compositions disclosed herein include renal cell carcinoma, melanoma, ovarian cancer, and lung cancer. In a particular specific instance, the cancer includes refractory solid tumors.

[0098] In a specific instance, the composition is administered subcutaneously.

[0099] In specific examples, the composition system is administered subcutaneously at a dose of about 1 mg to about 15 mg. In specific examples, the composition system is administered subcutaneously at a dose of about 1 mg. In specific examples, the composition system is administered subcutaneously at a dose of about 2 mg. In specific examples, the composition system is administered subcutaneously at a dose of about 3 mg. In specific examples, the composition system is administered subcutaneously at a dose of about 4 mg. In specific examples, the composition system is administered subcutaneously at a dose of about 5 mg. In specific examples, the composition system is administered subcutaneously at a dose of about 6 mg. In specific examples, the composition system is administered subcutaneously at a dose of about 7 mg. In specific examples, the composition system is administered subcutaneously at a dose of about 8 mg. In specific examples, the composition system is administered subcutaneously at a dose of about 9 mg. In specific examples, the composition system is administered subcutaneously at a dose of about 10 mg. In specific instances, the composition system is administered subcutaneously at a dose of approximately 11 mg. In specific instances, the composition system is administered subcutaneously at a dose of approximately 12 mg. In specific instances, the composition system is administered subcutaneously at a dose of approximately 13 mg. In specific instances, the composition system is administered subcutaneously at a dose of approximately 14 mg. In specific instances, the composition system is administered subcutaneously at a dose of approximately 15 mg.

[0100] In specific instances, the composition is administered subcutaneously once a week (Q1W), once every two weeks (Q2W), or once every three weeks (Q3W).

[0101] In specific instances, the composition is administered subcutaneously at a dose of about 1 mg to about 15 mg once a week (Q1W), once every two weeks (Q2W), or once every three weeks (Q3W).

[0102] In specific instances, the composition was administered subcutaneously once weekly (Q1W) at a dose of approximately 3 mg. In specific instances, the composition was administered subcutaneously once every three weeks (Q3W) at a dose of approximately 6 mg.

[0103] In a specific instance, the melanoma is either mucosal melanoma or advanced cutaneous melanoma, or both.

[0104] Those skilled in the art should readily understand that, without departing from the scope of the specific examples disclosed herein, suitable equivalents can be used to make other appropriate modifications and adjustments to the methods described herein. Specific examples have now been described in detail; referring to the following examples will provide a clearer understanding of these specific examples, which are included for illustrative purposes only and are not intended to be limiting. Examples

[0105] The present invention is further illustrated by the following examples, which should not be considered as further limitations. Unless otherwise indicated, the implementation of the present invention will utilize techniques of conventional organic synthesis, cell biology, cell culture, molecular biology, gene transfer biology, microbiology, and immunology within the scope of this art. Example 1 – Design and detection of polypeptide components

[0106] To determine the optimal subcutaneous formulation of peptide A (a circular recombinant IL-2 containing the amino sequence of SEQ ID NO: 1 fused to the extracellular portion of the IL-2Rα chain), several formulations of peptide A were tested for their protein stability, pH stability, physicochemical properties, lyophilized cake uniformity, and adhesion resistance to storage vials after lyophilization. This study sought two main objectives. The primary objective was to produce lyophilized cakes that, when reconstituted with readily available diluents (i.e., water for injection), produced an isotonic solution suitable for administration. Formulations that, when reconstituted with WFI, produced a non-isotonic solution were unsuitable for subcutaneous administration. The secondary objective was to produce lyophilized cakes with the optimal cake appearance and minimal cake shrinkage. An improved cake appearance could make the drug product more visually appealing to patients or healthcare professionals, potentially improving drug use compliance. Table 1 below illustrates the specific components and concentrations of peptide A formulations originally designed for intravenous administration. Table 1: Intravenous administration formulation of peptide A Components Function Concentration (mg / mL) Polypeptide A protein 1, 5 and 15 sucrose Protein stabilizers 50 Sodium citrate dihydrate buffer 2.03 Citric acid monohydrate buffer 0.97 Polysorbate 20 surfactants 0.1 Buffer pH 6.1 Osmotic pressure (mOsm / kg) when dissolved in WFI ~125 mOsm / kg The analytical method used was Fourier transform infrared (FTIR) spectroscopy.

[0107] FTIR analysis was performed using a PROTA FTIR protein analyzer equipped with CaF2 Biocells and an ATR tank for solid sample analysis. Approximately 10 μL of liquid sample or 10 mg of lyophilized powder sample was loaded for analysis. If appropriate, the absorption signal was processed by subtracting interference signals from the background lyophilized placebo and buffer. Finally, the processed data was converted to a second derivative signal using parameters set to 100 scans at 4 cm⁻¹ resolution to improve resolution. The percentage of different structural elements identifying native peptide A was obtained by subtracting from the spectrum processed using a protein secondary structure database. The percentage of similarity between native and dried states was calculated based on the overlap area of ​​1700–1600 cm⁻¹ or 1800–1400 cm⁻¹. Osmotic pressure analysis

[0108] The osmotic pressure of the prepared formulations was determined using a Wescor Vapro vapor osmometer. Approximately 10 μL of liquid sample was used for each analysis. Visual inspection was performed.

[0109] After digital photography, all vials were compared against a clear background for inspection. Sub-environmental and dry powder differential scanning calorimetry (DSC) was then performed.

[0110] DSC analysis was performed using a TA Q20 with refrigerated cooling system I. For this environment (frozen state) DSC, approximately 15 μL of prepared drug was loaded into the DSC tray and sealed. The sample was then cooled to -90°C at 10°C / min. The DSC tray was placed in the sample chamber for 2 minutes, and then heated to 30°C at 10°C / min. During the refrigeration period, after maintaining at -90°C for 2 minutes, the sample was heated to -10°C, then cooled back to -90°C, and then heated to 30°C at 10°C / min.

[0111] For high-temperature DSC of the dry powder, vials were placed in a drying oven with dry air at 8% RH or below. Equal aliquots of the lyophilized sample were removed and sealed in the DSC tray. A thermal scan was performed at 1°C / minute from 20°C to 180°C using a modulation program of ±1°C every 120 seconds, and the resulting reversible and irreversible heat fluxes were measured. Screening Study 1

[0112] The first screening involved various formulations to understand how different combinations of stabilizers, extenders, etc., affect the glass transition temperature or collapse temperature (Tg') and isotonicity. All formulations contained 12 mM sodium citrate buffer at pH 6.11 as a base formulation, followed by the addition of screening excipients. Tween 21 was used as a surface stabilizer. The formulations evaluated by sub-environmental DSC and osmolarity analysis in this study are summarized in Table 2. Table 2: Formulations from Screening Study 1 Ingredients# Polypeptide A (mg / mL) sucrose (mg / mL) Mannitol (mg / mL) osmotic pressure (mOsm / kg) Tg'°C 1 5 5 15 123 -28.81 2 5 5 10 Not measured -27.91 3 5 10 10 66 -28.00 4 5 5 - 59 -25.70

[0113] The osmotic pressure values ​​of formulations 1-4 were too low; ideally, they should be close to physiological values ​​(280-320 mOsm / kg). Formulation 1 showed signs of metastable behavior in the frozen phase; therefore, the lyophilization cycle was annealed at -10°C, which successfully transformed the metastable state into a stable eutectic phase. Glycine at a concentration of 30 mg / mL was added to formulation 4 in an attempt to increase the osmotic pressure of these formulations. This resulted in an osmotic pressure of 413 mOsm / kg. Based on this result, a glycine formulation was prepared to replace formulations 1 and 2, targeting an osmotic pressure of 290 mOsm / kg, with a DSC-corresponding glass transition temperature of 28.23°C. Additionally, the concentrations of sucrose and mannitol excipients in formulations 3 and 4 were adjusted to increase the osmotic pressure values. The four new formulations were lyophilized and analyzed in Screening Study 2. Screening Study 2

[0114] The formulations tested in Screening Study 2 and their associated osmolarity values ​​are shown in Table 3 below. All formulations contained 12 mM sodium citrate buffer at pH 6.11 as a base formulation, followed by the addition of screening excipients. Tween 21 was used as a surface stabilizer. Note that placebos were prepared for each formulation for secondary structure analysis after lyophilization in a VirTis Genesis SQ Super XL–70 freeze dryer. Table 3: Percentage of similarity between formulations from Screening Study 2 and secondary structures determined by osmolarity and FTIR. Ingredients# Polypeptide A (mg / mL) sucrose (mg / mL) Mannitol (mg / mL) Glycine (mg / mL) osmotic pressure (mOsm / kg) Compared to the original Similarity% 5 5 5 - 15 268 52.6 6 5 10 - 15 248 65.3 7 5 5 30 - 206 51.9 8 5 10 30 - 224 80.6

[0115] All samples were sterilized and filtered under aseptic conditions, and then filled into sterile 2 cc vials in 0.5 mL volumes. Table 4 details the lyophilization cycle parameters used in Screening Study 2. Table 4: Lyophilization Cycle Parameters for Screening Study 2 step temperature (°C) time (minute) heating and cooling rate (°C / min) tank pressure (mTorr) Bearing 5 - - - Freezing and annealing 5 to -40 100 0.5 - Stay at -40 30 - - -40 to -10 60 0.5 - Stay at -10 60 - - -10 to -40 60 0.5 - Stay at -40 60 - 100 One-time drying -40 to -25 30 0.5 100 Stay at -25 600 - 100 Secondary drying -25 to 10 100 0.5 100 Keep at 10 60 - 100 plug 10 Sealed under partial vacuum (nitrogen backfill).

[0116] After freeze-drying, all freeze-dried cakes showed no signs of remelting or collapse (no images were taken). The secondary structure of the samples was examined and compared with the original structure collected in the liquid bath. The FTIR spectrum (second derivative) of natural polypeptide A was overlaid with formulation 2 from screening study 2. The percentage of similarity between the original and dried states was calculated based on the area of ​​the overlaid second derivative at 1800 to 1400 cm⁻¹, as shown in Table 3. Formulation 8 showed the best retention of secondary structure after freeze-drying, however, with low tension. Screening Study 3

[0117] Based on the above results, a third screening study was conducted to explore different ratios of stabilizers and extenders. Furthermore, trehalose (a non-reducing disaccharide) and polyvinylpyrrolidone (PVP) (a polymer) were introduced to evaluate their effects as stabilizers and their effectiveness in improving Tg'. Eight formulations were prepared for screening study 3 and freeze-dried using the lyophilization cycle parameters described in Table 4. The formulation matrix for this study is detailed in Table 5 below. Formulations 9-16 in this study used 0.1 mg / mL Tween 20 (PS20). Table 5: Percentage of secondary structure similarity between formulations in screening study 3 and FTIR measurements. Ingredients# Polypeptide A (mg / mL) sucrose (mg / mL) Trehalose (mg / mL) Mannitol (mg / mL) PVP (mg / mL) Compared with the original Similarity% 9 5 5 - 30 - 40.3 10 5 10 - 30 - 73.7 11 5 15 - 20 - 61.5 12 5 15 - 20 10 45.4 13 5 20 - 20 - 58.4 14 5 - 15 15 - 81.2 15 5 - 15 15 10 76.0 16 5 - 20 15 - 71.8

[0118] As described above, FTIR spectroscopy was used to determine the percentage of similarity to natural polypeptide A. Besides formulation 8, formulations 10, 14, 15, and 16 showed the most promising results with >70% similarity. Based on this observation, screening study 4 was designed with five formulations prepared with low amounts of mannitol and high amounts of disaccharides. This combination appears to be advantageous for retaining a higher level of secondary structure. Screening Study 4

[0119] Table 6 describes the five formulations prepared in Screening Study 3 and freeze-dried using the lyophilization cycle parameters described in Table 4. Osmotic pressure measurements were not performed. Formulations 17-21 in this study used 0.1 mg / mL Tween 20 (PS20). Table 6: Percentage of secondary structure similarity between formulations from Screening Study 4 and those measured by FTIR. Ingredients# Polypeptide A (mg / mL) sucrose (mg / mL) Trehalose (mg / mL) Mannitol (mg / mL) Compared to the original Similarity% 17 5 15 - 10 78.4 18 5 15 - 5 84.5 19 5 - 15 10 64.5 20 5 - 15 5 67.5 twenty one 5 - 25 - 95.2

[0120] As described above, FTIR spectra were measured to determine the percentage of similarity to natural polypeptide A. Based on these results, it was confirmed that higher amounts of disaccharides have a greater impact on the retention of secondary structures. The next screening study is designed to explore combinations of higher concentrations of disaccharides and extenders. Fine-tuning tension.

[0121] Before the next screening, the isotonicity of various formulations was fine-tuned. Many formulations containing excipient combinations were prepared in 12 mM sodium citrate buffer at pH 6.1 with high amounts of disaccharides and varying amounts of extender, followed by osmotic pressure measurement. The different formulations and the resulting osmotic pressure values ​​are shown in Table 7. All formulations contained 5 mg / mL of peptide A. Table 7: Osmotic Pressure Screening Ingredients# sucrose (mg / mL) Trehalose (mg / mL) Mannitol (mg / mL) Glycine (mg / mL) osmotic pressure (mOsm / kg) twenty two 20 - - 40 612 twenty three 25 - - 15 303 twenty four 30 - - 10 258 25 - 25 10 - 164 26 - 25 20 - 209 27 - 30 30 - 289 28 - 30 - 20 372 29 - 20 - 20 293 30 - 25 - 15 303 Screening Study 5

[0122] Table 8 describes the five formulations prepared in Screening Study 3 as described above. Formulations 31-35 in this study used 0.1 mg / mL Tween 20 (PS20). Glycine in formulations 33 and 34 interfered with FTIR analysis, therefore no similarity percentage data were reported. Furthermore, it is more difficult to generate an effective lyophilization cycle with glycine than with mannitol. Therefore, formulations containing mannitol were further studied. Table 8: Percentage of similarity between formulations from Screening Study 5 and secondary structures measured by osmotic pressure and FTIR. Ingredients# sucrose (mg / mL) Trehalose (mg / mL) Mannitol (mg / mL) Glycine (mg / mL) osmotic pressure (mOsm / kg) Compared with the original Similarity% 31 - 30 30 - 299 80.8 32 30 - 30 - 300 77.9 33 - 30 - 10 252 - 34 - 20 - 15 292 - 35 - 20 35 - 282 89.0

[0123] All samples were sterilized and filtered under aseptic conditions, and then filled into sterile 2 cc vials in 0.5 mL volumes. Table 9 details the lyophilization cycle parameters used in Screening Study 5. Table 9: Lyophilization Cycle Parameters for Screening Study 5 step temperature (°C) time (minute) heating and cooling rate (°C / min) Tank pressure (mTorr) Bearing 5 - - - Freezing and annealing 5 to -45 100 0.5 - Stay at -45 30 - - -45 to -10 70 0.5 - Stay at -10 40 - - -10 to -45 70 0.5 - Stay at -45 30 - 90 One-time drying -45 to -30 30 0.5 90 Stay at -30 1260 - 90 Secondary drying -30 to 10 100 0.5 90 Keep at 10 60 - 90 plug 10 Sealed under partial vacuum (nitrogen backfill).

[0124] After freeze-drying, the secondary structure was determined and compared with the natural state as described above, and the percentage of similarity was calculated. Leading candidate formulations.

[0125] Based on FTIR and osmotic pressure results, the appearance, glass transition temperature (Tg') in the frozen state, and glass transition temperature (Tg) in the dry state of formulations 31 and 32 were tested. The intravenous administration formulation of peptide A was used as a comparison. The compositions of the three formulations evaluated in this screening are described in Table 10. Table 10: Formulations with Sub-environmental Glass Transition Temperatures Ingredients# Polypeptide A (mg / mL) Polysorbate 20 (mg / mL) sucrose (mg / mL) Trehalose (mg / mL) Mannitol (mg / mL) Tg'(°C) 36 5 0.1 50 - - -29.42 37 (31 in Table 8) 5 0.1 30 - 30 -38.39 38 (32 in Table 8) 5 0.1 - 30 30 -37.83

[0126] All formulations (placebo and active formulation) were prepared and sterilely filtered under aseptic conditions, then filled into sterile 5 cc vials at 2.28 mL volumes. A single aliquot (15 μL) of each candidate formulation was analyzed by sub-environmental DSC to evaluate its frozen state properties. The formulations were lyophilized using the cycle parameters listed in Table 11A, based on Tg' results. An annealing step was used to ensure complete crystallization of the extender mannitol. An alternative lyophilization cycle for similarly effective formulations is described in Table 11B below. Table 11A: Lyophilization Cycle Parameters for Leading Candidate Formulations step temperature (°C) time (minute) heating and cooling rate (°C / min) tank pressure (mTorr) Bearing 5 - - - Freezing and annealing 5 to -42 94 0.5 - Stay at -42 30 - - -42 to -10 64 0.5 - Stay at -10 60 - - -10 to -40 20 0.5 - Stay at -40 60 - 100 One-time drying -45 to -30 20 0.5 100 Stay at -30 2580 - 100 Secondary drying -30 to 25 110 0.5 100 Keep at 25 540 - 100 plug 25 Sealed under partial vacuum (nitrogen backfill). Table 11B: Another alternative freeze-drying cycle parameters step temperature (°C) time (minute) tank pressure (mTorr) Bearing 5 - 760 Bearing 5 30 550 Freezing and annealing 5 to -45 150 550 Stay at -45 120 550 -45 to -10 105 550 Stay at -10 180 550 -10 to -45 105 550 Stay at -45 120 550 One-time drying Stay at -45 30 0.1 -45 to -25 40 0.1 Stay at -25 3000 0.1 Secondary drying -25 to 30 220 0.1 Keep at 30 1440 0.1 Bearing 30 - 600

[0127] After freeze-drying, all samples were evaluated based on the appearance of the freeze-dried cakes, glass transition temperature (Tg) at the point of drying, pH, reconstruction time, osmotic pressure, concentration and water content, SEC, RP, and potency analysis. Appearance of freeze-dried cakes

[0128] The vial contained intact white cake pieces. The formulation containing 50 mg / mL sucrose (formulation 36) showed slight cake shrinkage. All other formulations showed no signs of shrinkage. High-temperature DSC of the freeze-dried cake pieces.

[0129] Table 12 summarizes the results of high-temperature DSC analysis. The glass transition temperature (Tg) values ​​of formulations 36 and 37 were determined to be around ~82°C, which is attributed to sucrose. The glass transition temperature of the formulation containing trehalose (formulation 38) could not be detected under these conditions. The detected melting peak of formulation 36 at 158°C is attributed to the melting of sucrose. The detected melting peaks of formulations 37 and 38 at 120-121°C are attributed to the melting of mannitol. Table 12: Summary of Tg and Melting Temperature Ingredients# Sample weight (mg) Glass transition temperature Tg (°C) melting temperature Tm (°C) 36 4.5 81.86 157.86 37 (31 in Table 8) 5.2 81.86 119.69 38 (32 in Table 8) 4.9 - 120.80 pH, regeneration time, osmotic pressure, concentration, and water content of leading candidate formulations

[0130] Reconstituted freeze-dried cakes using WFI. Table 13 summarizes the results for pH, reconstruction time, osmotic pressure, concentration, and moisture content. Table 13: Summary of Tg and melting temperature Ingredients# pH osmotic pressure (mOsm / kg) Stirring and vortex Reconstruction time (seconds) Moisture content (%) concentration (mg / mL) 36 6.18 188 20 1.55 5.127 37 (31 in Table 8) 6.20 296 twenty two 1.74 5.058 38 (32 in Table 8) 6.17 289 16 1.79 4.566 Size exclusion (SE) HPLC and reverse-phase (RP) HPLC of leading candidate formulations

[0131] SE-HPLC and RP-HPLC analyses were performed on formulations 36, 37, and 38. The combined results of the SE-HPLC analyses showed that each formulation had a maximum peak with a peak area >98%. The combined results of the RP-HPLC analyses showed that each formulation had a maximum peak with a peak area >85%. Potential analysis of leading candidate formulations.

[0132] To confirm whether the excipients affected the biological activity, formulations 36-38 were compared using pSTAT5 activity analysis. The dose-response curves were similar and equivalent to the reference standard for peptide A. Table 14 shows the data analysis of EC50 values, which, relative to the reference standard RT, showed a potency greater than 75%, which is acceptable. Table 14: pSTAT5 Activity Analysis Results parameter 36 ingredients 37 ingredients 38 ingredients Lower asymptote 0.116 0.109 0.072 Upward asymptote 0.757 0.786 0.755 slope 1.29 1.24 1.34 EC50 11.59 12.49 16.07 Relative effectiveness 84% 78% 78%

[0133] pSTAT5 activity was analyzed by measuring the binding of the formulations to HH cells (a human T lymphocyte line with a βγIL2 receptor isotype on its surface). After contact with each formulation, peptide A binding was measured using ELISA by measuring the amount of phosphorylated STAT5 (phosphate-STAT5 or pSTAT5) present in HH cells. ELISA analysis was performed using the Invitrogen InstantOne ELISA phosphate-STAT5 α / β (pTyr694 / pTyr699) kit.

[0134] A drug sample was prepared by reconstructing the sample with 2.2 mL of WFI. The sample was visually inspected to confirm that there were no visible particles in the contents.

[0135] A sample dilution with a final FBS concentration of 5% was prepared by adding 25 mL of fetal bovine serum (FBS) to 500 mL of Hank's balanced salt solution (HBSS) and warming to 37°C. A wash buffer containing 0.05% Tween 20 and phosphate-buffered saline (PBS) was used.

[0136] Dilute samples and standards to final analytical protein concentrations of 750 ng / mL, 250 ng / mL, 83 ng / mL, 28 ng / mL, 9.3 ng / mL, 3.1 ng / mL, 1.0 ng / mL, and 0.3 ng / mL. Prepare HH cell stock solution at a density of approximately 1.2 x 10⁶ cells / mL and add 50 µl of the cell stock solution to each well of a 96-well plate containing diluted samples or standards. Incubate cells at 37°C for 30 minutes. After incubation, dissociate cells with cell dissociation buffer for 10 minutes. After dissociation, transfer 50 µl of the dissociated cell mixture to an ELISA plate, followed by 50 µl of phosphate-STAT5 A / B antibody mixture. Incubate the mixture for 1 hour, then wash three times with washing buffer. Add 100 µl of the detection reagent to each well and incubate the plate for 15 minutes. Then, 100 µl of stop solution was added to each well, and the disc was read at 450 nm using a microdisc reader.

[0137] Measure the individual EC50 and calculate the % relative standard deviation (RSD) of the reference standard EC50 value and the control EC50 value.

[0138] Calculate the geometric mean of the three EC50 values ​​of the reference standard (reference standard EC50GM) and the geometric mean of the three EC50 values ​​of the control group (control EC50). Calculate the relative power of the control using the following equation: Relative power = (reference standard EC50GM) / (control EC50) × 100%.

[0139] The sample was calculated in the same manner. The analytical results were determined by the following equation: Relative power = (Reference standard EC50GM) / (Test sample EC50) × 100%. Initial conclusions

[0140] Based on the results obtained and their similarity to the intravenously administered peptide A formulation (which also contains sucrose as a stabilizer), formulation 37 was selected. The composition of the formulation is shown in Table 15. When reconstituted using WFI, the selected formulation produces an isotropic solution, thereby increasing its availability for direct subcutaneous drug delivery.

[0141] Screening studies showed that high levels of disaccharides, such as sucrose or trehalose, in the formulation significantly improved the preservation of the secondary structure of peptide A. Furthermore, mannitol was determined to be a superior extender and tension modifier compared to glycine. Table 15: Peptide A Formulation 37 Components Function Concentration (mg / mL) Polypeptide A protein 1 and 5 sucrose Protein stabilizers 30 Mannitol Incremental agent 30 Trisodium citrate dihydrate buffer 2.03 Citric acid monohydrate buffer 0.97 Polysorbate 20 surfactants 0.1

[0142] After identifying the above-mentioned formulation 37, the concentrations of trisodium citrate dihydrate and citrate monohydrate were optimized to avoid reaching pH 6.1 in the pH titration step. Table 16 below describes the modified formulation 37-2. Table 16: Polypeptide A formulation 37-2 Components Function Concentration (mg / mL) Polypeptide A protein 1 and 5 sucrose Protein stabilizers 30 Mannitol Incremental agent 30 Trisodium citrate dihydrate buffer 2.91 Citric acid monohydrate buffer 0.34 Polysorbate 20 surfactants 0.1 Further testing of mannitol / sucrose content

[0143] The amounts of sucrose and mannitol in formulation 37 were varied as shown in Table 17 to test osmotic pressure and the appearance of the lyophilized cake. Each formulation in Table 17 contained a 12 mM sodium citrate buffer at pH 6.1. After lyophilization, formulation 37 exhibited the best lyophilized cake appearance compared to other formulations, showing a more concave appearance and no shrinkage on the walls. This improved cake appearance can make the pharmaceutical product more visually appealing to patients and healthcare professionals, thus potentially improving drug use compliance. Table 17: Formulations with varied amounts of mannitol and sucrose sample# Ingredients# Polypeptide A (mg / mL) Polysorbate 20 (mg / mL) sucrose (mg / mL) Mannitol (mg / mL) osmotic pressure (mOsm / kg) 1 37 5 0.1 30 30 285 2 39 5 0.1 20 40 311 3 40 5 0.1 13.5 40 292 4 41 5 0.1 10 40 281 5 42 5 0.1 90 - 296 6 36 5 0.1 50 - 179

Claims

1. A composition comprising: a) A polypeptide of about 1 mg to about 50 mg, comprising a circular sequence recombinant IL-2 fused to the extracellular portion of the IL-2Rα chain; b) sucrose; c) mannitol; d) citrate buffer; and e) emulsifier.

2. The composition of claim 1, wherein the polypeptide comprises an amino acid sequence having at least 95% identity with SEQ ID NO:

1.

3. The composition of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

1.

4. A composition of any one of claims 1-3, wherein the composition comprises about 1 mg to about 15 mg of a polypeptide.

5. A composition of any one of claims 1-4, wherein the composition comprises about 1 mg of a polypeptide.

6. A composition of any one of claims 1-4, wherein the composition comprises about 5 mg of a polypeptide.

7. A composition of any one of claims 1-4, wherein the composition comprises about 15 mg of a polypeptide.

8. A composition of any one of claims 1-4, wherein the composition comprises about 20 mg of a polypeptide.

9. A composition of any one of claims 1-4, wherein the composition comprises about 30 mg of a polypeptide.

10. A composition as claimed in any of the preceding claims, wherein the composition comprises about 60 mg to about 72 mg of sucrose.

11. A composition as claimed in any of the preceding claims, wherein the composition comprises about 66 mg of sucrose.

12. A composition of any of the preceding claims, wherein the composition comprises about 60 mg to about 72 mg of mannitol.

13. A composition as claimed in any of the preceding claims, wherein the composition comprises about 66 mg of mannitol.

14. A composition of any of the preceding claims, wherein the composition comprises about 4.0 mg to about 6.0 mg of citrate anion.

15. A composition as claimed in any of the preceding claims, wherein the composition comprises about 5.0 mg of citrate anion.

16. A composition as claimed in any of the preceding claims, wherein the composition comprises citric acid and trisodium citrate dihydrate in a mass ratio of about 1:10 to about 1:

2.

17. A composition as claimed in any of the preceding claims, wherein the composition comprises citric acid and trisodium citrate dihydrate in a mass ratio of approximately 1:9 of citric acid to trisodium citrate dihydrate.

18. A composition as claimed in any of the preceding claims, wherein the composition comprises citric acid and trisodium citrate dihydrate in a mass ratio of about 1:2 of citric acid and trisodium citrate dihydrate.

19. A composition of any of the preceding claims, wherein the emulsifier comprises polysorbate 20.

20. The composition of claim 19, wherein the composition comprises about 0.10 mg to about 0.12 mg of polysorbate 20.

21. The composition of claim 19, wherein the composition comprises about 0.11 mg of polysorbate 20.

22. A component of any of the preceding claims, wherein the component is a freeze-dried cake block.

23. The composition of claim 22, wherein the freeze-dried cake dissolves in water to produce an aqueous solution with a pH of about 5.5 to about 6.

5.

24. The composition of claim 23, wherein the freeze-dried cake is dissolved in water to produce an aqueous solution with a pH of approximately 6.

1.

25. A composition of any one of claims 22-24, wherein the freeze-dried cake is dissolved in water to produce an aqueous solution having isotonic osmotic pressure.

26. A composition of any one of claims 22-25, wherein the freeze-dried cake is dissolved in water to produce an aqueous solution having an osmotic pressure of about 240 to about 340 mOsm / kg.

27. A composition of any one of claims 22-26, wherein the freeze-dried cake is dissolved in water to produce an aqueous solution having an osmotic pressure of about 280 to about 320 mOsm / kg.

28. The composition of claim 26, wherein the freeze-dried cake is dissolved in water to produce an aqueous solution having an osmotic pressure of about 285 mOsm / kg.

29. The composition of claim 26, wherein the freeze-dried cake is dissolved in water to produce an aqueous solution having an osmotic pressure of about 300 mOsm / kg.

30. A composition of any one of claims 1-29, wherein the composition is an aqueous solution.

31. The composition of claim 30, wherein the composition comprises a polypeptide of about 0.5 mg / mL to about 30 mg / mL.

32. The composition of claim 30, wherein the composition comprises about 1 mg / mL of a polypeptide.

33. The composition of claim 32, wherein the composition is a 1.1 ml aqueous solution comprising about 1.1 mg of polypeptide.

34. The composition of claim 31, wherein the composition comprises about 5 mg of polypeptide.

35. The composition of claim 34, wherein the composition is a 1.1 ml aqueous solution comprising about 15 mg of polypeptide.

36. The composition of claim 31, wherein the composition comprises about 20 mg / mL of polypeptide.

37. The composition of claim 31, wherein the composition comprises about 30 mg / mL of polypeptide.

38. A composition of any one of claims 30-37, wherein the composition comprises about 25 mg / mL to about 35 mg / mL of sucrose.

39. The composition of claim 38, wherein the composition comprises about 30 mg / mL sucrose.

40. A composition of any one of claims 30-37, wherein the composition comprises about 25 mg / mL to about 35 mg / mL mannitol.

41. The composition of claim 40, wherein the composition comprises about 30 mg / mL mannitol.

42. A composition of any one of claims 30-41, wherein the composition comprises about 10 mM to about 20 mM of citrate buffer.

43. The composition of claim 42, wherein the composition comprises about 12 mM citrate buffer.

44. A composition of any one of claims 30-43, wherein the citrate buffer is formed by mixing 2.03 mg / mL trisodium citrate dihydrate and 0.97 mg / mL citrate monohydrate in an aqueous solution.

45. A composition of any one of claims 30-43, wherein the citrate buffer is formed by mixing 2.91 mg / mL trisodium citrate dihydrate and 0.34 mg / mL citrate monohydrate in an aqueous solution.

46. ​​A composition of any one of claims 30-43, wherein the citrate buffer is formed by mixing 2.96 mg / mL trisodium citrate dihydrate and 0.30 mg / mL citrate monohydrate in an aqueous solution.

47. A composition of any one of claims 30-46, wherein the composition comprises about 0.09 mg / mL to about 0.11 mg / mL of polysorbate 20.

48. The composition of claim 47, wherein the composition comprises about 0.1 mg / mL polysorbate 20.

49. A composition of any one of claims 30-48, wherein the pH of the composition is about 5.5 to about 6.

5.

50. The composition of claim 49, wherein the pH of the composition is about 6.

1.

51. A composition of any one of claims 30-50, wherein the osmotic pressure of the composition is about 240 to about 340 mOsm / kg.

52. A composition of any one of claims 30-50, wherein the osmotic pressure of the composition is about 280 to about 320 mOsm / kg.

53. The composition of claim 51, wherein the osmotic pressure of the composition is about 285 mOsm / kg.

54. The composition of claim 51, wherein the osmotic pressure of the composition is about 300 mOsm / kg.

55. A composition of any one of claims 30-54, wherein the aqueous solution comprises about 0.03 mg / mL of a polypeptide to about 0.2 mg / mL of a polypeptide.

56. A freeze-dried composition prepared by freeze-drying a composition of any one of claims 30-55.

57. A composition of any of the preceding claims, wherein the composition is a single unit dose of a polypeptide.

58. An article comprising any of the preceding claims.

59. The product of claim 58 is a small glass vial.

60. A method for manufacturing a freeze-dried composition, the method comprising freeze-drying an aqueous solution of any one of claims 30-55.

61. A method for manufacturing an aqueous composition, the method comprising dissolving the composition as claimed in claim 22 or 56 in an aqueous solvent.

62. The method of claim 61, wherein the pH of the aqueous component is adjusted to approximately 6.

1.

63. The method of claim 61 or 62, wherein the pH of the aqueous component is adjusted to about 6.1 by using an alkali.

64. The method of claim 63, wherein the base is sodium hydroxide.

65. The method of any one of claims 61-64, wherein the aqueous composition is further diluted with an aqueous solution comprising about 1% (w / w) surfactant.

66. The method of claim 65, wherein the surfactant is polysorbate 20.

67. The method of claim 65, wherein the aqueous solution further comprises about 0.1% (w / w) citrate monohydrate, 0.2% (w / w) trisodium citrate dihydrate, and 98.7% (w / w) water for injection.

68. A method for activating natural killer (NK) cells in a subject, the method comprising administering to the subject an effective amount of any one of the components of claims 30-55.

69. A method for treating cancer in a subject in need, the method comprising administering to the subject an effective amount of any one of the components of claims 30-55.

70. The method of claim 69, wherein the cancer is renal cell carcinoma, melanoma, ovarian cancer, or lung cancer.

71. The method of claim 69 or 70, wherein the cancer includes refractory solid tumors.

72. The method of any one of claims 69-71, wherein the composition is administered subcutaneously.

73. The method of any one of claims 69-72, wherein the composition is administered subcutaneously at a dose of about 1 mg to about 15 mg.

74. The method of any one of claims 69-73, wherein the composition is administered subcutaneously at a dose of about 1 mg to about 15 mg once a week (Q1W), once every two weeks (Q2W), or once every three weeks (Q3W).

75. The method of any one of claims 69-73, wherein the melanoma is one or both of mucosal melanoma and advanced cutaneous melanoma.