WO2022100686A1

WO2022100686A1 - A pharmaceutical composition comprising human il-2 variant or derivative and use thereof

Info

Publication number: WO2022100686A1
Application number: PCT/CN2021/130277
Authority: WO
Inventors: 杨晓容; 陈钟威; 王淇
Original assignee: 江苏恒瑞医药股份有限公司; 上海恒瑞医药有限公司; 上海盛迪医药有限公司
Priority date: 2020-11-13
Filing date: 2021-11-12
Publication date: 2022-05-19
Also published as: TW202227122A

Abstract

The present disclosure relates to a pharmaceutical composition comprising a human IL-2 variant or derivative and use thereof. In particular, the disclosure relates to a pharmaceutical composition comprising an IL-2 variant or derivative and a pharmaceutically acceptable excipient. The pharmaceutical composition has improved high temperature stability, freeze-thaw stability and normal temperature stability as well as appearance formulation reproducibility.

Description

A kind of pharmaceutical composition comprising human interleukin 2 variant or derivative thereof and use thereof

This application claims the priority of the patent application (application number 202011269123.7) filed on November 13, 2020.

technical field

The present disclosure relates to the field of pharmaceutical preparations, and more particularly, to a pharmaceutical composition comprising a variant of human interleukin 2 or a derivative thereof.

Background technique

Human interleukin-2 (interleukin-2, IL-2), also known as T cell growth factor (TCGF). The IL-2 gene is located on chromosome 4 (4q27), including a sequence of about 7kb, consisting of about 133 amino acids, and a molecular weight of about 15kD.

In 1976 and 1977, Doris Morgan, Francis Ruscetti, Robert Gallo, Steven Gillis, Kendal Smith and others found that activated T cell culture medium could promote T cell proliferation. The stimulating factor in the culture medium was purified and identified as a single protein, IL-2. The ability of IL-2 to expand lymphocyte populations and enhance the effector function of these cells in vivo lends itself to its antitumor effect. IL-2 immunotherapy has emerged as a treatment option for some patients with metastatic cancer. Currently, high-dose IL-2 has been approved for the treatment of metastatic renal cell carcinoma and malignant melanoma.

A number of pharmaceutical companies are currently developing IL-2 variants, and related patent applications include WO2012062228, CN201280017730.1, US8906356, US9732134, US7371371, US7514073, US8124066, US7803361, WO2016014428, etc. For example, WO2020125743 relates to a new class of IL-2 variants and derivatives thereof, with higher stability and improved properties for use as immunotherapeutics.

IL-2 variants and their derivatives are protein drugs, which are easily degraded when taken orally, and are thermally unstable and easily hydrolyzed. It is of great significance to obtain stable formulations with good appearance. US4604377 describes freeze-dried IL-2 formulations comprising the stabilizer mannitol and the solubilizer sodium dodecyl sulfate (SDS) or sodium deoxycholate sulfate. US5417970 describes lyophilized formulations of IL-2 comprising hydrolyzed gelatin or human serum albumin and alanine. However, SDS may bind to proteins and be difficult to remove, which is not conducive to renaturation; serum albumin has a large market demand, is expensive, and has a complicated preparation process. ZL01814445.4 discloses formulations of IL-2 stabilized by histidine, sucrose and glycine, and it was found that Tween 80 promotes the formation of soluble IL-2 aggregates, which are evident before lyophilization. Wang et al. (Dual effects of Tween 80 on protein stability, W. Wang et al./International Journal of Pharmaceutics 347 (2008) 31–38) also found that although Tween 80 significantly inhibited shaking-induced aggregation of IL-2 muteins, But Tween 80 affects the storage stability of IL-2. This effect is temperature-related: during storage at 5°C for 22 months, aggregation was almost undetectable due to the presence of Tween 80; during storage at 40°C for 2 months, addition of 0.1% Tween 80 significantly increased IL- 2 Aggregation rates of mutant proteins.

In addition, appearance is one of the important quality attributes of freeze-dried products. The appearance of qualified freeze-dried products should be loose and porous, uniform in color and fine in texture. In large-scale production, the cakes of freeze-dried products sometimes have unqualified appearances such as shrinkage and cracking, resulting in great economic losses, and the auxiliary ingredients will affect the appearance of freeze-dried products.

In the present disclosure, according to the drug characteristics and dosage form characteristics of IL-2, through a large number of screening work, the pH, buffer, excipients and pH of the pharmaceutical composition are determined by indicators such as appearance, RP-HPLC\SE-HPLC\IE-HPLC purity and stability. Excipients, surfactants and other aspects were screened, and a pharmaceutical composition containing IL-2 was obtained. The pharmaceutical composition has improved high temperature, freeze-thaw, normal temperature stability, good appearance and good preparation reproducibility, and provides a product with better performance for actual production and clinical application.

SUMMARY OF THE INVENTION

The present disclosure provides a pharmaceutical composition comprising IL-2, mannitol and trehalose.

In some embodiments, the IL-2 is wild-type IL-2, an IL-2 variant, or a derivative thereof.

In some embodiments, the IL-2 concentration in the above pharmaceutical composition is about 0.1 mg/mL to 100 mg/mL, and in some specific embodiments, the IL-2 concentration is about 1 mg/mL to about 50 mg/mL, about 1 mg/mL to about 10 mg/mL, about 2 mg/mL to about 5 mg/mL.

In some embodiments, the concentration of IL-2 is about 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg /mL, 1.0mg/mL, 1.1mg/mL, 1.2mg/mL, 1.3mg/mL, 1.4mg/mL, 1.5mg/mL, 1.6mg/mL, 1.7mg/mL, 1.8mg/mL, 1.9mg /mL, 2.0mg/mL, 2.1mg/mL, 2.2mg/mL, 2.3mg/mL, 2.4mg/mL, 2.5mg/mL, 2.6mg/mL, 2.7mg/mL, 2.8mg/mL, 2.9mg /mL, 3.0mg/mL, 3.1mg/mL, 3.2mg/mL, 3.3mg/mL, 3.4mg/mL, 3.5mg/mL, 3.6mg/mL, 3.7mg/mL, 3.8mg/mL, 3.9mg /mL, 4.0mg/mL, 4.1mg/mL, 4.2mg/mL, 4.3mg/mL, 4.4mg/mL, 4.5mg/mL, 4.6mg/mL, 4.7mg/mL, 4.8mg/mL, 4.9mg /mL, 5.0mg/mL, 6.0mg/mL, 7.0mg/mL, 8.0mg/mL, 9.0mg/mL, 10mg/mL, 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL.

In some specific embodiments, the concentration of IL-2 is about 2 mg/mL ± 10%.

Mannitol, Trehalose:

In some embodiments, a saccharide is included in the pharmaceutical compositions of the present disclosure. The "sugar" includes conventional compositions (CH2O ₎ _n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like. The sugar can be selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol, Milibiose, meligotriose, raffinose, mannosetriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose, etc.

In some embodiments, mannitol, trehalose are included in the pharmaceutical compositions of the present disclosure.

In some embodiments, the concentration of mannitol is from about 5 mg/mL to about 100 mg/mL.

In some embodiments, the concentration of mannitol is about 10 mg/mL to about 50 mg/mL, about 15 mg/mL to 40 mg/mL, about 20 mg/mL to 35 mg/mL, about 25 mg/mL to 40 mg/mL, about 25 mg /mL to 35mg/mL. e.g. 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, 10mg/mL, 11mg/mL, 12mg/mL, 13mg/mL, 14mg/mL, 15mg/mL, 16mg/mL, 17mg /mL, 18mg/mL, 19mg/mL, 20mg/mL, 21mg/mL, 22mg/mL, 23mg/mL, 24mg/mL, 25mg/mL, 26mg/mL, 27mg/mL, 28mg/mL, 29mg/mL , 30mg/mL, 31mg/mL, 32mg/mL, 33mg/mL, 34mg/mL, 35mg/mL, 36mg/mL, 37mg/mL, 38mg/mL, 39mg/mL, 40mg/mL, 41mg/mL, 42mg /mL, 43mg/mL, 44mg/mL, 45mg/mL, 46mg/mL, 47mg/mL, 48mg/mL, 49mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL or 100mg/mL.

In some embodiments, the concentration of mannitol is about 30 mg/mL.

In some embodiments, the trehalose in the pharmaceutical compositions of the present disclosure comprises trehalose anhydrous or a hydrate thereof.

In some embodiments, the trehalose is trehalose dihydrate.

In some embodiments, trehalose is used in an amount of trehalose anhydrous at a concentration of about 10 mg/mL to about 100 mg/mL, about 20 mg/mL to about 80 mg/mL, about 20 mg/mL to about 60 mg/mL, about 30 mg /mL to about 50 mg/mL, about 30 mg/mL to 45 mg/mL, about 35 mg/mL to 40 mg/mL. For example, about 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, or 100 mg/mL.

In some specific embodiments, the amount of trehalose is based on anhydrous trehalose, and the concentration is about 10 mg/mL, 11 mg/mL, 12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, 16 mg/mL, 17 mg/mL. mL, 18mg/mL, 19mg/mL, 20mg/mL, 21mg/mL, 22mg/mL, 23mg/mL, 24mg/mL, 25mg/mL, 26mg/mL, 27mg/mL, 28mg/mL, 29mg/mL, 30mg/mL, 31mg/mL, 32mg/mL, 33mg/mL, 34mg/mL, 35mg/mL, 36mg/mL, 37mg/mL, 38mg/mL, 39mg/mL, 40mg/mL, 41mg/mL, 42mg/mL mL, 43mg/mL, 44mg/mL, 45mg/mL, 46mg/mL, 47mg/mL, 48mg/mL, 49mg/mL, 50mg/mL, 51mg/mL, 52mg/mL, 52.5mg/mL, 53mg/mL , 54mg/mL, 55mg/mL, 56mg/mL, 57mg/mL, 58mg/mL, 59mg/mL, 60mg/mL, 61mg/mL, 62mg/mL, 63mg/mL, 64mg/mL, 65mg/mL, 66mg /mL, 67mg/mL, 68mg/mL, 69mg/mL, 70mg/mL, 71mg/mL, 72mg/mL, 73mg/mL, 74mg/mL, 75mg/mL, 76mg/mL, 77mg/mL, 78mg/mL , 79mg/mL, 80mg/mL.

In some embodiments, trehalose is used in an amount of about 35 mg/mL based on anhydrous trehalose.

In some embodiments, the ratio of mannitol to trehalose (as trehalose anhydrous) is 1:10 to 10:1, 1:7 to 7:1, 1:6 to 6:1, 1:5 to 5:1 1, 1:4 to 4:1, 1:3 to 3:1, 1:2 to 2:1, 1:7 to 6:7, 1:7 to 1:1, 1:6 to 6:7, 1:6 to 1:1, 1:5 to 6:7, 1:5 to 1:1, 1:4 to 6:7, 1:4 to 1:1, 1:3 to 6:7, 1: 2 to 6:7, 1:2 to 1:1, 6:8 to 6:7. For example 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:9, 2:8, 2 :7, 2:6, 2:5, 2:4, 2:3, 2:2, 2:1, 3:9, 3:8, 3:7, 3:6, 3:5, 3:4 , 3:3, 3:2, 3:1, 4:9, 4:8, 4:7, 4:6, 4:5, 4:4, 4:3, 4:2, 4:1, 5 : 9, 5:8, 5:7, 5:6, 5:5, 5:4, 5:3, 5:2, 5:1, 6:9, 6:8, 6:7, 6:6 , 6:5, 6:4, 6:3, 6:2, 6:1, 7:9, 7:8, 7:7, 7:6, 7:5, 7:4, 7:3, 7 : 2, 7:1, 8:9, 8:8, 8:7, 8:6, 8:5, 8:4, 8:3, 8:2, 8:1, 9:9, 9:8 , 9:7, 9:6, 9:5, 9:4, 9:3, 9:2, 9:1, 10:1, 10:9, 10:8, 10:7, 10:6, 10 : 5, 10:4, 10:3, 10:2, 10:1.

The above ratio of mannitol and trehalose is a mass ratio, which can be obtained by actual conversion according to the concentrations of mannitol and trehalose, and this conversion method is well known in the art.

Similarly, in the pharmaceutical composition described in the present disclosure, the mass ratio of IL-2 to mannitol or trehalose can also be converted according to the concentration.

In some embodiments, the mass ratio of IL-2 to mannitol ranges from 1:1 to 1:50, eg, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1 : 7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50.

In some embodiments, the mass ratio of IL-2 to trehalose ranges from 4:50 to 4:150, eg, 4:50, 4:60, 4:70, 4:80, 4:90, 4:105, 4 : 120, 4: 140.

Surfactant:

In some embodiments, the pharmaceutical compositions of the present disclosure further comprise a surfactant. Surfactant can be selected from polysorbate 20, polysorbate 80, polyhydroxyalkene, Triton, sodium lauryl sulfonate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl-, meat Myristyl-, linoleyl-, stearyl-sulfobetaine, lauryl-, myristyl-, linoleyl-, stearyl-sarcosine, linoleyl-, myristyl-, Cetyl-Betaine, Lauramidopropyl-, Cocalamidopropyl-, Linoleamidopropyl-, Myristamidopropyl-, Palmamidopropyl-, Isostearamido Propyl-betaine, myristamidopropyl-, palmitamidopropyl-, isostearamidopropyl-dimethylamine, sodium methyl cocoayl, sodium methyl oleyl taurate, poly Ethylene glycol, polypropylene glycol, copolymers of ethylene and propylene glycol, and the like.

In some embodiments, the pharmaceutical compositions of the present disclosure comprise a polysorbate (eg, polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80).

In some embodiments, polysorbate 80 is included in the pharmaceutical compositions of the present disclosure. The present disclosure finds that polysorbate may act as a stabilizer by reducing the agglomeration of protein drugs. Polysorbate (eg, polysorbate 80) at concentrations of 0.01 mg/mL to 0.2 mg/mL, 0.02 mg/mL to 0.15 mg/mL, 0.02 mg/mL to 0.1 mg/mL, 0.03 mg/mL to 0.1 mg/mL mL, 0.04 mg/mL to 0.1 mg/mL, 0.02 mg/mL to 0.05 mg/mL, 0.05 mg/mL to 0.1 mg/mL; eg, about 0.01 mg/mL, about 0.02 mg/mL, about 0.03 mg/mL , about 0.04 mg/mL, about 0.05 mg/mL, about 0.06 mg/mL, about 0.07 mg/mL, about 0.08 mg/mL, about 0.09 mg/mL, about 0.1 mg/mL, about 0.15 mg/mL, about 0.2 mg/mL.

In some embodiments, the polysorbate (eg, polysorbate 80) concentration is about 0.05 mg/mL ± 10%.

Buffer system:

In some embodiments, the pharmaceutical compositions of the present disclosure further comprise a buffer. Said buffer includes, for example, citrate buffer, acetate buffer, histidine buffer, phosphate buffer, carbonate buffer, and succinate buffer. In some embodiments, the buffer is a phosphate buffer system.

In some aspects, the buffer concentration in the pharmaceutical composition is about 2 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 5 mM to about 20 mM, about 5 mM to about 15 mM, about 5 mM to about 10 mM , for example, about 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM.

In some embodiments, the buffer concentration in the pharmaceutical composition is about 10 mM ± 10%.

In some embodiments, the pH of the buffer in the pharmaceutical composition is about 5.5 to about 7.5, about 5.5 to about 7.0, about 6.0 to about 7.5, about 6.0 to about 7.0, eg, about 5.5, 6.0, 6.5, 7.0, 7.5 , in some embodiments, the buffer concentration of the drug from the composition is about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 , 7.1, 7.2, 7.3, 7.4, 7.5.

Solvent:

In some embodiments, the pharmaceutical compositions of the present disclosure further comprise a solvent.

The solvent in the pharmaceutical composition is selected from but not limited to non-toxic physiologically acceptable liquid carriers, such as physiological saline, water for injection, glucose solution (eg 5% glucose injection, glucose and sodium chloride injection) and the like.

In addition, the present disclosure also provides pharmaceutical compositions comprising IL-2, including but not limited to:

(1) a pharmaceutical composition comprising: IL-2, 30 mg/mL of mannitol, 35 mg/mL of trehalose, and 0.05 mg/mL of polysorbate, with a pH of about 6.5, wherein the IL-2 The concentration of 1mg/mL, 2mg/mL, 5mg/mL, 10mg/mL or 100mg/mL;

(2) a pharmaceutical composition comprising: IL-2, 30 mg/mL of mannitol, 35 mg/mL of trehalose, and 0.1 mg/mL of polysorbate, with a pH of about 6.5, wherein the IL-2 The concentration of 1mg/mL, 2mg/mL, 5mg/mL, 10mg/mL or 100mg/mL;

(3) a pharmaceutical composition comprising: 2 mg/mL IL-2, mannitol, 35 mg/mL trehalose, and 0.1 mg/mL polysorbate, pH is about 6.5, wherein the mannitol has a Concentrations of 10mg/mL, 20mg/mL, 30mg/mL, 35mg/mL or 50mg/mL;

(4) a pharmaceutical composition comprising: 2 mg/mL IL-2, mannitol, 35 mg/mL trehalose, and 0.05 mg/mL polysorbate, pH is about 6.5, wherein the mannitol has a Concentrations of 10mg/mL, 20mg/mL, 30mg/mL, 35mg/mL or 50mg/mL;

(5) A pharmaceutical composition comprising: 2 mg/mL IL-2, 30 mg/mL mannitol, trehalose, and 0.1 mg/mL polysorbate, the pH is about 6.5, wherein the trehalose has a Concentrations of 10mg/mL, 20mg/mL, 30mg/mL, 35mg/mL or 50mg/mL;

(6) A pharmaceutical composition comprising: 2 mg/mL of IL-2, 30 mg/mL of mannitol, trehalose, and 0.05 mg/mL of polysorbate, with a pH of about 6.5, wherein the trehalose has a pH of about 6.5. Concentrations are 10 mg/mL, 20 mg/mL, 30 mg/mL, 35 mg/mL or 50 mg/mL.

In some embodiments, the IL-2 is an IL-2 variant or derivative.

IL-2 as described in the present disclosure includes IL-2 variants or derivatives thereof. In an alternative embodiment, the IL-2 variant or derivative thereof in the aforementioned pharmaceutical composition is at the 11th, 20th, 26th, 27th, 29th, 30th, 31st, 32nd, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 70, 71, 72, 78, 82, 88, 125, 126, 132 have one or more amino acids mutation. The expression for mutation in the present disclosure is abc, or ab, or bc, wherein a is the amino acid type before mutation, b is the mutation site, and c is the amino acid type after mutation. For example, N26S, that is, the 26th position is mutated from asparagine (N) to serine (S); N26 is the mutation of the 26th asparagine (N); 26S is the 26th position mutated to serine (S).

Specifically, the IL-2 variants or derivatives thereof provided by the present disclosure contain one or more amino acid mutations or any combination thereof at the following positions: 26, 29, 30, 71, 11, 132, 70, 82, 27 , 78 bits. In some embodiments, the amino acid prior to mutation (eg, in wild-type human IL-2) is: asparagine (N) at position 26, asparagine (N) at position 29, and asparagine (N) at position 30 ( N), 71 is asparagine (N), 11 is glutamine (Q), 132 is leucine (L), 70 is leucine (L), 82 is proline ( P), glycine (G) at position 27, and phenylalanine (F) at position 78.

In some embodiments, the amino acid mutation of the IL-2 variant or derivative thereof comprises any one or any combination of the following: mutation at position 26 to Gln (Q), mutation at position 29 to serine (S), mutation at position 30 Ser(S), 71 mutated to Gln(Q), 11, 132, 70, 82, 27, 78 mutated to Cys(C).

In some specific embodiments, the IL-2 variant or derivative thereof comprises a first type of mutation represented by any one of (1) to (7) below, or any combination thereof:

(1) N26Q,

(2) N29S,

(3) N30S,

(4) N71Q,

(5) Q11C and L132C,

(6) L70C and P82C, and

(7) G27C and F78C.

In some specific embodiments, the IL-2 variants or derivatives thereof described above have increased stability, eg, increased deamination stability and/or thermal stability; in particular, the first type of mutation provided by the present disclosure is An IL-2 variant or derivative thereof comprising the mutation has increased stability, including, but not limited to, increased deamination stability and/or thermal stability compared to wild-type IL-2.

In another aspect, the IL-2 variants or derivatives thereof provided by the present disclosure further contain one or more amino acid mutations or any combination thereof at the following positions: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 72.

In some embodiments, the amino acid prior to mutation (eg, in wild-type human IL-2) is: asparagine (N) at position 29, asparagine (N) at position 30, tyrosine at position 31 ( Y), lysine (K) at position 32, asparagine (N) at position 33, proline (P) at position 34, lysine (K) at position 35, and leucine at position 36 ( L), threonine (T) at position 37, arginine (R) at position 38, methionine (M) at position 39, leucine (L) at position 40, and threonine at position 41 (T), 42 is phenylalanine (F), 43 is lysine (K), 44 is phenylalanine (F), 45 is tyrosine (Y), 72 is light Amino acid (L).

In some specific embodiments, the IL-2 variant or derivative thereof further comprises a second type of mutation selected from the mutations set forth in any one of (8) to (11) or (8) to any combination of (10):

(8) F42A,

(9) Y45A,

(10) L72G, and

(11) The amino acid residues at positions 29-44 were mutated to QSMHIDATL.

In some embodiments, the second type of mutation is capable of eliminating or reducing the affinity of IL-2 for high-affinity receptors (IL-2Rα/β/γ) and preserving IL-2 for medium-affinity receptors (IL-2Rβ/γ). γ) affinity; the second type of mutation can retain the function of IL-2 to induce proliferation and activation of effector cells (such as NK and T cells), but reduce the function of IL-2 to induce proliferation and activation of Treg cells.

In a third aspect, the IL-2 variants or derivatives thereof provided by the present disclosure contain one or more amino acid mutations at the following positions, or any combination thereof: 20, 88, 126. In some embodiments, the amino acid prior to mutation (eg, in wild-type human IL-2) is: aspartic acid (D) at position 20, asparagine (N) at position 88, and glutamine at position 126 ( Q).

In some embodiments, the IL-2 variant or derivative thereof is amino acid mutated in a manner comprising any one or any combination of the following:

Mutation at position 20 to alanine (A) or histidine (H) or isoleucine (I) or methionine (M) or glutamic acid (E) or serine (S) or valine ( V) or tryptophan (W) or tyrosine (Y);

Mutation at position 88 to alanine (A) or arginine (R) or glutamic acid (E) or leucine (L) or phenylalanine (F) or glycine (G) or isoleucine ( I) or methionine (M) or serine (S) or Y or valine (V), or aspartic acid (D);

Mutation at position 126 to asparagine (N) or leucine (L) or P or phenylalanine (F) or glycine (G) or isoleucine (I) or methionine (M) or sperm amino acid (R) or serine (S) or threonine (T) or tyrosine (Y) or valine (V).

In some specific embodiments, the IL-2 variant or derivative thereof further comprises a third type of mutation selected from the mutations set forth in any one of (12) to (14) or any combination thereof:

(12) N88 is mutated to A, R, E, L, F, G, I, M, S, Y, V or D,

(13) D20 is mutated to A, H, I, M, E, S, V, W or Y,

(14) Q126 is mutated to N, L, P, F, G, I, M, R, S, T, Y, V.

In some embodiments, the third type of mutation is capable of reducing the affinity of IL-2 for high-affinity receptors (IL-2Rα/β/γ) and medium-affinity receptors (IL-2Rβ/γ), but not for high-affinity receptors. The avidity of IL-2 is more reduced than that for intermediate-affinity receptors; the third type of mutation can retain the proliferation-inducing and activating function of IL-2 on Treg, but eliminate or reduce the effect of IL-2 on effector cells (such as NK and T cells) induced proliferation and activation functions.

In some embodiments, the IL-2 variant or derivative thereof contains a first type of mutation and a second type of mutation as described above, or a first type of mutation and a third type of mutation.

In some embodiments, the first type of mutation in the IL-2 variant or derivative thereof is selected from (15) to (17), or any one of (15) to (17) and the foregoing (5) to ( A combination of any of 7):

(15) N26Q and N29S,

(16) N26Q, N29S and N71Q, and

(17) N26Q and N30S;

The second type of mutation is selected from any one of (18) to (20) and (11):

(18) F42A and Y45A,

(19) F42A and L72G, and

(20) Y45A and L72G;

The third type of mutation is N88R or N88G or N88I or N88D.

In some embodiments, the IL-2 variant or derivative thereof comprises a mutation selected from any one of (21) to (29):

(21) N26Q, N29S, F42A, N71Q and L72G,

(22) N26Q, N29S and N88R,

(23) N26Q, N29S, F42A and L72G,

(24) N26Q, N30S, F42A and L72G,

(25) Q11C, N26Q, N30S, F42A, L72G and L132C,

(26) N26Q, N30S, F42A, L70C, L72G and P82C,

(27) N26Q, G27C, N30S, F42A, L72G and F78C,

(28) N29S, F42A and L72G, and

(29) Q11C, NNYKNPKLTRMLTFKF mutations at positions 29-44 to QSMHIDATL, and L132C.

In addition to the above (1) to (29), the present disclosure also provides IL-2 variants having any combination of mutation sites and mutation types in (1) to (20), including but not limited to those disclosed in WO2020125743A IL-2 variants.

Naturally expressed human IL-2 has a total of 153 amino acids, and amino acids 1-20 are signal peptides. A total of 133 amino acids (i.e. mature human IL-2) after excision of the signal peptide correspond to positions 2-134 of SEQ ID No. 2 of the present disclosure. For recombinant expression purposes, the artificially produced IL-2 carries an additional methionine in the first position (corresponding to the start codon AUG). In some embodiments, the above-mentioned mutation is a mutation relative to wild-type IL-2, the amino acid sequence of which is shown in SEQ ID NO:2. The mutated site numbering is counted from amino acid A at position 2 according to the amino acid sequence shown in SEQ ID NO: 2, the IL-2 variant comprising amino acid M at position 1 according to SEQ ID NO: 2 or Amino acid M at position 1 is not included.

In some embodiments, the IL-2 variant comprises amino acid M at position 1 according to SEQ ID NO:2.

In some embodiments, the IL-2 variant or derivative thereof comprises an amino acid selected from the group consisting of SEQ ID NO: 39 or SEQ ID NO: 40, the amino acid at position 1 is M, and the above-mentioned mutation sites are numbered from Amino acid A at position 2 starts counting.

In some embodiments, the IL-2 variant or derivative thereof comprises a compound selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 , SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 22 The amino acid set forth in any one of ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, and SEQ ID NO: 38. The amino acids and corresponding nucleotide sequence numbers of the polypeptides are shown in Table 1 (mutated amino acids are underlined):

Table 1. IL-2 variant amino acid and nucleic acid sequences

(Note: The number of the above-mentioned mutation sites is calculated according to the number of the human IL-2 mature protein shown in SEQ ID NO: 2. The human IL-2 mature protein does not contain the amino acid M at position 1, so the numbering starts from the amino acid at position 2. A starts counting. Wherein, "/" indicates that in the same IL-2 variant, the mutations are present simultaneously. All variants may contain or not contain C125A, which is to avoid dimerization.)

In some embodiments, derivatives of IL-2 variants include full-length, partial proteins related to the IL-2 variants of the present disclosure, or muteins obtained by further mutation on the basis of the IL-2 variants of the present disclosure, functional Derivatives, functional fragments, biologically active peptides, fusion proteins, isoforms or salts thereof. For example, fusion proteins comprising IL-2 variants, monomers or dimers or trimers or multimers of said IL-2 variants, various modified forms of said IL-2 variants (eg, PEG) ylation, glycosylation, albumin conjugation or fusion, Fc fusion or conjugation, hydroxyethylation, de-O-glycosylation, etc.), as well as homologues of the IL-2 variants in each species. The modification of IL-2 does not result in adverse effects on treatment-related immunogenicity.

In some embodiments, the IL-2 variant or derivative is PEGylated (may be denoted PEG-IL-2), eg, a mono- or di-PEGylated IL-2 variant or derivative. PEG-IL-2 variants or derivatives include SC-PEG linkers. In other embodiments, the PEG-IL-2 variant or derivative includes a methoxy-PEG-aldehyde (mPEG-ALD) linker. In certain embodiments, the relative molecular weight of the PEG moiety is from about 5KD to about 50KD, specifically 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 30, 35, 40, 45, 50KD; or between about 5KD and about 40KD, or between about 10KD and about 30KD, or between about 10KD and about 30KD, or between about 15KD and about 20KD. In certain embodiments, the mPEG-ALD linker comprises a PEG molecule having a relative molecular weight selected from the group consisting of about 5KDa, about 12KDa, or about 20KDa (with a quality control standard of 20±2KDa). In certain embodiments, the aldehyde group of mPEG-ALD can be acetaldehyde, propionaldehyde, butyraldehyde, and the like. In one embodiment, the IL-2 variant or derivative thereof has an increased serum half-life compared to wild-type IL-2 or a derivative thereof.

The relative molecular weight of PEG mentioned in the present disclosure refers to the relative molecular mass detected by MALDI-TOF, and the number of PEG repeating units can be obtained by inversely deducing the relative molecular weight of PEG.

In some embodiments, the IL-2 variant or derivative thereof is selected from:

The relative molecular weight of PEG is about 20KD; n is the number of repeating units of PEG, and n can be obtained by inversion of the relative molecular weight of PEG.

In some embodiments, the IL-2 variant or derivative thereof does not comprise amino acid M at position 1.

When the IL-2 variant or derivative thereof comprises a second class of mutations, in some embodiments, the IL-2 variant or derivative thereof is capable of eliciting one or more cellular responses selected from the group consisting of activated T lymphocytes Proliferation in cells, differentiation in activated T lymphocytes, cytotoxic T cell (CTL) activity, proliferation in activated B cells, differentiation in activated B cells, proliferation in natural killer (NK) cells, NK Differentiation in cells, cytokine secretion by activated T cells or NK cells, and NK/lymphocyte activated killer (LAK) antitumor cytotoxicity. In some embodiments, the IL-2 variant or derivative thereof has a reduced ability to induce IL-2 signaling in regulatory T cells as compared to a wild-type IL-2 polypeptide. In one embodiment, the IL-2 variant or derivative thereof induces less activation-induced cell death (AICD) in T cells than wild-type IL-2 or derivative thereof. In some embodiments, the IL-2 variant or derivative thereof has reduced toxicity in vivo compared to wild-type IL-2 or derivative thereof.

When the IL-2 variant or derivative thereof comprises a third class of mutations, in some embodiments, the IL-2 variant or derivative thereof is capable of reducing the effect of IL-2 on high affinity receptors (IL-2Rα/β/γ) and medium-affinity receptors (IL-2Rβ/γ), but the affinity for high-affinity receptors is more reduced than that for medium-affinity receptors. In some embodiments, IL-2 variants or derivatives thereof are capable of retaining the proliferation-inducing and activating functions of IL-2 on Tregs, but eliminating or reducing the proliferation-inducing and activation of effector cells (eg, NK and T cells) by IL-2 Features.

In another aspect, the present disclosure provides linkers or conjugates in which the IL-2 variant or derivative thereof is attached directly or indirectly through a linker to a non-IL-2 moiety. In some embodiments, it is an immunoconjugate, wherein the non-IL-2 moiety is an antigen binding moiety. In some embodiments, the antigen binding moiety targets an antigen presented on tumor cells or in the tumor cell environment.

In some embodiments, the IL-2 variant (or derivative thereof) is linked to at least one non-IL-2 moiety. In some embodiments, the IL-2 variant (or derivative thereof) and the non-IL-2 moiety form a fusion protein, ie, the IL-2 variant (or derivative thereof) shares a peptide bond with the non-IL-2 moiety.

In some embodiments, the IL-2 variant (or derivative thereof) is linked to at least one non-IL-2 module, eg, a first and a second non-IL-2 module. In some embodiments, the non-IL-2 moiety is an antigen binding moiety. In some embodiments, the IL-2 variant (or derivative thereof) shares an amino- or carboxy-terminal peptide bond with the first antigen-binding moiety, and the second antigen-binding moiety shares an amino- or carboxy-terminal peptide bond with: i) IL- 2 the variant (or derivative thereof); or ii) the first antigen binding moiety. In some specific embodiments, the IL-2 variant (or derivative thereof) shares a carboxy-terminal peptide bond with the first non-IL-2 moiety and an amino-terminal peptide bond with the second non-IL-2 moiety . In some embodiments, the non-IL-2 moiety is an antigen binding moiety. The antigen-binding moiety can be an antibody or antigen-binding fragment, including but not limited to immunoglobulin molecules (eg, IgG (eg, IgGl) class immunoglobulin molecules), antibodies or antigen-binding fragments thereof. In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a polypeptide complex comprising an antibody heavy chain variable region and an antibody light chain variable region, Fab, Fv, sFv, F(ab')2, linear antibody, single antibody Chain antibodies, scFvs, sdAbs, sdFvs, Nanobodies, peptibodies, domain antibodies and multispecific antibodies (bispecifics, diabodies, triabodies and tetrabodies, tandem di-scFv, tandem tri-scFv). Where the IL-2 variant (or derivative thereof) is linked to more than one antigen binding moiety, eg, a first and a second antigen binding moiety, each antigen binding moiety can be independently selected from various forms of antibody and antigen binding Fragments, for example, the first antigen binding moiety can be a Fab molecule and the second antigen binding moiety can be a scFv molecule, or each of the first and second antigen binding moieties is a scFv molecule, or the first and second antigen Each of the binding modules is a Fab molecule. In some embodiments, where the IL-2 variant (or derivative thereof) is linked to more than one antigen binding moiety, eg, a first and a second antigen binding moiety, the antigen to which each antigen binding moiety is directed can be independently selected For example, the first and the second antigen binding moieties are directed against different antigens or against the same antigen.

In some embodiments, the antigen bound by the antigen binding moiety may be selected from the group consisting of the A1 domain of tenascin C (TNC A1), the A2 domain of tenascin C (TNC A2), the extradomain of fibronectin (Extra Domain B) (EDB), carcinoembryonic antigen (CEA), and melanoma-associated chondroitin sulfate proteoglycan (MCSP). In some embodiments, tumor antigens include, but are not limited to, MAGE, MART-1/Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin b. Colorectal-associated antigen (CRC)-C017-1A/GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aML1, prostate specific antigen (PSA) and Its immunogenic epitopes PSA-1, PSA-2 and PSA-3, prostate specific membrane antigen (PSMA), T cell receptor/CD3-zeta chain, MAGE family of tumor antigens (eg MAGE-A1, MAGE- A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2(MAGE-B2) , MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE family of tumor antigens (eg GAGE-1 , GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM- 1. CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin , p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products (such as human papilloma virus proteins), Smad family of tumor antigens, lmp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylation Enzymes, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2.

In some embodiments, non-limiting examples of viral antigens include influenza virus hemagglutinin, Epstein-Barr virus LMP-1, hepatitis C virus E2 glycoprotein, HIV gp160, and HIV gp120.

In some embodiments, non-limiting examples of ECM antigens include syndecan, heparanase, integrin, osteopontin, cadherin, laminin, EGF Types of laminin, lectins, fibronectin, notch, tenascin and matrix proteins.

In some embodiments, the IL-2 variant or derivative thereof comprising the above-described antigen binding moiety comprises the second type of mutation and does not comprise the third type of mutation.

In some embodiments, a method for increasing the stability of IL-2 or a derivative or conjugate thereof is provided, the method comprising introducing any of 1) to 7) in IL-2 or a derivative or conjugate thereof. or any combination of the mutations shown:

1) N26Q,

2) N29S,

3) N30S,

4) N71Q,

5) Q11C and L132C,

6) L70C and P82C, and

7) G27C and F78C.

The IL-2 variants or derivatives thereof, related sequences, and methods of preparation thereof in WO2020/125743 are incorporated herein in their entirety.

Freeze-dried preparation:

The present disclosure also provides a method for preparing a lyophilized formulation of a pharmaceutical composition comprising IL-2, the method comprising the step of lyophilizing the aforementioned pharmaceutical composition.

The present disclosure also provides a freeze-dried preparation of the pharmaceutical composition comprising IL-2, the freeze-dried preparation is obtained by freeze-drying any of the aforementioned IL-2 pharmaceutical compositions.

In some embodiments, the lyophilized formulation is stable for at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 18 months when stored in the dark at 2-8°C 30 months.

In some embodiments, the lyophilized formulation is stable at 25°C for at least 1 month, at least 3 months, at least 6 months, at least 12 months.

In some embodiments, the lyophilized formulation is stable at 40°C for at least 7 days, at least 14 days, or at least 30 days.

The present disclosure also provides a reconstituted solution comprising IL-2, which is prepared by reconstituting the aforementioned lyophilized preparation.

The present disclosure also provides a method for preparing the above-mentioned reconstituted solution, which includes the step of reconstituting the aforementioned freeze-dried preparation, and the solution used for the reconstitution is selected from but not limited to: water for injection, physiological saline or glucose solution such as 5% glucose injection , Glucose and Sodium Chloride Injection, etc.

The present disclosure also provides an article of manufacture comprising one or more containers, each container independently containing the aforementioned pharmaceutical composition, lyophilized formulation, or reconstitution solution. The labeled volume of the container can be adjusted as needed, and can be selected from 0.5mL, 0.6mL, 0.7mL, 0.8mL, 0.9mL, 1.0mL, 1.1mL, 1.2mL, 1.3mL, 1.4mL, 1.5mL, 1.6mL, 1.7mL, 1.8mL, 1.9mL, 2.0mL, 2.1mL, 2.2mL, 2.3mL, 2.4mL, 2.5mL, 5.0mL, 10mL. In the process of clinical administration of injections, there may be residues and losses of preparations. Therefore, in order to ensure the labeled filling volume, according to the requirements in the four general rules of injections of the Chinese Pharmacopoeia 2020 edition, the preparation should be appropriately increased during actual production, and every 1mL of drug combination The target filling volume during actual filling production is set to be about 1.15mL, and the filling volume range is controlled from about 1.10 to about 1.20mL.

In some embodiments, the inner packaging materials of the container are medium borosilicate glass control injection bottles, parylene-coated bromobutyl rubber stoppers for freeze drying for injection, and aluminum-plastic composite caps for antibiotic bottles.

Preparation:

The preparation of the pharmaceutical composition of the present disclosure may adopt a common method in the art, including the step of mixing an appropriate amount of IL-2 with the aforementioned pharmaceutical excipients.

In some embodiments, after IL-2 is prepared, a pharmaceutical formulation containing it is prepared, including adding appropriate amounts of trehalose, mannitol, and optionally other adjuvant ingredients to the pharmaceutical formulation.

use:

In another aspect, the present disclosure provides pharmaceutical uses of the above-described pharmaceutical compositions, lyophilized formulations or reconstituted solutions comprising IL-2.

When IL-2 is wild-type or a variant comprising a second class of mutations, it is used in the treatment of proliferative disorders, immune disorders, modulation of T cell-mediated immune responses, methods of stimulating the immune system of an individual, and related pharmaceutical uses. The proliferative disease may be a tumor or carcinoma (eg, metastatic tumor or carcinoma), and may be a solid tumor (eg, metastatic renal cell carcinoma and malignant melanoma).

In some embodiments, the pharmaceutical compositions, lyophilized formulations or reconstituted solutions of the present disclosure may be used to treat disease conditions in which stimulation of the host's immune system for benefit, particularly conditions in which it is desirable to enhance cellular immune responses, may include insufficient host immune responses or defective disease conditions. In some embodiments, the disease situation in which the pharmaceutical composition, lyophilized formulation, or reconstituted solution is administered includes tumors or infections in which cellular immune responses are key mechanisms of specific immunity, such as cancer (eg, renal cell carcinoma or melanoma), immune Deficiencies (eg, in HIV-positive patients, immunosuppressed patients), chronic infections, etc. In some embodiments, enhancing a cellular immune response may include any one or more of the following: general increase in immune function, increase in T cell function, increase in B cell function, restoration of lymphocyte function, IL-2 receptor expression Increased, increased T cell responsiveness, increased natural killer cell activity or lymphokine-activated killer (LAK) cell activity, etc. In some embodiments, the disease for which the pharmaceutical composition, lyophilized formulation or reconstituted solution of the present disclosure is used to treat is a proliferative disorder, such as cancer. Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, Rectal, gastric, prostate, blood, skin, squamous cell, bone and kidney cancers. Other cell proliferative disorders that can be treated using the pharmaceutical compositions, lyophilized formulations, or reconstituted solutions of the present disclosure include, but are not limited to, neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, chest, and genitourinary system. Also included are precancerous conditions or injuries and cancer metastases. In certain embodiments, the cancer is selected from the group consisting of renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer. Similarly, other cell proliferative disorders may also be treated with the pharmaceutical compositions, lyophilized formulations or reconstituted solutions of the present disclosure, including but not limited to: hypergammaglobulinemia, lymphoproliferative disorders, pathoproteinemia ( paraproteinemias), purpura, sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, and any other Cell proliferative disorders other than neoplasia in the organ systems listed herein. In other embodiments, the disease involves autoimmunity, transplant rejection, post-traumatic immune responses, and infectious diseases (eg, HIV).

When the IL-2 variant or derivative thereof comprises a third class of mutations, it is used to treat autoimmune diseases or to alleviate/treat/prevent autoimmune responses following organ transplantation. The autoimmune disease may be selected from the group consisting of: type I diabetes mellitus, rheumatoid arthritis, multiple sclerosis, chronic gastritis , Crohn's disease, Basedow disease, Bechterew disease, psoriasis, myasthenia gravis, autoimmune hepatitis ), APECED, Chrug-Strauss syndrome, ulcerative colitis, glomerulonephritis, Guillain-Barré syndrome, Hashimoto’s Hashimoto thyroiditis, lichen sclerosus, systemic lupus erythematodes, PANDAS, rheumatic fever, sarcoidosis, syndrome , Stiff-Man syndrome, scleroderma, Wegener's granulomatosis, vitiligo, autoimmune enteropathy, pulmonary hemorrhagic nephritis syndrome (Goodpasturesyndrome), dermatomyositis (dermatomyositis), polymyositis (polymyositis), autoimmune allergy (autoimmune allergy), asthma (asthma). In some embodiments, the IL-2 variant or derivative thereof can be used in combination with an immunosuppressive agent. In some embodiments, the immunosuppressant is selected from the group consisting of: glucocorticoids, including decortin, prednisol; azathioprine; cyclosporin A ; mycophenolatemofetil; tacrolimus; anti-T lymphocyte globulin, anti-CD3 antibodies, including muromonab; anti-CD25 antibodies, including basiliximab ( basiliximab and daclizumab; anti-TNF-alpha antibodies, including infliximab and adalimumab; azathioprine; methotrexate; cyclosporine (cyclosporin); sirolimus; everolimus; fingolimod; CellCept; myfortic; and cyclophosphamide ( cyclophosphamide).

In some embodiments, the pharmaceutical compositions, lyophilized formulations or reconstitution solutions of the present disclosure provide no cure but only partial benefit. In some embodiments, physiological changes with some benefit are also considered therapeutically beneficial. As such, in some embodiments, the amount of the IL-2 variant, or derivative, immunoconjugate thereof, that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount." The subject, patient or individual in need of treatment is usually a mammal, more specifically a human.

In some embodiments, there is provided wherein at least 2 times a day, at least once a day, at least once every 48 hours, at least once every 72 hours, at least once a week, at least once every 2 weeks, at least once a month, every A method of administering a pharmaceutical composition, lyophilized formulation, or reconstituted solution of the present disclosure to a subject at least once every 2 months or at least once every 3 months.

Pharmaceutical compositions, lyophilized formulations or reconstituted solutions can be administered by any effective route. For example, by injection, administration into the bloodstream of a patient may be in a form suitable for injection (eg, by bolus injection) or continuous infusion, or intravenous, subcutaneous, intradermal, intraperitoneal, and the like. Alternatively, local administration (intratumoral or peritumoral administration) can be performed at the tumor site. In the case of intratumoral or peritumoral administration, administration can be via any route, such as by topical administration, regional administration, local administration, systemic administration, convection-enhanced delivery, or a combination thereof Apply.

Injectable forms, including sterile aqueous solutions or dispersions. Further, the above-mentioned pharmaceutical compositions can be prepared in sterile powder form for the extemporaneous preparation of sterile injectable solutions or dispersions. In any event, the final injectable form must be sterile and, for ease of injection, must be readily flowable. Furthermore, the pharmaceutical compositions must be stable during manufacture and storage. Therefore, preferably, the pharmaceutical composition is to be preserved under conditions that are resistant to contamination by microorganisms such as bacteria and fungi.

the term

For easier understanding of this application, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this application belongs.

The present disclosure incorporates the entire contents of the published patent text WO2020/125743A1.

The three-letter and one-letter codes for amino acids used in this application are as described in J. Biol. Chem, 243, p3558 (1968).

"Interleukin-2" or "IL-2" refers to any native IL-2 from any vertebrate source, including mammals such as primates (eg, humans) and rodents (eg, mice and rats). The term encompasses unprocessed IL-2 as well as any form of IL-2 derived from processing in a cell. The term also encompasses naturally occurring variants of IL-2, such as splice variants or allelic variants. The amino acid sequence of an exemplary wild-type human mature IL-2 is set forth in SEQ ID NO: 2 (or without the leading M). Unprocessed human IL-2 additionally contains an N-terminal 20 amino acid signal peptide (as shown in SEQ ID NO: 272 in WO2012107417), which is absent in the mature IL-2 molecule.

"Amino acid mutation" includes amino acid substitutions, deletions, insertions, modifications, and any combination thereof, to achieve a final construct such that the final construct possesses desired properties, such as enhanced stability. Amino acid sequence deletions and insertions include amino- and/or carboxy-terminal deletions and amino acid insertions. An example of a terminal deletion is the deletion of an alanine residue at position 1 of full-length human IL-2. Preferably amino acid mutations are amino acid substitutions. For example, in order to alter the binding properties of an IL-2 polypeptide, non-conservative amino acid substitutions, ie replacing one amino acid with another amino acid with different structural and/or chemical properties, can be made. Preferred amino acid substitutions include the replacement of hydrophobic amino acids with hydrophilic amino acids. Amino acid substitutions include substitutions from non-naturally occurring amino acids or from naturally occurring amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine) amino acid) replacement. Amino acid mutations can be generated using genetic or chemical methods known in the art, including methods such as site-directed mutagenesis, PCR, gene synthesis, chemical modification, and the like.

A "wild-type IL-2" is a form of IL-2 that is otherwise identical to a variant IL-2 polypeptide, except that the variant IL-2 polypeptide has a wild-type amino acid at each amino acid position. For example, if the IL-2 variant is full-length IL-2 (ie, IL-2 that is not fused or conjugated to any other molecule), then the wild-type form corresponding to this variant is full-length native IL-2. If the IL-2 variant is a fusion between IL-2 and another polypeptide encoded downstream (eg, an antibody chain), then the corresponding wild-type form of the IL-2 variant is a wild-type fused to the same downstream polypeptide type amino acid sequence of IL-2. Furthermore, if the IL-2 variant is a truncated form of IL-2 (a sequence that is mutated or modified within a non-truncated portion of IL-2), then the wild-type form of the IL-2 variant has the wild-type sequence similar to truncated IL-2. For the purpose of comparing IL-2 receptor binding affinity or biological activity for various forms of IL-2 variants with the corresponding wild-type form of IL-2, the term "wild-type" encompasses, as compared to naturally occurring IL-2, including A form of IL-2 with one or more amino acid mutations that do not affect binding to the IL-2 receptor (eg, a cysteine substitution to alanine C125A at a position corresponding to residue 125 of human IL-2) . In some embodiments, wild-type IL-2 comprises the amino acid sequence set forth in SEQ ID NO:2.

"Derivative" is intended to be construed broadly to include any IL-2 related product. Including but not limited to human and non-human IL-2 homologs, fragments or truncations, fusion proteins (such as fusions with signal peptides or other active or inactive ingredients, such as antibodies or antigen-binding fragments thereof), Modified forms (eg, PEGylation, glycosylation, albumin conjugation/fusion, Fc conjugation and/fusion, hydroxyethylation, etc.), and conservatively modified proteins, and the like.

"High-affinity IL-2 receptor" refers to the heterotrimeric form of the IL-2 receptor consisting of the receptor gamma subunit (also known as the universal cytokine receptor gamma subunit, gammac or CD132), the receptor beta subunit (also known as CD122 or p70) and receptor alpha subunit (also known as CD25 or p55). In contrast, a "medium affinity IL-2 receptor" refers to an IL-2 receptor that contains only gamma and beta subunits and no alpha subunit (see, eg, Olejniczak and Kasprzak, MedSci Monit 14, RA179-189 (2008) ).

"Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (eg, a receptor) and its binding partner (eg, a ligand). Unless otherwise indicated, "binding affinity" herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, receptor and ligand). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (KD), which is the ratio of dissociation to association rate constants (K _dissociation and K _binding , respectively). As such, equal affinities may contain different rate constants, as long as the ratio of rate constants remains the same. Affinity can be measured by methods routine in the art, including those described herein.

"Regulatory T cell" or "T regulatory cell" or "Treg" means a specialized CD4+ T cell type capable of suppressing the response of other T cells. Tregs are characterized by expressing the alpha subunit of the IL-2 receptor (CD25) and the transcription factor forkhead box P3 (FOXP3), and are involved in the induction and maintenance of peripheral autotolerance to antigens, including those expressed by tumors plays a key role. Tregs require IL-2 for their function and development as well as the induction of their inhibitory characteristics.

"Effector cells" refer to the population of lymphocytes that mediate the cytotoxic effects of IL-2. Effector cells include effector T cells such as CD8+ cytotoxic T cells, NK cells, lymphokine-activated killer (LAK) cells and macrophages/monocytes.

An "antigen binding moiety" refers to a polypeptide molecule that binds an antigenic determinant. In some embodiments, an antigen binding moiety is capable of directing its linker (eg, a cytokine (IL-2 or a variant thereof) and/or other antigen binding moiety) to a target site, such as to a particular type of tumor cell or Tumor stroma for specific epitopes. The antigen binding moiety includes an antibody or antigenic fragment thereof, preferably the antigen binding moiety includes the antigen binding domain of an antibody, which may comprise an antibody heavy chain variable region and an antibody light chain variable region. The antigen binding moiety may comprise antibody constant regions. Useful heavy chain constant regions include any of the following 5 isotypes: alpha, delta, epsilon, gamma, or mu, as known in the art. Useful light chain constant regions include any of the following 2 isotypes: kappa and lambda. IL-2 or a variant thereof can be linked to one or more antigen binding moieties via one or more linker sequences to form an "immunoconjugate".

"Specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antigen binding moiety to bind a specific epitope can be determined by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

"Antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bi specific antibodies) and antigen-binding fragments. Antibodies can include murine antibodies, human antibodies, humanized antibodies, chimeric antibodies, camelid antibodies. Illustratively, the antibody may be an immunoglobulin, which is a tetrapeptide chain structure consisting of two heavy chains and two light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ, δ, and γ chains, respectively. , alpha chains and epsilon chains. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into kappa chains or lambda chains by the difference in the constant region. Each of the five classes of Ig can have a kappa chain or a lambda chain.

"Conservative modifications" apply to amino acid and nucleotide sequences. With respect to nucleotide sequences, conservative modifications refer to those nucleic acids that encode the same or substantially the same amino acid sequence, or, where the nucleotides do not encode an amino acid sequence, to substantially the same nucleotide sequence. With respect to amino acid sequences, "conservative modifications" refer to the replacement of amino acids in a protein by other amino acids with similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that frequent changes can be made without Does not alter the biological activity of the protein. Those skilled in the art know that, in general, single amino acid substitutions in non-essential regions of polypeptides do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecμlar Biology of the Gene, The Benjamin/Cummings Pub. Co., 224, (4th ed.).

"PEGylation" refers to the attachment of at least one PEG molecule to another molecule (eg, an IL-2 variant of the present disclosure). For example, Adagen (a PEGylated formulation of adenosine deaminase) is approved for the treatment of severe combined immunodeficiency. Linking of polyethylene glycol has been shown to prevent proteolysis (see, eg, Sada et al. (1991) J. Fermentation Bioengineering 71:137-139). In the most common form, PEG is a linear or branched polyether linked at one end to a hydroxyl group and has the following general structure: HO-(CH2CH2O)n-CH2CH2-OH. To couple PEG to molecules (polypeptides, polysaccharides, polynucleotides, and small organic molecules), PEG can be activated by preparing derivatives of PEG with functional groups on some or both ends. A common approach to PEG conjugation of proteins is to activate PEG with functional groups suitable for reaction with lysine and N-terminal amino acid groups. In particular, a common reactive group involved in conjugation is the alpha or epsilon amino group of lysine. Reaction of the PEGylated linker with the protein can result in attachment of the PEG moiety primarily at the following sites: the alpha amino group at the N-terminus of the protein, the epsilon amino group on the side chain of a lysine residue, or a histidine residue imidazolyl on the side chain. Since most recombinant proteins have a single alpha and many epsilon amino and imidazole groups, many positional isomers can be generated depending on the chemistry of the linking group.

"Administering," "administering," and "treating," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to exogenous drugs, therapeutic agents, diagnostic agents, or compositions that interact with the animal. , contact of humans, subjects, cells, tissues, organs or biological fluids. "Administering," "administering," and "treating" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, wherein the fluids are in contact with cells. "Administering," "administering," and "treating" also mean in vitro and ex vivo treatment of, eg, cells by an agent, diagnostic, binding composition, or by another cell. "Administering," "administering," and "treating," when applied to human, veterinary, or research subjects, refer to therapeutic treatment, prophylactic or prophylactic measures, research and diagnostic applications.

"Treatment" means administering to a subject an internal or external therapeutic agent, such as a composition comprising any of the IL-2 variants of the present disclosure and derivatives thereof, or compositions comprising the variants or derivatives, to the subject A person diagnosed with, suspected of having, or susceptible to one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect. Typically, a therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in a subject or population being treated, whether by inducing regression of such symptoms or inhibiting progression of such symptoms to any clinically unmeasurable extent.

The amount of a therapeutic agent effective to alleviate symptoms of any particular disease (also referred to as a "therapeutically effective amount") may vary depending on a variety of factors, such as the subject's disease state, age, and weight, and the level of the drug that produces the desired effect in the subject. ability. Whether symptoms of a disease have been alleviated can be assessed by any clinical test commonly used by doctors or other health care professionals to assess the severity or progression of the symptoms. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating symptoms of the target disease that each patient has, it is possible to use any statistical test known in the art, such as Student's t-test, chi-square test, based on Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determine that it should reduce target disease symptoms in a statistically significant number of subjects.

An "effective amount" includes an amount sufficient to ameliorate or prevent a medical condition or condition thereof. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular subject or veterinary subject may vary depending on factors such as the condition being treated, the general health of the subject, the method, route and dosage of administration, and the severity of the side effects. An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

"Buffer" refers to a buffer that resists changes in pH through the action of its acid-base conjugated component. Examples of buffers to control pH in the appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumaric acid Salt, glycylglycine and other organic acid buffers.

A "histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate and the like, preferably histidine-acetate Buffer or histidine-hydrochloric acid buffer, histidine-acetate buffer is prepared from histidine and acetic acid, and histidine buffer is prepared from histidine and hydrochloric acid.

A "citrate buffer" is a buffer that includes citrate ions. Examples of citrate buffers include citrate-sodium citrate, citrate-potassium citrate, citrate-calcium citrate, citrate-magnesium citrate, and the like. A preferred citrate buffer is citrate-sodium citrate.

A "succinate buffer" is a buffer that includes succinate ions. Examples of succinate buffers include succinate-sodium succinate, succinate-potassium succinate, succinate-calcium succinate, and the like. A preferred succinate buffer is succinate-sodium succinate.

"Phosphate buffer" is a buffer including phosphate ions. The phosphate buffer can be selected from any phosphate buffer known to those skilled in the art that is suitable for the system of the present disclosure. The phosphate buffer component preferably contains a Mixtures of one or more phosphate buffers, eg, monobasic phosphate, dibasic phosphate, and the like. Particularly useful phosphate buffers are selected from alkali metal and/or alkaline earth metal phosphates. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, Tris-HCl buffer, sodium phosphate-phosphoric acid solution, hydrogen phosphate Disodium-phosphoric acid solution, sodium phosphate-sodium dihydrogen phosphate solution, etc. A preferred phosphate buffer is disodium hydrogen phosphate-sodium hydrogen phosphate. According to needs, sodium dihydrogen phosphate and disodium hydrogen phosphate can be anhydrous or hydrated, such as anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate monohydrate, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate heptahydrate, decahydrate Disodium hydrogen phosphate dihydrate, etc.

An "acetate buffer" is a buffer that includes acetate ions. Examples of acetate buffers include acetate-sodium acetate, acetate-histidine, acetate-potassium acetate, calcium acetate, acetate-magnesium acetate, and the like. A preferred acetate buffer is acetic acid-sodium acetate.

"Pharmaceutical composition" means a mixture comprising one or more of the compounds described herein (or physiologically/pharmaceutically acceptable salts or prodrugs thereof) and other chemical components, such as physiological/pharmaceutically acceptable salts or prodrugs. Pharmaceutical carriers and excipients. The purpose of the pharmaceutical composition is to maintain the stability of the active ingredient, facilitate the administration to the organism, and facilitate the absorption of the active ingredient to exert biological activity.

Herein, "pharmaceutical composition" and "formulation" are not mutually exclusive.

"Lyophilized formulation" means a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a liquid or solution formulation has been subjected to a freeze-drying step.

As used herein, the terms "about", "approximately" refer to an index value within an acceptable error range of the particular value determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (ie, the limits of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" or "consisting essentially of" may mean a range of up to 20%, such as between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% ranging from 0.5% to 1%, in this disclosure, each instance of a number or range of values preceded by the term "about" also includes embodiments of the given number. Unless otherwise stated, when a specific value appears in this disclosure, the meaning of "about" or "substantially comprising" should be within an acceptable error range of the specific value.

The numerical values in the present disclosure are the values measured by the instrument or the calculated values after the measurement by the instrument, and there is a certain degree of error. In general, ±10% is within a reasonable error range, which can be ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1 %, for example, the relative molecular weight of PEG is 20KD, and the relative molecular weight of 20KD±2KD is within a reasonable error range.

The lyophilized formulation described in the present disclosure can achieve a stable effect: a pharmaceutical composition in which the active ingredient substantially retains its physical stability and/or chemical stability and/or biological activity after storage. For example, a pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The shelf life is generally selected based on the intended shelf life of the pharmaceutical composition. There are various analytical techniques for measuring protein stability, including RP-HPLC, SE-HPLC, IE-HPLC, which measure stability after storage at selected temperatures for selected periods of time.

In the present disclosure, the skilled artisan will understand that "stability" of IL-2 (IL-2 variant or derivative thereof) includes various aspects of stability, such as, but not limited to, long-term stability of the formulations evaluated in specific embodiments stability (also used as shelf stability, long-term tracking evaluation under storage conditions), thermal stability (also used as accelerated stability; aging test at high temperature), airborne stability (evaluated in actual operation under machine conditions) stability), reconstitution stability (stability after re-dissolution of dry powder formulations), freeze-thaw stability (under repeated freeze-dissolution conditions). The stability of these different aspects is reflected in the different kinetic characteristics of IL-2 variants or their derivatives; in different stability evaluations, IL-2 variants or their derivatives face different physical, chemical, and biological pressures . This means that, prior to reading the teachings of this disclosure, the skilled artisan would not have the ability to extrapolate from one stability result of an IL-2 variant or derivative thereof to another.

A stable formulation is one in which no significant change is observed: in some embodiments, the lyophilized formulation is stable at 2-8°C for at least 1 month, eg, at least 3 months, at least 6 months, at least 12 months months, at least 18 months, at least 24 months, at least 30 months. In some embodiments, the lyophilized formulation is stable at 25°C for at least 1 month, eg, at least 3 months, at least 6 months, at least 12 months. In some embodiments, the lyophilized formulation is stable at 40°C for at least 7 days, eg, at least 14 days or at least 30 days.

Typical acceptable criteria for stability are as follows: typically no more than about 10%, eg no more than about 5% of the IL-2 variant or derivative aggregates as measured by RP-HPLC, SE-HPLC or IE-HPLC or degradation. By visual analysis, the formulation was a pale yellow near colorless clear liquid or colorless, or clear to slightly opalescent. The formulations had no more than ±10% variation in concentration, pH and osmolality. A reduction of no more than about 10%, eg, no more than about 5%, is generally observed. If the active ingredient does not show a significant increase in aggregation, precipitation and/or after visual inspection of color and/or clarity, or as measured by UV light scattering, size exclusion chromatography (SEC) and dynamic light scattering (DLS) Denatured, then the active ingredient "retains its physical stability" in the pharmaceutical formulation. Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).

An IL-2 variant or derivative "retains its chemical stability" in a pharmaceutical formulation if it does not exhibit a significant chemical change. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that frequently alter the chemical structure of proteins include hydrolysis or truncation (as assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (by peptide mapping such as combined with mass spectrometry or MALDI/TOF/MS, etc.) methods), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartic acid measurement, etc.), and isomerization (by measuring isoaspartic acid content, Peptide Mapping, etc.).

An IL-2 variant or derivative "retains it" in a pharmaceutical formulation if its biological activity at a given time is within a predetermined range of the biological activity exhibited at the time of preparation of the pharmaceutical formulation biological activity". Biological activity can be determined, for example, by antigen binding assays.

specific implementation

The following examples are used for further description, but these examples are not intended to limit the scope.

The experimental methods that do not specify specific conditions in the examples or test examples are usually in accordance with conventional conditions, or in accordance with conditions suggested by raw material or commodity manufacturers. See Sambrook et al., Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory; Contemporary Methods in Molecular Biology, Ausubel et al., Greene Publishing Association, Wiley Interscience, NY, or as suggested by the raw material or commercial manufacturer. Reagents with no specific source indicated are conventional reagents purchased in the market.

Example 1. Study on the stability of IL-2 as a bulk drug

1. API 1 is a PEGylated recombinant human IL-2 variant (SEQ ID No. 34) with the structural information and amino acid sequence shown below.

The molecular weight was detected by SDS-polyacrylamide gel electrophoresis (reducing type): 35.4±3.5KD;

Appearance: colorless or slightly yellow clear liquid;

pH: 6.0 to 7.0.

The results of the investigation of the influencing factors showed that the stock solution of API 1 should be stored at low temperature and protected from light.

Wherein: the solution system where the analyte is located is the same as the solution system obtained by screening in Example 5.

1) High temperature conditions: when the analyte is placed at 40℃±2℃ for 30 days, the purity of RP-HPLC decreases by 16.1%, the purity of SE-HPLC decreases by 46.9%, the purity of IE-HPLC decreases by 37.1%, and the purity of electrophoresis method decreases by 17.2% , has exceeded the quality standard range, the biological activity is 58% beyond the quality standard range, and the rest of the detection indicators have no significant changes. The analyte was placed under the condition of 25℃±2℃ for 30 days, the purity of RP-HPLC decreased by 3.4%, the purity of SE-HPLC decreased by 0.7%, the purity of IE-HPLC decreased by 3.7%, the purity of electrophoresis method decreased by 1.6%, and the other detection indicators were not Significant changes. It shows that the high temperature condition has a great influence on the test object.

2) Lighting conditions: when the substance to be tested is placed under the condition of light intensity of 4500lx ± 500lx for 30 days, the purity of RP-HPLC drops by 6.8%, which has exceeded the quality standard range, the purity of SE-HPLC drops by 2.9%, and the purity of IE-HPLC drops by 4.1%. The purity of the electrophoresis method decreased by 4.3%, and the other detection indexes did not change significantly. Indicates that light conditions have an impact on the test object.

3) Freeze-thaw cycle: After the object to be tested undergoes 5 freeze-thaw cycles at -35°C±5°C/5°C±3°C, there is no significant change in each test item, and the results meet the quality standards. It shows that the analyte has good freeze-thaw stability.

4) The inspection results of the accelerated test show that there is no obvious change in the product quality for 1 month under accelerated conditions. Three batches of analytes were placed under accelerated (5°C ± 3°C) conditions for one month, and there was no significant change in each test index, and all the results were within the quality standard range.

2. API 2 is a PEGylated recombinant human interleukin-2 variant (SEQ ID No. 38) with the structural information and amino acid sequence shown below.

Contains a pair of disulfide bonds (Cys59-Cys106);

Molecular weight detected by SDS-polyacrylamide gel electrophoresis (reducing type): 35.5±3.6KD;

Appearance: colorless or slightly yellow clear liquid; pH: 4.5 to 6.0.

The microbial and bacterial endotoxin levels of API 2 will have an impact on the quality of the finished product. Therefore, it is necessary to control the microbial and bacterial endotoxin levels of API 2, which are in line with the provisions of the 2015 edition of the Chinese Pharmacopoeia.

Example 2. Screening of buffer systems in IL-2 formulations

In order to screen the appropriate buffer system, under the premise of keeping the IL-2 variant concentration at 2 mg/mL and 5% mannitol as excipient, the buffer systems were prepared as 10 mM glycine-hydrochloric acid (pH 4.5 and 5.0), 10 mM succinate, respectively. Lyophilized formulations of sodium-hydrochloric acid (pH 5.0), 10 mM histidine-hydrochloric acid (pH 5.5 and 6.0), 10 mM phosphate (pH 6.0, 6.5, 7.0 and 7.5). Then, the above preparations with different buffer systems were placed at 40 °C for 30 d, and relevant indicators were investigated at 0 d, 7 d, 14 d, and 30 d. During the sample preparation process, it was found that API 1 was unstable in the 10 mM glycine-hydrochloric acid buffer system and was easily precipitated, so the sample preparation of this buffer system was abandoned. The investigation results of the samples of sodium succinate-hydrochloric acid, histidine-hydrochloric acid and phosphate buffer system are shown in Table 2.

Table 2. Inspection results of API 1 preparations with different buffer systems at 40°C for 30d

According to the above investigation results, compared with the phosphate buffer system samples, the RP-HPLC purity of the pH6.0 histidine-hydrochloric acid buffer system samples decreased significantly, and the stability was poor; while the pH5.0 sodium succinate-hydrochloric acid and pH5. Although the stability of the .5 histidine-hydrochloric acid buffer system sample is basically the same as that of the phosphate buffer system sample, the pH range that can be buffered by these two buffer systems is narrow, and there are certain deficiencies. In summary, the final choice is to use a phosphate buffer system.

Example 3. Screening of excipients in IL-2 formulations

Excipients can affect the appearance and stability of the drug. In view of the fact that mannitol can be used as a freeze-dried skeleton to obtain a good appearance, and trehalose and sucrose are commonly used protective agents for protein drugs, which are beneficial to improve the stability of drugs. Therefore, mannitol or/and trehalose and sucrose were initially selected as excipients, and the dosage of excipients was investigated. Specifically, using a relatively stable freeze-drying process (the primary drying temperature is -29°C), 5% mannitol, 1% mannitol + 7% trehalose, 1% mannitol + 7% sucrose, and 2% mannitol were prepared respectively. +5.25% trehalose and 3% mannitol + 3.5% trehalose samples (formulation numbers 1 to 4, respectively). Then, the samples of the above different preparations were placed at 40°C for 14d, and the relevant indicators were investigated at 0d, 7d, and 14d. The inspection results of API 1 are shown in Table 3:

Table 3. Effects of different ratios of excipients on 14d stability at 40°C

According to the test results, the RP-HPLC and SEC-HPLC purity of preparation No. 1 sample decreased significantly after being placed at high temperature of 40 °C for 14 days, while the stability trends of preparation No. 2 to 4 samples were similar and had no significant difference. In addition, the samples of Formulation Nos. 1 and 4 had good appearance, while the samples of Formulation Nos. 2 and 3 had a slight shrinkage in appearance after lyophilization. Under the same experimental conditions, that is, after being placed at a high temperature of 40 °C for 14 days, the formulation of 1% mannitol + 7% sucrose as an excipient was also tested in this study, and it was found that there was a relatively obvious bottom shrinkage phenomenon, so this excipient was not selected. To sum up, considering the stability and appearance of the sample, the dosage of the excipient composition in the preparation of the test substance is finally determined as 3% mannitol + 3.5% trehalose, that is, the preparation concentration of mannitol is 30 mg/mL, and the seaweed The sugar was formulated at a concentration of 35 mg/mL (38.68 mg/mL as dihydrate). The mass ratio of mannitol and trehalose was controlled in the range of 6:6 to 6:8.

Example 4. Screening of surfactants in IL-2 formulations

Adding a surfactant to the preparation is beneficial to improve the stability of the drug. In this example, the effect of polysorbate 80 on improving the stability of the drug is preliminarily evaluated. Specifically, with 5% mannitol as an excipient, samples without polysorbate 80 and containing 0.05 mg/mL polysorbate 80 were prepared, respectively. Then the samples of the above two preparations were placed at 40°C for 30d, and the relevant indicators were investigated at 0d, 7d, 14d, and 30d. The inspection results of API 1 are shown in Table 4:

Table 4. Investigation results of API 1 sample without and with surfactant at 40℃ for 30d

It can be seen from the inspection results in the above table that, relative to the preparation without polysorbate 80 at high temperature, the decreasing trend of the SE-HPLC purity of the preparation containing polysorbate 80 is significantly slower, indicating that polysorbate 80 has indeed improved the preparation to a certain extent. Because of the effect of stability, 0.05 mg/mL of polysorbate 80 was selected as the surfactant of the test substance.

Similarly, 1% mannitol + 7% trehalose and 3% mannitol + 4% trehalose samples were prepared under the premise that the active ingredient concentration of API 2 was 2 mg/mL, and polysorbate 80 was selected as the surface Active agent, and then the samples of the different formulations mentioned above were placed at 40°C, and the relevant indicators were investigated for 14 days. The inspection results are shown in Table 5.

Table 5. Effects of different ratios of excipients on the stability of API 2 at 40°C for 14 days

According to the test results, the stability of 1% mannitol + 7% trehalose and 3% mannitol + 4% trehalose is basically the same when the polysorbate 80 is 0.05mg/mL after being placed at a high temperature of 40 °C for 14 days. 7% Trehalose + 1% Mannitol Appearance is slightly collapsed. In summary, considering the stability, appearance and process time of the sample, the dosage of the excipient composition in the preparation of the test substance is finally determined to be 3% mannitol + 4% trehalose, that is, the preparation concentration of mannitol is 30 mg/ mL, the formulation concentration of trehalose was 40 mg/mL, and polysorbate 80 was 0.05 mg/mL.

Example 5. Determination of IL-2 formulation components

Based on the results of the screening study, the final formulation components are shown in Table 6.

Table 6. Formulation (strength: 2 mg)

组分名称component name	每毫升用量(mg)Dosage per ml (mg)	每瓶用量(mg)Dosage per bottle (mg)
原料药1API 1	22	2.32.3
甘露醇Mannitol	3030	34.534.5
海藻糖 ^[1] Trehalose ^[1]	38.6838.68	44.48244.482
聚山梨酯80Polysorbate 80	0.050.05	0.05750.0575
磷酸二氢钠 ^[2] Monosodium Phosphate ^[2]	1.0521.052	1.20981.2098
磷酸氢二钠 ^[3] disodium hydrogen phosphate ^[3]	0.3370.337	0.38760.3876
注射用水 ^[4][5] Water for Injection ^[4][5]	加至1mLAdd to 1mL	加至1.15mLAdd to 1.15mL

Remark:

[1] Trehalose, the molecular formula is C ₁₂ H ₂₂ O ₁₁ ·2H ₂ O;

[2] Sodium dihydrogen phosphate, the molecular formula is NaH ₂ PO ₄ ·H ₂ O;

[3] disodium hydrogen phosphate, the molecular formula is Na ₂ HPO ₄ ;

[4] Water for injection is removed in the freeze-drying process;

[5] The declared filling volume of the preparation is 1 mL, and the target filling volume during actual production is set to 1.15 mL.

Example 6. Detection of relevant properties of IL-2 preparations

Relevant properties of IL-2 variant formulations (Table 6) include appearance, solution clarity and color, insoluble particles, visible foreign matter, moisture, pH, protein content, purity, biological activity, sterility and bacterial endotoxin, etc. Quality requirements for applying for clinical registration approval. Referring to Table 7, the quality of the samples from each batch was comparable.

Table 7. Formulation sample related properties - content, purity and biological activity

Example 7. Adsorption detection of IL-2 preparation filter membrane

Prepare a 2 mg/mL liquid preparation according to the preparation components in Table 6, and then filter the above-mentioned liquid preparation with a filter membrane made of 0.22 μm PVDF material. According to 1 unit per 3 ml of filtrate, 4 filtration samples were continuously collected for content detection, and Compare with the content of the liquid formulation before filtration. The results are shown in Table 8. After the liquid preparation was filtered with a PVDF membrane, the protein content and polysorbate 80 content of each sampling point did not change significantly compared with those before filtration, and a PVDF membrane could be selected for filtration.

Table 8. Filter membrane adsorption investigation

过滤体积(mL)Filtration volume (mL)	蛋白含量(mg/mL)Protein content (mg/mL)	聚山梨酯80含量(mg/mL)Polysorbate 80 content (mg/mL)
未过滤液体制剂Unfiltered liquid preparations	2.082.08	0.0510.051
33	2.052.05	0.0540.054
66	2.062.06	0.0570.057
99	2.072.07	0.0470.047
1212	2.062.06	0.0460.046

Example 8. Lyophilization of IL-2 Formulations

The IL-2 preparation was filled into vials according to the preparation components in Table 6, placed in a freeze-drying box, and freeze-dried. The lyophilization procedure is pre-freezing, primary drying and secondary drying. After the lyophilization procedure is complete, vacuum stopper. The reconstituted samples were compared before and after lyophilization. The results show that the reconstituted solution can maintain the good properties of the liquid formulation.

Although the foregoing invention has been described in detail with the aid of examples for a clear understanding, the description and examples should not be construed as limiting the scope of the present disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

A pharmaceutical composition comprising IL-2, mannitol, trehalose and polysorbate;

The IL-2 is an IL-2 variant or a derivative thereof,

Said IL-2 variant or derivative thereof comprises a mutation selected from N26Q and/or N29S.
The pharmaceutical composition according to claim 1, wherein the mass ratio of mannitol and trehalose is 1:10 to 10:1, preferably 1:7 to 1:1, more preferably 1:7 to 6:7, and most preferably Preferably it is 6:8 to 6:7.
The pharmaceutical composition according to claim 1 or 2, wherein:

The IL-2 is at a concentration of about 0.1 mg/mL to about 100 mg/mL, preferably about 1 mg/mL to about 10 mg/mL, more preferably about 2 mg/mL; and/or,

The mannitol concentration is about 10 mg/mL to about 100 mg/mL, preferably about 10 mg/mL to about 50 mg/mL, more preferably about 30 mg/mL; and/or,

The trehalose concentration is about 10 mg/mL to about 100 mg/mL, preferably about 30 mg/mL to about 60 mg/mL; more preferably about 35 mg/mL; and/or,

The polysorbate is polysorbate 80; the concentration is about 0.01 mg/mL to about 0.2 mg/mL, preferably about 0.02 mg/mL to about 0.1 mg/mL, and most preferably about 0.05 mg/mL.
The pharmaceutical composition according to any one of claims 1 to 3, further comprising a buffer selected from the group consisting of citrate buffer, acetate buffer, histidine buffer, phosphate Buffer, carbonate buffer, succinate buffer; preferably phosphate buffer.
The pharmaceutical composition of claim 4, the concentration of the buffer is about 2 mM to about 50 mM, preferably about 5 mM to about 20 mM, more preferably about 10 mM.
The pharmaceutical composition of any one of claims 1 to 5, which has a pH of 5.5 to 7.5, preferably a pH of 6.0 to 7.0, more preferably a pH of about 6.5.
According to the pharmaceutical composition of any one of claims 1 to 6, the IL-2 is dissolved in a physiologically acceptable solvent, preferably physiological saline, water for injection, and glucose solution.
The pharmaceutical composition of any one of claims 1 to 7, comprising:

a) 0.1 mg/mL to 100 mg/mL IL-2,

b) 10 mg/mL to 100 mg/mL of mannitol,

c) 10 mg/mL to 100 mg/mL trehalose, and

d) 0.01 mg/mL to 0.2 mg/mL polysorbate,

The pH of the pharmaceutical composition is 5.0 to 7.5;

Preferably, the pharmaceutical composition comprises:

a) 1 mg/mL to 10 mg/mL IL-2,

b) 10 mg/mL to 50 mg/mL of mannitol,

c) 30 mg/mL to 60 mg/mL trehalose,

d) 0.02 mg/mL to 0.1 mg/mL polysorbate 80,

e) 5 mM to 20 mM phosphate buffer,

The pH of the pharmaceutical composition is 6.0 to 7.0;

More preferably, the pharmaceutical composition comprises:

a) about 2 mg/mL of IL-2,

b) about 30 mg/mL of mannitol,

c) about 35 mg/mL trehalose,

d) about 0.05 mg/mL polysorbate 80,

e) about 10 mM phosphate buffer,

The pH of the pharmaceutical composition is about 6.5.
The pharmaceutical composition according to any one of claims 1 to 8, the IL-2 variant or derivative thereof comprising a first type of mutation, further comprising a second type of mutation and/or a third type of mutation, and any Optionally, the IL-2 variant or derivative thereof may or may not comprise C125A;

wherein, the first type of mutation is selected from N26Q and/or N29S,

Preferably, the first type of mutation further comprises N71Q;

The second type of mutation is selected from any one of 1) to 3):

1) F42A and Y45A,

2) F42A and L72G, and

3) Y45A and L72G;

The third type of mutation is N88R or N88G or N88I or N88D;

Preferably, the IL-2 variant or derivative thereof comprises the mutation shown in any one of 4) to 6):

4) N26Q, N29S, F42A, N71Q and L72G,

5) N26Q, N29S and N88R,

6) N26Q, N29S, F42A and L72G.
The pharmaceutical composition according to claim 9, said IL-2 variant or derivative thereof comprising a compound selected from the group consisting of SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:38 The polypeptide shown in any one of ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 30;

Optionally, the IL-2 variant or derivative thereof may or may not comprise amino acid M at position 1.
The pharmaceutical composition of any one of claims 1 to 10, wherein the IL-2 is monomeric, and/or PEGylated, and/or glycosylated, and/or albumin conjugated or fused and/or Fc-fused, and/or hydroxyethylated, and/or de-O-glycosylated;

Preferably, the PEG is attached to the N-terminus of the IL-2 variant;

More preferably, the relative molecular weight of PEG is 5KD to 50KD;

Most preferably, the relative molecular weight of the PEG is about 20 KD.
The pharmaceutical composition according to claim 9, the IL-2 variant or derivative thereof is selected from the group consisting of:

Wherein the relative molecular weight of PEG is about 20KD;

Optionally, the IL-2 variant or derivative thereof may or may not comprise amino acid M at position 1.
A freeze-dried preparation obtained by freeze-drying the pharmaceutical composition according to any one of claims 1 to 12; and/or, the freeze-dried preparation can be reconstituted to form as claimed in claim 1 The pharmaceutical composition of any one of to 12.
A reconstituted solution prepared by reconstituting the lyophilized preparation of claim 13.
Use of the pharmaceutical composition according to any one of claims 1 to 12, or the lyophilized preparation according to claim 13, or the reconstituted solution according to claim 14 in the preparation of a medicine;

When IL-2 contains the first type of mutation, or contains the first type and the second type of mutation, the pharmaceutical composition is used for the treatment of proliferative diseases or metastatic proliferative diseases, immune diseases, regulatory T cell mediators induced immune response, stimulate the individual's immune system;

Preferably, the proliferative disease is a tumor or carcinoma,

Preferably, the immune disease is diabetes;

More preferably, the tumor or carcinoma is selected from the group consisting of epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, carcinoma caused by papilloma virus, adenocarcinoma, carcinoma, melanoma, sarcoma, teratoma, lung Tumors, metastatic lung cancer, lymphoma and metastatic renal cell carcinoma;

When IL-2 contains the first type of mutation, or contains the first and third types of mutations, the drug is used for the treatment of autoimmune diseases or autoimmune reactions after organ transplantation;

Preferably, the autoimmune disease is selected from the group consisting of type I diabetes, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, eczema, asthma.
A method for preparing the pharmaceutical composition of any one of claims 1 to 12, the method comprising the step of mixing IL-2 with each pharmaceutical excipient.