A composition of pegylated IL-2 as a Treg-targeting modulator and methods of making and uses thereof

Abstract

The present application relates to a PEG-modified IL-2 composition as a targeted modulator for regulating T cells (Treg), which comprises one or more of a mono-modified PEG-IL-2 molecule, a double-modified PEG-IL-2 molecule, a triple-modified PEG-IL-2 molecule and a quadruple-modified PEG-IL-2 molecule, wherein the PEG modification of the mono-modified PEG-IL-2 molecule occurs at the alpha-NH2 position at the N-terminal of the IL-2 amino acid chain, and the PEG modification of the double-modified PEG-IL-2 molecule, the triple-modified PEG-IL-2 molecule and the quadruple-modified PEG-IL-2 molecule occurs at the alpha-NH2 position at the N-terminal of the IL-2 amino acid chain and / or the epsilon-NH2 position of the internal lysine of the IL-2 amino acid chain. The present application also relates to the use of the above-mentioned PEG-modified IL-2 composition in the preparation of a medicament for treating autoimmune diseases and other immune-related diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biopharmaceuticals, and specifically relates to a composition of PEG-modified IL-2 as a Treg-targeting modulator and a preparation method thereof. The present application also relates to the use of the above-mentioned PEG-modified IL-2 composition as a Treg-targeting modulator in the preparation of a medicament for treating autoimmune system diseases. BACKGROUND

[0002] Interleukin-2 (IL-2) is an important cytokine in the human body, which plays an important role in T cell proliferation and differentiation. At a high dose, it can stimulate the activation and proliferation of effector T cells, activate the immune response, and is clinically used to treat diseases such as renal cell carcinoma. At present, high-dose IL-2 has been approved by regulatory authorities for the clinical treatment of tumors. Low-dose IL-2 can activate Treg, inhibit Th17 and Tfh differentiation, and suppress the immune response of the body. At present, low-dose IL-2 has multiple clinical studies in the treatment of autoimmune diseases and their complications. In addition, based on the same mechanism of action, IL-2 has also been reported in the treatment of immune-related diseases such as acne, recurrent oral ulcer, uveitis, skin rash, miscarriage, asthma, and urticaria.

[0003] Low-dose IL-2 as a new means of treating autoimmune diseases needs to solve two main problems: short half-life and stimulation of effector T cells. The in vivo half-life of IL-2 is less than 15 min, and it needs to be administered daily or every other day, which has poor patient compliance. At present, the molecular weight of the molecule is mainly increased by PEG modification and fusion protein methods to reduce the glomerular filtration effect and prolong the half-life. For the stimulation of effector T cells, the current methods mainly change the receptor preference of the molecule by means of steric hindrance effect or key interaction site mutation brought by PEG modification, etc. to achieve. Biasing IL-2Rαβγc can selectively stimulate Treg cells to treat autoimmune diseases.

[0004] The long-acting IL-2 molecules currently under development for autoimmune diseases mainly include PEG-modified molecules and fusion protein molecules. Among them, PEG modification includes IL-2 modified molecules or IL-2 mutant (IL-2v) modified molecules. The initial research represented by Cetus Company randomly modified IL-2 with the aim of prolonging the drug half-life. With the in-depth study of the mechanism of action of IL-2 molecules, in addition to prolonging the half-life, another main purpose of IL-2 modification is to regulate its receptor preference, which can reduce the side effects of drug use. Among them, Nektar Company randomly modified NTKR-358 (see, for example, KIRK PETER BENEDICT, et al. (2019). SELECTIVE TREG STIMULATOR RUR20kD-IL-2 AND RELATED COMPOSITIONS and WO2019226538A1), through the modification process to control the relatively specific modification of PEG molecules on several sites, to affect the binding of IL-2 to IL-2Rβ subunit, and then to make it more specifically act on Treg cells, to achieve the purpose of treating diseases. THOR-707 and THOR-809 of Sanofi Company use non-natural amino acid substitution to introduce PEG site-specific modification (see, for example, Ptacin J, et al. (2019). THOR-809: An IL-2 Engineered from an Expanded Genetic Alphabet for the Potential Treatment of Autoimmune Disorders. Arthritis Rheumatol. 2019; 71 (suppl 10)), which respectively interfere with the binding of IL-2 to IL-2Rα and IL-2Rβ. The molecules obtained can be used for the treatment of tumors and autoimmune diseases, respectively. Dual-31 / 51-20K developed by the team of Professor ZHOU Demin of the School of Pharmacy of Peking University and Professor ZHANG Xue of Peking Union Medical College also belongs to IL-2v modified molecules. The representative variety of fusion protein is AMG-592 of Amgen Company, which is an Fc-fused IL-2v molecule, and RG7835 molecule of Roche, which is an IgG-fused IL-2v molecule. The present inventors have also developed a method for preparing PEG site-specific modified IL-2 conjugates using denaturant, and have synthesized a new class of PEG site-specific modified IL-2 molecules using this method (see CN114392348A).

[0005] However, among the above-mentioned molecules, NKTR-358 randomly modifies the epsilon-NH2 modification of the Lys residue in the IL-2 molecule with a Y-type PEG molecule (20K), obtains a molecule mainly with double modification and triple modification, and shows certain efficacy in preclinical animal and phase I clinical studies. However, based on the literature data, the activity of NKTR-358 is greatly lost relative to the IL-2 molecule; THOR-809 and Dual-31 / 51-20K both use non-natural amino acid mutations to reduce the affinity of the IL-2 molecule for IL-2Rβγ, and then extend the half-life by PEG modification, which has obvious shortcomings. The non-natural amino acid substitution is the first to increase the production cost, and the introduction of non-natural amino acids will increase the risk of immunogenicity. In addition to protein mutation, fusion protein molecules increase the risk of immunogenicity, and the fusion protein leads to an increase in molecular weight, and Fc or IgG fusion is not suitable for E. coli expression, and generally needs to be produced by CHO cell lines, which has high production cost.

[0006] In view of the unsatisfied clinical needs of low-dose IL-2 in the treatment of autoimmune diseases, the development of new IL-2-based biotechnology drugs with long-acting and low adverse reaction characteristics and the ability to target specific immune cell subpopulations is a scientific problem that has been concerned in this field for a long time, and has important scientific significance and clinical transformation value. SUMMARY

[0007] In order to solve the above technical problems, the inventors have developed an IL-2 composition with Treg-targeted regulation by PEG modification at certain sites of IL-2, and found that the PEG-modified IL-2 composition has a significantly prolonged half-life in vivo compared with natural IL-2, and can significantly induce a significant increase in the level of Treg in animals or humans in a specific dose range, and the effect is better than that of natural IL-2 at the same dose. Therefore, it can be applied to the treatment of various autoimmune diseases and their complications, as well as immune-related diseases such as acne, recurrent oral ulcer, alopecia areata, skin rash, miscarriage, asthma, and urticaria in the future.

[0008] Specifically, the present application is realized by the following technical solutions:

[0009] In the first aspect, the present application provides a composition of PEG-modified IL-2 as a Treg-targeting modulator, which comprises one or more of a mono-modified PEG-IL-2 molecule, a di-modified PEG-IL-2 molecule, a tri-modified PEG-IL-2 molecule and a tetra-modified PEG-IL-2 molecule, wherein the PEG modification of the mono-modified PEG-IL-2 molecule occurs at the a-NH2 position at the N-terminal of the IL-2 amino acid chain, and the PEG modification of the di-modified PEG-IL-2 molecule, the tri-modified PEG-IL-2 molecule and the tetra-modified PEG-IL-2 occurs at the a-NH2 position at the N-terminal of the IL-2 amino acid chain and at the ε-NH2 position of lysine inside the IL-2 amino acid chain.

[0010] As an optional way, in the above composition, the PEG is linear, or the PEG is branched, and the average molecular weight of the PEG is 1 kDa or more, usually 1 k-100 kDa.

[0011] Preferably, the average molecular weight of the PEG is 10 kDa-60 kDa.

[0012] More preferably, the average molecular weight of the PEG is 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa or 60 kDa.

[0013] As an optional way, in the above composition, the end-capping group of the PEG is selected from H, alkyl, cycloalkyl, aryl and the like, and can be methyl, ethyl, propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, cyclopentyl, n-hexyl, isohexyl, cyclohexyl, benzyl or naphthyl and the like.

[0014] As an optional way, in the above composition, the linking group between the PEG and the IL-2 is selected from one or more combinations of -(CH2) n -, -(CR1CR2) n -, -CO(CH2) n -, -CO(CR1CR2) n -, -(CH2) n COO(CH2) n -, -CONH(CH2) n -, -CONH(CR1CR2) n -, -N(R1R2)(CH2) n CONH(CH2) n -.

[0015] n is an integer of 0-10, preferably, the n is an integer of 1-6. More preferably, the n is an integer of 1-4.

[0016] R1and R2are independently selected from one or more of: hydrogen atom, hydroxyl group, amino group, imino group, alkyl group or substituted alkyl group, alkoxy group, ester group, acyl ester group, halogen atom. Preferably, R1and R2are independently selected from one or more of: -H, -OH, -OCH3, -CH3, -CH2CH3, -CH2-, -CH2CH2-, -CH2CO-, -CH2CO-, F, -Cl, -Br, -I.

[0017] More preferably, the linker between PEG and IL-2 is selected from one or more of: -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CON(CH2CH2)CH2CONHCH2CH2-.

[0018] Preferably, the IL-2 is one or more of native IL-2, recombinant IL-2, or IL-2 gene mutation product with IL-2 function.

[0019] As an alternative, in the above composition, the composition targets the activation of Treg, and the half-life of the composition is prolonged compared to wild-type IL-2.

[0020] Preferably, the half-life of the composition is prolonged for 1-120 hours or more.

[0021] Further preferably, the half-life of the composition is prolonged for 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 48 hours, 3 days, 4 days, 5 days or more.

[0022] As an alternative, in the above composition, the composition contains 10 mol% or less of mono-modified PEG-IL-2 molecules, 20-60 mol% of di-modified PEG-IL-2 molecules, 20-60 mol% of tri-modified PEG-IL-2 molecules, and / or 20 mol% or less of tetra-modified PEG-IL-2 molecules in terms of molar percentage.

[0023] As an alternative, in the above composition, the composition contains 10 mol% or less of mono-modified PEG-IL-2 molecules, 30-55 mol% of di-modified PEG-IL-2 molecules, 35-50 mol% of tri-modified PEG-IL-2 molecules, and / or 20 mol% or less of tetra-modified PEG-IL-2 molecules in terms of molar percentage.

[0024] As an alternative, the composition according to the first aspect of the present application can be prepared by any conventional method of PEG modification known to those skilled in the art of biopharmaceuticals.

[0025] As an alternative, in the above composition, the N-terminus of substantially all of the modified molecules contains PEG modification.

[0026] As an alternative, in the above composition, the modification of ε-NH2 within IL-2 can occur at any amino acid site.

[0027] Preferably, the modification of ε-NH2 within IL-2 occurs mainly at Lys residues.

[0028] More preferably, the modification of ε-NH2 within IL-2 can occur mainly at Lys residues such as K33, K44, and K77.

[0029] In a second aspect, the present application provides a method for preparing the composition according to the first aspect of the present application, the method comprising the steps of:

[0030] reacting IL-2, a PEG modification agent, and sodium cyanoborohydride at 4-30°C and pH 4-7 to obtain a PEG-modified IL-2 composition in which the α-NH2 at the N-terminus of the IL-2 amino acid chain and / or the ε-NH2 of Lys within the IL-2 amino acid chain is modified.

[0031] As an alternative, in the above method, the PEG modification agent can be PEG-aldehyde, and the aldehyde group can be formaldehyde, acetaldehyde, propionaldehyde, isopropyl aldehyde, butyl aldehyde, isobutyl aldehyde, pentyl aldehyde, isopentyl aldehyde, hexyl aldehyde, isohexyl aldehyde, heptyl aldehyde, isoheptyl aldehyde, octyl aldehyde, isooctyl aldehyde, nonyl aldehyde, isononyl aldehyde, decyl aldehyde, isodecyl aldehyde, and substituted aldehydes. Preferably, it can be mPEG-formaldehyde, mPEG-acetaldehyde, mPEG-propionaldehyde, mPEG-butyl aldehyde, mPEG-pentyl aldehyde, etc.

[0032] As an alternative, in the above method, the method further comprises a step of purifying the modified product by chromatography.

[0033] In a third aspect, the present application provides a pharmaceutical preparation as a Treg-targeting modulator, the pharmaceutical preparation comprising the composition according to the first aspect of the present application, and a pharmaceutically acceptable carrier.

[0034] As an alternative, in the above pharmaceutical preparation, the dosage form of the pharmaceutical preparation is an oral dosage, a liquid dosage, an emulsion, a coating, an ointment, a plaster, a transdermal absorption type dosage form, an aerosol, an injection, or a suppository.

[0035] Preferably, the dosage form of the pharmaceutical preparation is an injection.

[0036] In a fourth aspect, the present application provides use of the composition of the first aspect above or the pharmaceutical preparation of the second aspect above in the manufacture of a medicament for treating autoimmune diseases and related diseases thereof.

[0037] Alternatively, in the use above, the autoimmune disease is one or more of graft versus host disease, rheumatoid arthritis, systemic lupus erythematosus, Sjogren’s syndrome, type I diabetes, type II diabetes, autoimmune liver disease, hyperthyroidism / Graves’ disease, hypothyroidism / Hashimoto’s disease, psoriasis, dermatomyositis, scleroderma, multiple sclerosis, myasthenia gravis, demyelinating disease, uveitis, factor VIII inhibitor syndrome, ulcerative colitis, Crohn’s disease, vitiligo, lupus nephritis, polymyositis, psoriatic arthritis, juvenile idiopathic arthritis, autoimmune glomerulonephritis, primary biliary cholangitis, Goodpasture’s syndrome, Behcet’s disease, ankylosing spondylitis, granulomatosis with polyangiitis, nephrotic syndrome, alopecia areata, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia, recurrent miscarriage, relapsing polychondritis, or autoimmune alopecia, immune-related adverse events, hepatitis C-induced vasculitis, systemic vasculitis, rheumatic fever, sarcoidosis, autoimmune myocarditis, autoimmune lymphoproliferative syndrome, autoimmune giant scaly cutaneous lupus, encephalomyelitis, various dermatitis, Addison’s disease, chronic fatigue syndrome, Kawasaki disease, Guillain-Barre syndrome, Wegener’s disease, myelodysplastic syndrome, Evan’s syndrome, Parkinson’s disease, anti-phospholipid syndrome, mixed connective tissue disease, idiopathic pulmonary fibrosis, and complications thereof.

[0038] Preferably, the autoimmune disease is systemic lupus erythematosus, rheumatoid arthritis, Sjogren’s syndrome, acne, uveitis, atopic dermatitis, or type I diabetes.

[0039] Alternatively, in the use above, the immune-related disease comprises acne, recurrent oral ulceration, skin rash, alopecia areata, acute coronary syndrome, amyotrophic lateral sclerosis, ischemic heart disease, hepatitis C, AIDS, bipolar depression, novel coronavirus infection, atherosclerosis, asthma, urticaria, allergic rhinitis, allergic retinitis.

[0040] In some embodiments, the compositions, pharmaceutical compositions, or formulations described herein are administered to a subject by a variety of routes of administration, including, but not limited to, parenteral or transdermal routes of administration. In some cases, parenteral administration includes intravenous, subcutaneous, intramuscular, intracerebral, intranasal, intraarterial, intraarticular, intradermal, intravitreal, intraosseous infusion, intraperitoneal, or intrathecal administration. In some cases, the compositions or pharmaceutical compositions are formulated for local administration. In other cases, the compositions or pharmaceutical compositions are formulated for systemic administration.

[0041] In some embodiments, the pharmaceutical formulations include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, liposomal dispersions, aerosols, immediate release formulations, controlled release formulations, delayed release formulations, extended release formulations, pulsatile release formulations, and mixed immediate release and controlled release formulations.

[0042] In some embodiments, the pharmaceutical formulations include one or more carrier materials selected based on compatibility with the conjugate or pharmaceutical composition disclosed herein and release characteristics of the desired dosage form. Exemplary carrier materials include, for example, binders, surfactants, solubilizers, stabilizers, lubricants, humectants, diluents, and the like.

[0043] The present application has the following beneficial effects over the prior art:

[0044] The inventors have developed an IL-2 composition of a Treg-targeting modulator with PEG modification at certain specific sites of IL-2, and found that the PEG-modified IL-2 composition provided by the present application has a significantly prolonged half-life in vivo compared to native IL-2, and can significantly induce a significant increase in the level of Tregs in animals or humans in a specific dose range, with better effects than the same dose of native IL-2. Therefore, it can be applied to the treatment of various autoimmune diseases in the future.

[0045] And, compared with PEG modification at the ε-NH2 position of the internal lysine of IL-2, the loss of activity at the N terminus is less, so that, at the same modification degree, the modification method of the N terminus plus the internal modification has better effects than the internal modification alone (see Effectiveness Example 2). It has also been found that, due to molecular structure problems, Y-type PEG modification is prone to breakage, and has poor stability compared with straight-chain type PEG modification.

[0046] In addition, the present application avoids the use of site mutations, thereby reducing the risk of immunogenicity; the present application uses an IL-2 molecule expressed by E. coli recombinant as a modification raw material, which does not require mammalian system expression, and the cost is significantly lower than that of fusion proteins. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 : Representative liquid chromatogram of PEG-modified IL-2 molecules.

[0048] Figure 2 Comparison of Treg induction ability of different molecular weight PEG modified double modified PEG-IL-2 molecules.

[0049] Figure 3 Figure of peripheral blood Treg level of healthy mice after single subcutaneous administration of different site double PEG modified molecules.

[0050] Figure 4 Figure of peripheral blood Treg level of healthy mice after single subcutaneous administration of PEG modified IL-2 composition of the present application.

[0051] Figure 5 Figure of Treg level of MRL / lpr animals after 12 weeks of administration. Figure 5 A is the peripheral blood Treg level of MRL / lpr mice after 12 weeks of administration, Figure 5 B is the spleen Treg level of MRL / lpr mice after 12 weeks of administration.

[0052] Figure 6 Figure of urine protein level of MRL / lpr animals after 12 weeks of administration.

[0053] Figure 7 Figure of Treg level of Sjogren's syndrome model animals.

[0054] Figure 8 Figure of α-fodrin antibody level of Sjogren's syndrome model animals.

[0055] Figure 9 Figure of therapeutic effect of PEG modified IL-2 composition of the present application in rheumatoid arthritis model.

[0056] Figure 10 Figure of Treg induction ability of diPEG-IL-2v (IL-2 mutant). DETAILED DESCRIPTION

[0057] The present application will be further described below with reference to specific examples. It should be understood that the specific examples described herein are intended to explain the present application and are not intended to limit the scope of the present application.

[0058] Unless otherwise specified, the techniques or conditions in the examples are in accordance with those described in the literature or in accordance with the product instructions. Unless otherwise specified, the reagents or instruments used are conventional products that can be purchased through regular channels.

[0059] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0060] Preparation Example

[0061] Different PEG modified molecules were prepared. The composition of different molecules was confirmed by liquid detection method, Figure 1 The representative liquid phase diagram of PEG modified IL-2 molecules. Table 1 shows the composition of different PEG modified IL-2 molecules.

[0062] Wherein IL-2 is a natural IL-2 molecule, nPEG-IL-2 is a N-terminal single PEG modified IL-2 molecule, diPEG-IL-2 is a double PEG modified molecule, triPEG-IL-2 is a three PEG modified IL-2 molecule, PEG-IL-2(n+di), PEG-IL-2(di+tri) and PEG-IL-2(tri+tet) represent single+double modified PEG-IL-2 molecule, double+three modified PEG-IL-2 molecule and three+four modified PEG-IL-2 molecule as the main PEG-IL-2 molecule.

[0063] CNPEG-IL-2 molecule is a N-terminal and C125 site modified PEG-IL-2 molecule, K2PEG-IL-2 molecule is a random modification of internal lysine (K) of the protein, and the double modified molecule obtained by purification.

[0064] Table 1: Composition of different PEG modified IL-2 molecules (unit: mol%)

[0065]

[0066] In particular, for the PEG-IL-2(di+tri) described above, the present application prepared multiple batches of PEG-IL-2(di+tri) samples, and analyzed the composition of different modification degree molecules by reverse chromatography, as shown in Table 2, all batches of samples were mainly double modified PEG-IL-2 and triple modified PEG-IL-2, the sum of the two was more than 80mol%, the single modified PEG-IL-2 was between 0-10mol%, and the four or higher modified PEG-IL-2 accounted for between 0-20mol%.

[0067] Table 2: Composition of different batches of PEG-IL-2(di+tri) PEG modified IL-2 molecules (unit: mol%)

[0068]

[0069]

[0070] Preparation Example 1: Preparation of N-terminal α-NH2 PEG modified IL-2 molecule

[0071] Reaction system: IL-2, 0.5 mg / mL; mPEG-ALD, 6.5 mg / mL; sodium cyanoborohydride, 3.3 mM / L; 20 mM phosphate buffer, pH 6.0. The above reaction system was reacted at 4°C for 16 h, with constant stirring, and the reaction was terminated by adjusting the pH to about 3.0 with acetic acid. The reaction solution was purified by chromatography to obtain the modified sample, which was detected as an α-NH2 modified molecule, designated as nPEG-IL-2.

[0072] Reaction system: IL-2, 0.8 mg / mL; mPEG-ALD, 10.4 mg / mL; sodium cyanoborohydride, 5.3 mM / L; 20 mM phosphate buffer, pH 6.0. The above reaction system was reacted at 4°C for 16 h, with constant stirring, and the reaction was terminated by adjusting the pH to about 3.0 with acetic acid. The modified product was detected as a mixed product, containing an α-NH2 modified molecule (nPEG-IL-2) and an α-NH2 and ε-NH2 modified double molecule (dPEG-IL-2), and the reaction solution was purified by chromatography to obtain nPEG-IL-2 and dPEG-IL-2 molecules, respectively.

[0073] Reaction system: IL-2, 1.0 mg / mL; mPEG-ALD, 13 mg / mL; sodium cyanoborohydride, 6.6 mM / L; 20 mM phosphate buffer, pH 6.0. The above reaction system was reacted at 4°C for 16 h, with constant stirring, and the reaction was terminated by adjusting the pH to about 3.0 with acetic acid. The modified product was detected as a mixed product, containing an α-NH2 modified molecule (nPEG-IL-2) and an α-NH2 and ε-NH2 modified double molecule (dPEG-IL-2), and the reaction solution was purified by chromatography to obtain nPEG-IL-2 and dPEG-IL-2 mixed molecules (PEG-IL-2 (n+di)).

[0074] Preparation Example 4: Preparation of α-NH2 and ε-NH2 combined molecules (double and triple modifications, small amount of single and quadruple modifications)

[0075] Preparation Example 4: Preparation of di- and tri-modified molecules

[0076] Preparation Example 5: Preparation of di-modified molecules with ε-NH2

[0077] Methoxypolyethylene glycol succinimidyl carbonate (mPEG-SC) is the most commonly used reagent for PEG modification of proteins. It reacts with ε-NH2 of Lys residues in proteins under alkaline conditions to form PEG-modified molecules. Reaction system was prepared as follows: IL-2, 0.8 mg / mL; mPEG-SC, 10.4 mg / mL; 20 mM phosphate buffer, pH 8.0. The reaction system was stirred at 4°C for 6 h, and the reaction was terminated by adjusting the pH to about 3.0 with acetic acid. The modified product was a mixture containing mono- and di-ε-NH2 modified molecules. The reaction solution was purified by chromatography to obtain the di-ε-NH2 modified molecule K2-PEG-IL-2.

[0078] Preparation Example 6: Preparation of C125 and α-NH2 combined modified molecules

[0079] The methoxy polyethylene glycol maleimide (mPEG-Maleimide, mPEG-MAL) is generally used as a thiol modifier to modify the thiol group on the Cys residue inside the protein under certain conditions. The reaction system is prepared as follows: 1 mg / mL IL-2, 4 M urea, 0.05 w / v (mg / mL) % SDS, mPEG-MAL 5 mg / mL, 1x PBS, pH 7.4. After thorough mixing, the reaction is carried out at room temperature for 6 hours. The reaction product is purified by hydrophobic chromatography and gel filtration system to obtain the Cys site-modified IL-2 molecule C-PEG-IL-2. The C-PEG-IL-2 is further reacted with mPEG-ALD to prepare the reaction system as follows: C-PEG-IL-2, 0.8 mg / mL; mPEG-ALD, 10.4 mg / mL; sodium cyanoborohydride, 5.3 mM / L; 20 mM phosphate buffer, pH 6.0. The above reaction system is reacted at 4°C for 16 h, during which it is constantly stirred, and the reaction is terminated by adjusting the pH to about 3.0 with acetic acid. The reaction product is purified by chromatography to obtain the double-PEG-modified IL-2 molecule CN-PEG-IL-2 which is co-modified at the Cys and N-terminal α-NH2.

[0080] Effect Example

[0081] Effect Example 1: Comparison of Treg induction ability of double-modified PEG-IL-2 molecules with different molecular weight PEG

[0082] Linear 5KD, 10KD, 20KD, 30KD, 40KD and branched 20KD, 30KD, 40KD double-PEG-modified IL-2 molecules are prepared respectively.

[0083] The above molecules are compared in terms of Treg induction ability according to the method of Effect Example 2. The results, as shown in Table 1, show that the linear 20K double-PEG-modified IL-2 molecule has the highest Treg induction ability. Figure 2 Effect Example 2: Comparison of Treg induction ability of double-modified PEG-IL-2 molecules with different modification sites

[0084] C57BL / 6 mice (10 weeks old) are randomly divided into a control group, an IL-2 group, a diPEG-IL-2 group, a CN-PEG-IL-2 group and a K2-PEG-IL-2 group, with 5 mice in each group. The control group is subcutaneously injected with PBS once. The IL-2 group is subcutaneously injected with 0.3 mg / kg IL-2 (dosing volume 10 mg / mL) once a day for 5 consecutive days. The rest of the double-PEG-IL-2 molecule groups are subcutaneously injected with 0.3 mg / kg of the corresponding drug (dosing volume 10 mg / mL) once.

[0085] The day of administration was recorded as day 0 (D0), and the blood was taken from the orbital (about 200 μL) on day 1-7 (D1-D7) and day 10 (D10) after administration for the detection of lymphocyte subgroups, including Treg cells, CD4 + T cells. The growth ratio of various T cell subgroups was determined according to the above results.

[0086] As shown in Figure 3 , the results showed that the induction ability of the double-modified PEG-IL-2 molecules of several different modification sites was better than that of the same dose of IL-2 multiple administrations. Among the double-modified PEG-IL-2, the double PEG-modified molecule composed of α-NH2 and single ε-NH2 modification (diPEG-IL-2) was better than the double ε-NH2 modified molecule (K2-PEG-IL-2) and the double PEG-modified IL-2 molecule co-modified by C125 and α-NH2 (CN-PEG-IL-2).

[0087] Example 3: Detection of T cell changes in healthy mice by subcutaneous injection of single administration

[0088] C57BL / 6 mice (10 weeks old) were randomly divided into nPEG-IL-2 group, diPEG-IL-2 group, PEG-IL-2 (n+di) group, PEG-IL-2 (di+tri) group and triPEG-IL-2 group, 5 mice in each group. Subcutaneous injection of 0.3 mg / kg of the corresponding drug (dosing volume 10 mg / mL), administration once.

[0089] The day of administration was recorded as day 0 (D0), and the blood was taken from the orbital (about 200 μL) on day 1-7 (D1-D7) and day 10 (D10) after administration for the detection of lymphocyte subgroups, including Treg cells, CD4 + T cells. The growth ratio of various T cell subgroups was determined according to the above results.

[0090] As shown in Figure 4 , the results showed that with the increase of the degree of PEG modification, the ability of equal amount of PEG-IL-2 molecules to induce Treg in healthy animals gradually increased, but PEG-IL-2 (di+tri) and triPEG-IL-2 were similar in effect.

[0091] Example 4: Systemic lupus erythematosus, Sjogren's syndrome and rheumatoid arthritis animal pharmacodynamic study of different modification degree molecules

[0092] Female MRL / lpr mice were randomly divided into PBS group, nPEG-IL-2 group, diPEG-IL-2 group, PEG-IL-2(n+di) group, PEG-IL-2(di+tri) and triPEG-IL-2 group according to body weight and urine protein level before administration, 5 mice in each group, 0.3 mg / kg was subcutaneously injected, the administration frequency was 3 days once. The administration was started at the 12th week and lasted to the 24th week. All mice were sacrificed at the 3rd day after the last administration, and urine protein detection was performed. The results are shown in Table 3:

[0093] Table 3: Urine protein level of systemic lupus erythematosus model mice in each group before and after treatment (mg / mL, x±s)

[0094] Group Before treatment After treatment PBS group 0.91±0.42 2.74±0.56 nPEG-IL-2 group 0.79±0.39 2.12±0.85 diPEG-IL-2 group 0.87±0.23 1.55±0.70* PEG-IL-2 (n+di) group 0.81±0.51 1.72±0.47* PEG-IL-2 (di+tri) group 0.88±0.55 0.82±0.37*** triPEG-IL-2 group 0.97±0.32 1.03±0.33***

[0095] Note: t test, compared with PBS group, *P<0.05; ***P<0.001.

[0096] The submandibular glands (both sides) of Balb / c mice were cut into pieces in PBS, then homogenized on an ultrasonic homogenizer, and then centrifuged in a centrifuge. The supernatant was used to immunize 6-8 week old female BALB / c mice, with immunization every 2 weeks, for a total of 3-4 times of subcutaneous injection in the back of the neck.

[0097] The Sjogren's syndrome model mice were randomly divided into PBS group, nPEG-IL-2 group, diPEG-IL-2 group, PEG-IL-2(n+di) group, PEG-IL-2(di+tri) and triPEG-IL-2 group, 5 mice in each group, 0.3 mg / kg was subcutaneously injected, the administration frequency was 3 days once. The administration was continuously given for 4 weeks, and then the serum α-fodrin antibody level was detected. The results are shown in Table 4:

[0098] Table 4: α-fodrin antibody level of Sjogren's syndrome model mice in each group before and after treatment (A450, x±s)

[0099] Group Before treatment After treatment PBS group 1.48±0.15 1.91±0.21 nPEG-IL-2 group 1.53±0.08 1.53±0.19 diPEG-IL-2 group 1.62±0.18 1.37±0.23* PEG-IL-2 (n+di) group 1.44±0.26 1.51±0.32 PEG-IL-2 (di+tri) group 1.49±0.30 0.85±0.35** triPEG-IL-2 group 1.37±0.15 0.82±0.23**

[0100] Note: t test, compared with PBS group, *P<0.05, **P<0.01.

[0101] 8 weeks of male DBA / 1 mice tail vein injection of 400 μg / mL bovine type II collagen and equal volume of complete Freund's adjuvant suspension emulsion 100 μL, 1 week later, the same method of booster immunization, to observe the change of joint swelling of four limbs. The mice with arthritis were randomly divided into PBS group, nPEG-IL-2 group, diPEG-IL-2 group, PEG-IL-2(n+di) group, PEG-IL-2(di+tri) and triPEG-IL-2 group, 5 mice in each group, subcutaneous injection of 0.3 mg / kg, the administration frequency is 3 days once. A total of 4 weeks of administration. After treatment, observe the change of joint swelling, record the arthritis score (no joint swelling is 0 points, one joint swelling is 1 point, two joint swelling is 2 points, more than two joint swelling is 3 points, all joint swelling is 4 points, the joint swelling of four limbs is added as the joint score). The results are shown in Table 5:

[0102] Table 5: Inflammation score of rheumatoid arthritis model mice in each group after treatment (x ± s)

[0103]

[0104]

[0105] Note: t test, compared with PBS group, *P<0.05, ***P<0.001.

[0106] The above animal studies show that PEG modified IL-2 molecules of different degrees and different compositions all show certain therapeutic effect compared with the control group (PBS group), among which PEG-IL-2(di+tri) and triPEG-IL-2 group have the most significant effect, which is consistent with the Treg induction ability.

[0107] Effect example 5: single dose pharmacokinetics of healthy mice subcutaneously injected

[0108] Wistar rats (10 weeks old) were randomly divided into nPEG-IL-2 group, diPEG-IL-2 group, PEG-IL-2(n+di) group and PEG-IL-2(di+tri) group, 5 rats in each group. Subcutaneous injection of 0.3 mg / kg of the corresponding drug (dosing volume 10 mg / mL), administration once.

[0109] Orbital blood was taken before administration, recorded as 0 point, and 100 μL of orbital blood was taken at 5 min, 15 min, 30 min, 1 h, 4 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h after administration, centrifuged, and the plasma was collected. IL-2 ELISA was used to detect the concentration of IL-2 in the blood, and the half-life of each molecule was calculated.

[0110] As shown in Table 6, the results show that with the increase of the degree of PEG modification, the molecular weight of PEG-IL-2 molecule gradually increases, the in vivo metabolic rate gradually slows down, and the metabolic rate has little to do with the modification method, which is consistent with the results reported in the literature.

[0111] Table 6: Pharmacokinetic results of PEG-modified IL-2 compositions of the present application in healthy mice after single subcutaneous administration

[0112]

[0113] Effect Example 6: Modification site identification

[0114] The freeze-dried IL-2 and PEG-modified molecules were dissolved with 0.1 M NH4HCO3 and adjusted to the same protein concentration. 1 mL of protein solution was added to an EP tube containing 20 μL of trypsin, mixed thoroughly, and incubated at 37°C for 20 hours. Centrifugation was performed at 13400 rpm for 50 minutes. The supernatant was freeze-dried. The dried sample was quantitatively added with 0.1% formic acid solution to the required sample concentration, and the supernatant was injected into MALDI-TOF and LC-MS / MS for analysis.

[0115] The freeze-dried IL-2 and PEG-modified molecules were dissolved with 0.1 M NH4HCO3 and adjusted to the same protein concentration. 1 mL of protein solution was added to an EP tube containing 20 μL of trypsin, mixed thoroughly, and incubated at 37°C for 20 hours. Centrifugation was performed at 13400 rpm for 50 minutes. The supernatant was freeze-dried. The dried sample was quantitatively added with 0.1% formic acid solution to the required sample concentration, and the supernatant was injected into MALDI-TOF and LC-MS / MS for analysis.

[0116] The modification degree of the site is equal to 100% minus the ratio of the response value of the peptide segment of the PEG-modified molecule containing the modification site to the response value of the IL-2 peptide segment, and the calculation method is shown in the following formula.

[0117]

[0118] As shown in Table 7, the results show that the N-terminus of substantially all modified molecules contains PEG modification, and the modification of IL-2 internal ε-NH2 mainly occurs on Lys residues such as K33, K44 and K77.

[0119] Table 7: Modification site identification results of PEG-modified IL-2 compositions of the present application

[0120]

[0121] Effect Example 7: Systemic lupus erythematosus animal efficacy study

[0122] Female MRL / lpr mice were randomly divided into PBS group, PEG-IL-2(di+tri) (low dose group, 0.1 mg / kg, 3 days once) group and PEG-IL-2(di+tri) (high dose group, 0.3 mg / kg, 3 days once) group, IL-2 group (0.3 mg / kg, once a day, 5 days a week) according to the body weight and urine protein level before administration. The administration was started at the 12th week and lasted to the 24th week. All mice were sacrificed at the 3rd day after the last administration, and the abdominal cavity blood was taken, the Treg cell level was detected by flow cytometry, and the urine protein was detected.

[0123] The experimental results are shown in Figure 5 and Figure 6 The results show that, similar to the results of healthy animals, both low dose and high dose PEG-IL-2(di+tri) can induce the increase of Treg level in peripheral blood and spleen of MRL / lpr mice, and can reduce the urine protein level of MRL / lpr mice, which indicates that PEG-IL-2(di+tri) has a therapeutic effect on systemic lupus erythematosus.

[0124] Example 8: Sjogren's syndrome animal efficacy study

[0125] The submandibular glands (both sides) of Balb / c mice were cut into pieces in PBS, then homogenized on an ultrasonic grinder, and then centrifuged in a centrifuge. The supernatant was used to immunize 6-8 week old female BALB / C mice, and the immunization was performed every 2 weeks, for a total of 3-4 times of subcutaneous injection in the back of the neck.

[0126] The Sjogren's syndrome model mice were randomly divided into PBS group, IL-2 group (0.3 mg / kg, once a day, 5 days a week), PEG-IL-2(di+tri) (low dose group, 0.1 mg / kg, 3 days once) group and PEG-IL-2(di+tri) (high dose group, 0.3 mg / kg, 3 days once) group. The administration was continuously performed for 4 weeks, and then the peripheral blood Treg level of the model animals and the serum alpha-fodrin antibody level were determined.

[0127] The experimental results are shown in Figure 7 and Figure 8 The results show that the administration of PEG-IL-2(di+tri) can significantly inhibit the production of serum alpha-fodrin antibody, which indicates that PEG-IL-2(di+tri) has a therapeutic effect on Sjogren's syndrome.

[0128] Example 9: Rheumatoid arthritis animal efficacy study

[0129] Eight-week-old male DBA / 1 mice were injected intravenously via the tail vein with a suspension of 400 μg / mL bovine type II collagen and an equal volume of complete Freund's adjuvant (100 μL). One week later, a booster immunization was administered using the same method, and changes in joint swelling of the limbs were observed. Mice developing arthritis were randomly divided into four groups: PBS group, IL-2 group (0.3 mg / kg, once daily, 5 days a week), PEG-IL-2 (di+tri) (low-dose group, 0.1 mg / kg, once every 3 days), and PEG-IL-2 (di+tri) (high-dose group, 0.3 mg / kg, once every 3 days), for a total of 4 weeks. Changes in joint swelling were observed daily, and arthritis scores were recorded (0 points for no joint swelling, 1 point for one swollen joint, 2 points for two swollen joints, 3 points for more than two swollen joints, 4 points for all swollen joints, and the scores for all limb joint swelling were added together to obtain the joint score).

[0130] Experimental results are as follows Figure 9 As shown, the results indicate that PEG-IL-2(di+tri) administration significantly reduced joint inflammation scores in model mice, suggesting that PEG-IL-2(di+tri) has a therapeutic effect on rheumatoid arthritis. Example 10: Animal Efficacy Study of Atopic Dermatitis

[0131] Eight-week-old female Balb / c mice were used. One day prior to the experiment, the back hair of the mice (approximately 1.5cm × 2.5cm) was shaved using an electric shaver, and the shaved area was repeatedly taped to disrupt the skin barrier. On days 1 and 2 of the first week of the experiment, 100μL of 0.5% DNFB solution (acetone / olive oil = 3:1) was pipetted and evenly applied to the shaved area on the backs of the treatment and negative control mice. Starting from the second week, 100μL of 0.2% DNFB solution (acetone / olive oil = 3:1) was evenly applied to the backs of the sensitized mice twice a week (approximately 3 days apart) for 3 consecutive weeks, for a total of 4 weeks.

[0132] Mice were randomly divided into four groups: PBS group, IL-2 group (0.3 mg / kg, once daily, 5 days a week), PEG-IL-2 (di+tri) (low-dose group, 0.1 mg / kg, once every 3 days), and PEG-IL-2 (di+tri) (high-dose group, 0.3 mg / kg, once every 3 days). Treatment began in the third week of the experiment and lasted for two weeks. Skin lesions were scored before and after treatment in each group as follows: (1) erythema / hemorrhagic rash; (2) epidermal peeling / erosion; (3) edema; (4) dryness / scaling. Each symptom was scored from 0 to 3: 0 (none), 1 (mild), 2 (moderate), 3 (severe). A score of 0.5 was given for symptoms falling between two possible scores, for a total score of 12.

[0133] The experimental results are shown in Table 8, and the results show that PEG-IL-2(di+tri) administration can significantly reduce the skin lesion score of the model mice, indicating that PEG-IL-2(di+tri) has a therapeutic effect on atopic dermatitis.

[0134] Table 8: Skin lesion scores of mice in each group before and after treatment (x ± s)

[0135] Group Before treatment lesion score After treatment lesion score PBS group 8.91±0.88 8.74±0.56 IL-2 group 8.64±0.71 6.62±0.85 PEG-IL-2 (di+tri) 0.1 mg / kg 9.03±1.24 6.55±1.10 PEG-IL-2 (di+tri) 0.3 mg / kg 8.87±0.69 4.23±0.49

[0136] Effect Example 11: Pharmacodynamic study of I type diabetes animal

[0137] 7-8 week old male Kunming mice were used, and streptozotocin (STZ) was injected intraperitoneally at 40 mg / kg for 5 consecutive days to induce the preparation of I type diabetes model. Blood glucose was measured rapidly after 3 days of injection, and the model was considered successful if the blood glucose was >11.1 mmol / L for 3 consecutive days.

[0138] The successful model mice were randomly divided into PBS group, IL-2 group (0.3 mg / kg, once a day, 5 days a week), PEG-IL-2(di+tri) (low dose group, 0.1 mg / kg, 3 days once) group and PEG-IL-2(di+tri) (high dose group, 0.3 mg / kg, 3 days once) group, a total of 4 weeks of administration. During the administration period, the blood glucose of the mice was detected at the same time every week, and the results are shown in Table 9, which shows that PEG-IL-2(di+tri) administration can significantly reduce the skin lesion score of the model mice, indicating that PEG-IL-2(di+tri) has a therapeutic effect on I type diabetes.

[0139] Table 9: Blood glucose detection values of mice in each group (mmol / L, x ± s)

[0140]

[0141] Effect Example 12: Induction of Treg ability of diPEG-IL-2v (IL-2 mutant)

[0142] It has been reported in the literature that mutation of Cys125 of natural IL-2 to Ser or Ala, and mutation of Y3 to C do not affect the activity of IL-2, so whether these IL-2v after PEG modification is similar to the effect of natural IL-2 modified sample was studied. diPEG-IL-2(125S), diPEG-IL-2(125A) and diPEG-IL-2(3C) were prepared, and the induction of Treg ability of diPEG-IL-2 and K2PEG-IL-2 was compared.

[0143] The experimental results are shown in Table 8, and the results show that PEG-IL-2(di+tri) administration can significantly reduce the skin lesion score of the model mice, indicating that PEG-IL-2(di+tri) has a therapeutic effect on atopic dermatitis. Figure 10As shown, the results indicate that diPEG-IL-2v produced by the same process as diPEG-IL-2 has similar effect as diPEG-IL-2, both of which are superior to K2PEG-IL-2.

[0144] Obviously, various modifications and changes are possible in the present application without departing from the spirit and scope of the application. It is to be understood that the above description is intended to be illustrative and not restrictive. The application is not to be limited to the details given herein, but can be modified within the scope and range of equivalents of the claims.

Claims

1. A composition of PEG-modified IL-2 as a Treg-targeting modulator, characterized in that: The composition is a tri-modified PEG-IL-2 molecule, or the composition contains 10 mol% or less of a mono-modified PEG-IL-2 molecule, 20 mol% to 60 mol% of a di-modified PEG-IL-2 molecule, 20 mol% to 60 mol% of a tri-modified PEG-IL-2 molecule, and 20 mol% or less of a tetra-modified PEG-IL-2 molecule, the PEG modification of the mono-modified PEG-IL-2 molecule occurs at the alpha-NH2 position at the N-terminal of the IL-2 amino acid chain, the PEG modification of the di-modified PEG-IL-2 molecule, the tri-modified PEG-IL-2 molecule and the tetra-modified PEG-IL-2 molecule occurs at the alpha-NH2 position at the N-terminal of the IL-2 amino acid chain and at the epsilon-NH2 position of lysine inside the IL-2 amino acid chain, the epsilon-NH2 modification inside includes the modification at K33, K44 and / or K77 lysine residues, and the IL-2 is a natural IL-2 or a recombinant IL-2 having IL-2 function.

2. The composition of claim 1, wherein: The PEG is linear, or the PEG is branched, and the average molecular weight of the PEG is 10 kDa to 40 kDa.

3. The composition of claim 1 or claim 2, wherein: The composition targets the activation of Treg, the half-life of the composition is prolonged compared with wild-type IL-2, and the half-life of the composition is prolonged for 1 to 120 hours or more.

4. The composition of claim 1, wherein: The composition contains 10 mol% or less of a mono-modified PEG-IL-2 molecule, 30 mol% to 55 mol% of a di-modified PEG-IL-2 molecule, 35 mol% to 50 mol% of a tri-modified PEG-IL-2 molecule, and / or 20 mol% or less of a tetra-modified PEG-IL-2 molecule.

5. Process for the preparation of a composition according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: The IL-2, the PEG modification agent, and sodium cyanoborohydride are reacted at 4-30°C and pH 4-7 to obtain the composition.

6. The method of claim 5, wherein: The preparation method further comprises the step of purifying the modification product by chromatography.

7. A pharmaceutical preparation as a Treg targeting modulator, characterized by: The pharmaceutical preparation comprises the composition of any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

8. The pharmaceutical preparation according to claim 7, characterized in that: The dosage form of the pharmaceutical preparation is an oral dosage, a liquid dosage, an emulsion, a coating, an ointment, a plaster, a transdermal absorption type dosage form, an aerosol, an injection or a suppository.

9. Use of a composition according to any one of claims 1 to 4 or a pharmaceutical preparation according to claim 7 or claim 8 for the manufacture of a medicament for the treatment of an autoimmune disease, characterised in that: The autoimmune disease is selected from systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, type I diabetes or atopic dermatitis.

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