CN110642934B

CN110642934B - Long-acting interleukin-2 of target regulatory T cell and application thereof in treating autoimmune disease

Info

Publication number: CN110642934B
Application number: CN201911120718.3A
Authority: CN
Inventors: 张博; 周德敏; 张烜; 王妍; 孙家琦
Original assignee: Peking University; Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Current assignee: Peking University; Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Priority date: 2019-09-10
Filing date: 2019-11-15
Publication date: 2022-08-23
Anticipated expiration: 2039-11-15
Also published as: WO2021093633A1; CN114853874A; CN110642934A

Abstract

The invention relates to modification sites capable of enabling human interleukin-2 to activate Tregs in a targeting mode, human interleukin-2 subjected to site-specific mutagenesis at the sites, and human interleukin-2 modified at the sites in a site-specific mode. The modified long-acting interleukin-2 can activate regulatory T cells in a targeted manner within a wide treatment window, and less or even no other effector cells are activated, so that the long-acting systemic immunosuppression effect is achieved. The invention further relates to the use of such site-directed mutated or modified interleukin-2, e.g. as a stable, long-acting immunosuppressant for the treatment of various autoimmune diseases.

Description

Long-acting interleukin-2 of target regulatory T cell and application thereof in treating autoimmune disease

Technical Field

The invention belongs to the field of biological pharmacy, and particularly relates to site-directed modified interleukin-2, such as a derivative of the interleukin-2 subjected to site-directed pegylation, so that the derivative has the effect of regulating T cells in a targeted manner. The invention further relates to the application of the site-specific modified interleukin-2 in treating autoimmune diseases, such as safe and effective rheumatoid arthritis, systemic lupus erythematosus, autoimmune diabetes and the like.

Background

Interleukin-2 (Interleukin-2) is the first type of cytokine, and as one of the first discovered and pharmaceutically acceptable cytokines, IL-2 has a vital biological role in immune regulation. Human IL-2, with the signal sequence cleaved, has a molecular weight of 15418.02 daltons and consists of 133 amino acid residues.

IL-2 exerts its biological effects by binding to the IL-2 receptor (IL-2R) on the cell membrane, IL-2R being composed of α, β, γ trimolecular subunits, where the α chain (CD25) has no signaling function and can only form high affinity binding with IL-2 as a trimolecular complex (Kd ═ 10 pM); whereas the beta chain (CD122) and the gamma (CD132) both belong to the type I cytokine receptor superfamily, both can form affinity binding with IL-2 (Kd ═ 1nM, which in turn conducts downstream signals (R. spolski et al, 2018) under the precondition of alpha chain deletion, three receptor subunits are expressed in different cell subsets in different amounts, for example, IL-2 ra can be expressed on the surface of regulatory T cells (tregs) continuously and for a long time, natural killer cells (NK) and CD8+ T killer cells express IL-2 ra at a low level under normal conditions and conventionally express IL-2R β and γ, therefore, the sensitivity of Treg cells to IL-2 is greater than NK without external antigen stimulation on the body, CD8+ T equivalent cells (Boyman et al, 2006), IL-2 serves as a "double-cutting edge" of immune regulation, and the steady-state level concentration of IL-2 CD 3526 + CD25 and Treg can be ensured to maintain survival of CD4+ 25+ Treg cells and Treg in vivo, but not sufficient to activate other effector cells; and because the number of effector cells is greatly higher than that of Tregs, excessive IL-2 can activate a certain amount of effector cells, so that the effect of enhancing immunity is caused. Thus, the complex biological effects of IL-2 determine the complexity and challenges of using it as a drug for the treatment of immune-based diseases.

Treg cells are used as one of key effector cells of immunosuppression, and have important significance for the occurrence and development of autoimmune diseases. The existing research shows that IL-2 can transmit a downstream signal path by combining with three-molecule IL-2Rs on Tregs to convert immature CD4+ CD25 ^low Foxp3 ^low Cells are converted into functional Tregs, and the immunosuppressive effect of the Tregs is exerted by up-regulating CD25 and Foxp3 functional molecules; on the other hand, IL-2 can also maintain the functional homeostasis of tregs in the periphery by inducing differentiation and growth of Treg cells. IL-2 has great potential in the treatment of immune diseases as a key molecule for the activation and regulation of Treg cells. As described above, due to the sustained high expression of IL-2 α on the surface of Treg cells, Treg cells are more sensitive to IL-2 in the absence of stimulation by foreign antigens in the body. Low doses of IL-2 have gained increasing acceptance and use in the treatment of autoimmune diseases through precise design of dosing and treatment periods. Existing studies have shown that after 5 consecutive days of IL-2 injection in patients with malignant SLE, the Treg cell levels and function in the patients can be significantly increased and symptoms significantly improved (c.von Spee-Mayer et al, 2016). Subsequent further studies show that in the treatment of SLE by low-dose IL-2, besides the improvement of the quantity and functions of Tregs, Tfh and Th17 differentiation can be inhibited to regulate immune inflammatory response so as to restore the immune homeostasis of the organism, and a theoretical basis (J.He et al.,2016) is laid for the treatment of lupus by low-dose IL-2Feasibility of immune diseases.

Based on the wide and high-efficiency biological effect of IL-2, the IL-2 selectively activates target cells, improves the therapeutic window of the IL-2 on specific diseases, and has important significance for improving the safety and effectiveness of the IL-2 in treating the diseases. Due to the high flexibility of binding of IL-2 to a receptor complex, the change of IL-2 conformation or the shielding of IL-2 at different positions can obviously influence the activation effect of IL-2 on different cell subsets, thereby achieving the effect of targeted activation. The formation of IL-2 antibody complexes by coupling IL-2 to specific antibodies is one of the effective means of achieving this strategy. Results from Boyman et al show that by combining murine IL-2 with specific antibody JES6-1 to form a complex, a more pronounced activation of Treg and a lower level of activation of other effector cells can be achieved (Boyman et al, 2006); binding of IL-2 to the other antibody, S4B6, may favor the activation of effector cells over all relevant immune cells. Further studies show that after JES6-1 is combined with IL-2, the combination of IL-2 with IL-2R beta and gamma is blocked spatially, and the combination of IL-2 with IL-2 alpha is reduced, so that IL-2 activates Treg more preferentially; the binding of antibody S4B6 to IL-2 not only sterically hinders the binding of IL-2 to IL-2 ra, but also allows the bound IL-2 conformation to form a more stable complex with IL-2R β, thus allowing the IL-2/S4B6 antibody complex to have the effect of targeted activation of effector cells (j.b. spangler et al, 2015). Based on the above research work, Eleonora et al screened and optimized a series of IL-2 antibodies directed against human origin and finally determined that F5111.2 can form an antibody complex with IL-2 for the biased activation of tregs to treat various autoimmune diseases such as graft versus host disease and autoimmune diabetes (e.trotta et al, 2018). On the other hand, IL-2 and an antibody or an antibody Fc segment are subjected to fusion expression, fine multipoint mutation is carried out on multiple sites on the IL-2, and structural-based redesign synthesis and the like of the IL-2 (J.B.Spangler et al, 2018; L.B.Peterson et al, 2018; A.B.Shanafelt et al, 2000; D.A.Silva et al, 2019) can obviously increase the targeting activation effect of the IL-2 on specific immune cells, so that the more effective activation or inhibition of the immunity is achieved, and the antibody or the antibody Fc segment is further used for treating specific diseases.

Although the above studies have achieved certain clinical effects, there are still significant practical problems. The treatment scheme of the low-dose IL-2 has great difference aiming at different diseases and different individuals, and is difficult to predict and verify the dosage in a human body; on the other hand, for chronic diseases such as autoimmune diseases, IL-2 needs to be administered by multiple injections for a long time, and the compliance of patients is also seriously reduced; from the aspect of antibody application, the chemical dose ratio and the compound stability of the IL-2/antibody compound are unfavorable for industrial preparation and clinical practical use, and the immunogenicity brought by the application of the antibody or other IL-2 mutants also limits the wide clinical application of the method of the type. Therefore, the development of a novel biotechnology drug based on IL-2 to enable the drug to activate specific immune cell subsets in a targeting manner is a key scientific problem of continuous research in the field and has important clinical transformation significance.

Disclosure of Invention

In order to solve the above problems, the present invention provides long-acting interleukin-2 targeting regulatory T cells and its use in treating autoimmune diseases.

First, the present invention provides a modification site and a combination of modification sites that allow targeted activation of tregs by human interleukin-2, the selected modification site being selected from the group consisting of: shown in SEQ ID NO: 1, position H16, position D20, position Y31, position N33, position R38, position T51, position a73, position K76, position H79, position N88, position D109 or other combination with one or more sites having modification sites targeted to activate Treg activity that, in terms of modification effect, will affect more the binding of IL-2 to IL-2 receptor β and less the binding to IL-2 receptor α.

The invention also provides a site-directed mutant human interleukin-2, wherein one or more amino acids at the specific site are mutated into unnatural amino acids

At least one of the shown unnatural amino acids or other unnatural amino acids containing an azide structure; the specific site is selected from: display deviceIn SEQ ID NO: 1 at position H16, D20, Y31, N33, R38, T51, A73, K76, H79, N88 and D109, or a combination thereof.

Preferably, the site-directed mutagenesis of human interleukin-2 is compared to the sequence shown in SEQ ID NO: 1 are distinguished by: in SEQ ID NO: 1 is mutated into an unnatural amino acid, wherein the mutated amino acid is similar to the amino acid sequence shown in SEQ ID NO: 1 is represented by the following formula (II):

the direction from R1 to R2 is the direction from the N-terminus to the C-terminus of the amino acid sequence, wherein the amino acid at the N-position is selected from one or more of the amino acids at positions H16, D20, Y31, N33, R38, T51, A73, K76, H79, N88 and D109,

r1 is SEQ ID NO: 1 from the 1 st to the N-1 st amino acid residues of the sequence shown in 1,

r2 is SEQ ID NO: 1 from the N +1 th to the C-terminal amino acid residue,

r4 is

The invention also provides the modified human interleukin-2 with site-directed mutation, wherein the connection mode containing unnatural amino acids is shown as the following formula (III) or (IV): wherein R3 can be PEG, fatty acid chain, cyclodextrin, sugar, nucleic acid, amino acid, polypeptide or carboxyl terminal modifying group with same or different molecular weight,

(III) a molecular formula of point coupling of non-natural amino acid containing azide group inserted into human interleukin-2 with a modifier requiring a catalyst;

(IV) formula of site-directed coupling of non-natural amino acids containing azide groups after insertion into human interleukin-2 with modifying agents containing cyclooctyne functional groups.

When more than one amino acid site is substituted, the modified compounds coupled with different substitution sites can be the same or different.

The invention also provides the human interleukin-2 modified by PEG, fatty acid chain and sugar and mutated at the specific site, and the molecular weight range of the modifier introduced at the modification position is 1k Da-100 kDa.

Wherein the PEG preferably has a molecular weight of 5kDa, 10kDa, 20kDa, 30kDa, or 40 kDa.

Specifically, the human interleukin-2 modified by PEG and mutated at a specific site is preferably H16-5K, H16-10K, H16-20K, D20-5K, D20-10K, D20-20K, Y31-5K, Y31-10K, Y31-20K, R38-5K, R38-10K, R38-20K, T51-5K, T51-10K, T51-20K, A51-5K, A51-10K, A51-20K, K51-5K, K51-10K, K51-20K, H51-5K, H51-10K, H51-20K, Y51/T51-20K or other modified products of PEG with 20kDa size combined at any two positions, more preferably H51-51K, D20-20K, Y31-20K, T51-20K, A73-20K, K76-20K, H79-10K, H79-20K, Y31/T51-10K or Y31/T51-20K.

The invention also provides a pharmaceutical composition, which contains an effective amount of the human interleukin-2 or the modified interleukin-2 and a pharmaceutically acceptable carrier.

The invention also provides the application of the interleukin-2 or the modified interleukin-2 in preparing medicines for immune regulation and various autoimmune diseases.

The invention also provides the application of the interleukin-2 or the modified interleukin-2 in preparing medicaments for treating graft-versus-host diseases, rheumatoid arthritis, systemic lupus erythematosus, autoimmune diabetes, dermatomyositis, scleroderma, multiple sclerosis, myasthenia gravis, demyelinating diseases, primary adrenal cortical atrophy, chronic thyroiditis, chronic nonspecific ulcerative colitis, chronic active hepatitis, pernicious anemia and atrophic gastritis, autoimmune glomerular nephritis, pneumonephrohemorrhagic syndrome, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia and autoimmune alopecia.

Through thinking and research on the prior art, the inventor carries out modification of a specific site, such as polyethylene glycol (PEG), on the specific site of the IL-2, so that the IL-2 has the effects of activating and regulating T cells in a targeting mode, prolonging the half-life period of the IL-2, and playing a safer and more effective role in treating autoimmune diseases.

Compared with the existing research medicaments, the IL-2 derivative obtained based on the invention has the technical effects and advantages mainly embodied in one or more of the following:

the IL-2 derivative obtained by the invention has stronger treatment effectiveness and safety: compared with antibody complex-based shielding methods, the shielding site and effect are often determined by a large number of random screens; and site screening is carried out on IL-2, site-directed modification is carried out, for example, PEG modification is carried out, modification sites are directly obtained from the structural level of IL-2, modification design is carried out aiming at the binding region of IL-2 and receptor IL-2R alpha/beta purposefully, then IL-2 modification products with stronger targeting effect are obtained, and the treatment window of IL-2 in autoimmune diseases is greatly improved.

The IL-2 derivative obtained by the invention has long-acting property and low immunogenicity: the product obtained by the invention carries out site-specific modification on a specific site of the IL-2, improves the IL-2 targeting property, obviously improves the IL-2 half-life period, reduces the immunogenicity, further reduces the dosage and the injection times of the IL-2 for long-term administration, further improves the effectiveness and the safety of IL-2 treatment, and ensures that patients have better compliance. For the sites mentioned above, the greater the molecular weight of the modified PEG, the greater the targeting of the Treg, with consequent lower retention of biological activity, but greater in vivo stability, the overall therapeutic effect depending on the balance between the two. Specifically, (1) the Y31 and T51 sites have better tolerance to PEG modification and are suitable for modification with large molecular weight, such as more than 20 kDa-PEG; (2) h16, D20, A73, K76 and H79 modification can cause obvious activity influence, and the modification effect of 10kDa or 20kDa is optimal; (3) compared with Y31 and T51 single sites, the simultaneous modification of Y31 and T51 double sites can greatly improve the targeting of the modified product to Tregs, and the simultaneous modification of 10kDa and 20kDa PEG molecules at two sites can obviously improve the half-life period in vivo while improving the targeting of the Tregs, and retain acceptable biological activity, thereby achieving the best treatment effect.

The IL-2 derivatives obtained by the invention are IL-2 analogues modified at fixed points, and have the characteristics of uniform chemical properties and biological activity.

Drawings

FIG. 1 shows the modification site design and verification of unnatural amino acid insertion efficiency for IL-2. Mutating the corresponding position of the selected site into a TAG stop codon, preparing a mutant expression vector, co-transferring the mutant expression vector and an auxiliary plasmid pSUVR-YAV into Escherichia coli origamB (DE3), adding unnatural amino acid into a culture medium, and performing induced expression to obtain the IL-2 inserted with the unnatural amino acid at the site for modification. The upper diagram shows the relationship between selected different sites and IL-2R alpha, beta, gamma; the following figure shows the verification of unnatural amino acid insertion, and it can be seen that different sites have different insertion efficiencies.

FIG. 2 shows the preparation of IL-2 with different PEG molecular sizes modified at different sites. Mixing mutant IL-2 containing azide group unnatural amino acid with DIBO-PEG (5, 10 and 20kDa) with different molecular weights, and performing cation exchange purification (RESOURCE 15S) and molecular exclusion purification (Superdex200 Increate) on the modified product to obtain the site-directed PEGylated IL-2 modified derivative with the purity of more than 95%.

FIG. 3 shows the detection of pSTAT5 phosphorylation activity of PEGylated IL-2 modified at different sites and with different molecular weights on Treg and CD8+ T in human PBMC. A.20kDa-PEG modifies different selected sites, and influences the phosphorylation of Treg and CD8+ T; it can be seen that wild type IL-2(WT) is prone to tregs only at low concentrations (1.6ng/ml), and modifications at

positions

31,51 and 73 can increase the prone concentration to 40ng/ml with higher retained activity; the sites 76 and 79 can increase the tendency concentration to 200ng/ml, but the influence on the activity is more obvious than that of the rest sites; B. the PEG modification with different molecular weights at different sites has influence on the targeting of the Tregs; yt cell line model validation, wild-type WT cells express only IL-2R β and γ for simulation of effector cells; IL-2R alpha (YT-CD25low and YT-CD25high) with different degrees of expression is used for simulating Treg through gene modification; d.20 site modification of PEG with different molecular weights has influence on Treg targeting and IL-2 activity. It can be seen that the 20-site modification can simultaneously and obviously reduce the response of Treg and CD8+ T, but more affect CD8+ T, and the modified product has Treg targeting property in an extremely high concentration range (0.2-5 ug/ml).

FIG. 4 shows a multi-site combinatorial modification of IL-2. A. SDS-PAGE and Coomassie blue staining analysis of the 10kDa-, 20kDa-PEG at position 31/51 and the 5kDa-PEG combinatorial modification at position 20/31/51 of IL-2; B. the combination modifications were monitored for pSTAT5 phosphorylation in human peripheral blood. It can be seen that the combined modification at position 31/51, the PEG modified products of 10kDa and 20kDa increased the Treg to significantly biased concentrations of 1 and 5ug/ml, respectively, and the modified products had some retained activity.

FIG. 5 shows the detection of Treg and CD8+ T activation markers by the modification different modified IL-2 derivatives were treated with PBMC for 72 hours, and CD25 and Foxp3 of Treg and CD25 and CD49d of CD8+ T cells were detected by multi-color flow. Targeting results were consistent with phosphorylation.

FIG. 6 shows the activation assay of isolated pure subsets of immune cells by modified PEGylated IL-2 derivatives. The detection result is consistent with that of CD8+ T, 20-20K is insensitive to various immune cells and has higher correspondence to Treg; the response of 31-20K to Treg is higher than that of other various immune cells, and is consistent with the response degree of WT to Treg; 31/51-20K combined modified product can not activate other immune cells obviously, and simultaneously maintain higher level of activation on Tregs.

FIG. 7 shows the in vitro activity assay of pSTAT5 on murine splenic lymphocytes with human and murine IL-2 modification products. A. Detecting the activation of Treg and CD8+ T cells in mouse spleen lymphocytes by human and mouse wild type IL-2; B. detecting the activation of Treg and CD8+ T cells in mouse spleen lymphocytes by products of different site modified 20 kDa-PEG; C. analyzing the cell number and cell proportion of Treg, CD8+ T and Tconv in splenic lymphocytes of mice of different treatment groups; D. activation marker analysis on Treg and CD8+ T in splenic lymphocytes of mice of different treatment groups.

Figure 8 shows the Treg activation targeting assay of 20-20K in mice a. the Treg activation ratio assay of different dose 20-20K treatment groups; B. analyzing the cell number and cell proportion in Treg, CD8+ T and Tconv of different 20-20K administration dose treatment groups; C. activation marker analysis on Treg and CD8+ T in splenic lymphocytes of mice in different 20-20K dose-treated groups.

FIG. 9H 16-20K is tested for the targeting activation of Treg activity in mice. Flow data of percentage of tregs activated in CD4+ T cells and percentage of CD3+ CD8+ T cells activated in vivo for h16-20K, PBS, and WT-IL-2; B.16-20K, PBS, and WT-IL-2 in vivo activation of Treg percentage, CD8+ T cell percentage, and Treg/CD8+ T cell analysis.

Figure 10 shows the use of IL-2 modifications in a model of graft versus host disease. Heavily immunodeficient mice were given stimulated human PBMCs the first day. WT-IL-2 was administered once a day, 5 times; PEGylated IL-2 was administered once every other day for 3 times; B. detecting human Treg, CD8+ T and Tconv in the spleen in a flow type gate-looping mode; 2 μ g Treg ratio, CD8+ T number, and Treg and CD8+ T surface activator analysis in spleen of mice in the group administered; D. different doses of each sample treatment group activated Treg, CD8+ T, Tconv cell number and cell proportion analysis in the graft; E. therapeutic efficacy of graft versus host disease. The effect of WT-IL-2 and 31/51-20K (D-20K) on survival of model mice with graft-versus-host-immune disease was observed at different doses.

FIG. 11 is a graph showing the efficacy of IL-2 modifications in a CIA model A. different groups of mice are dosed with a design pattern; B. ankle joint section analysis at observation endpoint for different sample treatment groups; C. analyzing the number and proportion of Treg, CD8+ T and TH17 cells in different sample treatment groups; D. arthritis scores for different sample treatment groups; the prevention and treatment effect of the PEG-IL-2 product on the CIA. WT-IL-2 and 31/51-20K (D-20K) were administered one week before the second immunization of collagen, and it was shown that D-20K was more effective in preventing the occurrence and development of CIA than WT-IL-2.

FIG. 12 shows the evaluation of IL-2 modifications in lupus model mice A.WT-IL-2 and 31/51-20K (D-20K) at different doses in 9 week old MRL/lpr spontaneous lupus mice with antinuclear antibodies, anti-double-stranded DNA antibodies, and proteinuria profile; B. and (4) evaluating immunogenicity. ELISA method detects IL-2 specific antibody production in mice of different samples and dose treatment groups.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

EXAMPLE 1 selection of modifiable sites and preparation of hIL-2 containing an azido group unnatural amino acid at the site

Based on the crystal structure of IL-2, the binding site of IL-2 to its receptor, the inventors of the present invention selected several suitable sites for modification, which are based primarily on the following factors 1. exposure of amino acids to the protein surface for ease of coupling; 2. shielding an immunogen region; 3. shielding the proteolytic region. Consult by more detailed literature [ Eleonora Trotta et al, Nat med.2018jul; 24(7) 1005-1014; spangler jb.et al, immunity.2015may 19; 42(5):815-25]The inventors selected binding sites that affect IL-2 to various degrees with the IL-2R α, β, γ subunits, including H16, D20, Y31, T51, A73, K76, H79 (FIG. 1). For each site, the inventors designed primers capable of mutating the codon encoding the amino acid to amber codon, respectively, and then used a site-directed mutagenesis kit (

Lightning Site-Directed Mutagenesis Kits, Catalog #210518), according to the instructions, and using a wild-type IL-2 expression vector pET21a-IL-2(WT) as a template, and mutating the corresponding position of IL-2 to an amber stop codon, to obtain an expression plasmid of mutant IL-2. The above expression with ampicillin resistanceThe plasmid and the auxiliary plasmid with chloramphenicol resistance are simultaneously transformed into Escherichia coli OrigamiB (DE3), and a cotransformed positive strain is screened out by a chloramphenicol and ampicillin double resistant plate, thus obtaining an expression strain simultaneously transformed with two plasmids. Culturing the expression strain obtained in the step in a 2Y T culture medium containing 34 mu g/ml chloramphenicol and 100 mu g/ml ampicillin at 37 ℃ for 12-16 hours, performing secondary amplification until the OD value of the bacterial liquid reaches 0.6-1.0, adding unnatural amino acid to the final concentration of 1mM, continuing amplification at 37 ℃ for 30 minutes, adding IPTG to the final concentration of 0.5mM and arabinose to the final concentration of 0.1%, inducing expression at 24 ℃ for 12 hours, and collecting the bacterial cells. And (2) carrying out balanced resuspension on the collected thalli by using Ni-NTA-Bind-Buffer, carrying out 1200bar circulation crushing by using an ultrahigh pressure homogenization crusher, removing cell fragments by high-speed centrifugation after 2-cycle crushing, carrying out Ni-NTA metal chelating affinity chromatography, fully washing by using Ni-NTA-Wash-Buffer, and finally eluting by using Ni-NTA-Elute-Buffer to obtain a primarily purified interferon sample with the purity of about 90%. The product was examined by SDS-PAGE and Coomassie blue to confirm that the unnatural amino acid was inserted into the specific site.

EXAMPLE 2 preparation of IL-2 with site-directed modification of PEG

And (2) coupling the IL-2 mutant containing the azide group unnatural amino acid with a PEG modifier containing cyclooctyne, and performing coupling reaction of ring tension by using the azide group on the unnatural amino acid and the cyclooctyne to obtain the IL-2 derivative of PEG modified at a specific site. The reacted complex was desalted and purified by ion exchange (Source 15S, 20mM sodium acetate PH 4.5, 0-250mM NaCl gradient) and size exclusion chromatography to obtain > 95% pure IL-2 derivatives of site-specific modified PEG as shown in figure 2.

Example 3 enhancement of modification sites of IL-2-targeted activation tregs, and acquisition of optimal modifiers corresponding thereto

The affinity of the modified IL-2 derivative to different IL-2R receptor subunits determines the targeting property of the modified IL-2 derivative to specific immune cells, and the optimal modification site for targeting Treg and the corresponding PEG modifier are determined by respectively detecting the affinity change of different sites, IL-2 derivatives modified by different PEG sizes and IL-2R alpha and IL-2R beta.

(1) Biofilm interference experiment (BLI):

IL-2 exerts its biological effects by binding to the cell surface IL-2R receptor complex, IL-2R being composed of α, β, γ trimolecular subunits, where the α chain (CD25) has no signaling function and can only form high affinity binding with β and γ chains as a trimolecular complex with IL-2 (Kd ═ 10 pM); the beta chain (CD122) and the gamma chain (CD132) both belong to the I-type cytokine receptor superfamily, and both can form a medium affinity binding (Kd ═ 1nM) with IL-2 in the absence of the alpha chain, thereby conducting downstream signals. The principle of the biomembrane interference technology is that a beam of visible light passes through an optical fiber, two beams of reflection spectra are formed on two interfaces of an optical film layer at the tail end of a sensor, and the two beams of reflection spectra are superposed to form a beam of interference spectra; and the binding of molecules can cause the thickness of the film layer to change, and the change is reflected by the displacement value of the interference spectrum, so that information such as the concentration, the affinity, the kinetic constant, the specificity and the like of the analyte can be obtained. The specific process is as follows: fc-fused IL-2R α and IL-2R β were coupled to an SA sensor and flowed through a PEG-IL-2s sample with a dissociation time of 120 s. By flowing through samples of different concentrations, the influence of different sites modifying different molecular weight PEG on the affinity of IL-2 to the receptor was quantified by software fitting KD values, as shown in Table 1. The results indicate that site-directed PEGylation of IL-2 at the selected sites described above affected the binding of IL-2 to both IL-2R β and IL-2R α, with greater effect on IL-2R β than on IL-2R α. The degree of influence of the detected site as IL-2R alpha affinity is as follows: d20, T51, A73-Y31, H79 and K76; the degree of influence on the affinity of IL-2R beta is Y31> T51> A73> H79-K76 > 20. For

sites

31, 73 and 76, the effect of modification on IL-2R alpha is obviously increased along with the increase of the molecular weight of PEG; for position 73, the effect of the modification on IL-2R β increased significantly with increasing PEG molecular weight.

TABLE 1 Effect of different site modification of different molecular weight PEG on affinity of IL-2 to receptor

(2) Experiment of phosphorylation of pSTAT5 of human peripheral blood PBMCs:

human Peripheral Blood Mononuclear Cells (PBMCs) were isolated, stimulated in vitro with the sample to be tested for 50 minutes, fixed to rupture the membrane, and surface antibody stained with fluorescent antibodies: anti-CD3-APC/Cy7, anti-CD4-PE/Cy7, anti-CD25-APC, anti-CD8-FITC, anti-CD127-PE, anti-pSTAT5-Pacific blue. Detecting the phosphorylation levels of Treg and CD8+ T cells, and evaluating the activation levels of Treg/CD8+ T cells of different samples, wherein the specific cell types are as follows: treg (CD3+ CD8+ CD25hi-CD127low), CD8+ T (CD3+ CD8 +). We first evaluated the effect of modifying the 20kDa-PEG molecule at the same site. The results show (fig. 3A) that the modification of 20kDa-PEG at each site can significantly improve the targeting of tregs: unmodified wild-type IL-2 has Treg-targeted activation only at the lowest concentration of 1.6 ng/ml; the 20K PEG modified at Y31, T51 and A73 increased targeting at 1.6ng/ml and increased targeting to 40 ng/ml; compared with the rest sites, the PEG modified by 20K at K76 and H79 can greatly improve the targeting of Tregs and increase the targeting concentration to 200 ng/ml. On the other hand, the Y31 and T51 sites have better tolerance to PEG modification and retain higher biological activity. The influence of different sites on the targeting property by modifying PEG with different molecular weights is detected, and the result shows that the effect of the targeting property on the Treg is obviously improved along with the improvement of the molecular weight of the PEG, and Y31 and T51 have better tolerance on the modification of the PEG, are suitable for the modification of PEG with high molecular weight and have the highest targeting property on the Treg; the activities of a73, K76 and H79 were sensitive to PEG modification, and were suitable for PEG modifications of 10K or 20K molecular weight (fig. 3B). We also performed validation of the YT cell model, with wild-type YT cells expressing IL-2R β and γ for mimicking effector cells, and engineered YT cells transformed with IL-2R α for mimicking tregs. The results indicate that the increase in PEG molecular weight does contribute to the improvement in targeting of the modified product Treg and is caused by the difference in the amount of IL-2 ra expression on the cells (fig. 3C). Furthermore, the modification at the 20-position has a greater effect on IL-2 activity, which significantly affects its Treg activity, but is more severe and even blocks IL-2 activity on effector cells (fig. 3D).

Example 4 combinatorial modifications and Effect of improving the ability of IL-2 to Targeted activate Tregs

Based on the screening site and effect of the embodiment 3, the Treg targeting effect of the IL-2 can be more finely adjusted and improved by carrying out multi-point combined modification on the specific site on the IL-2. The inventor carries out multi-site combined modification on 31/51 sites and 20/31/51 sites of IL-2 to obtain a combined modified product of 31/51 sites respectively coupled with 10kDa and 20kDa-PEG modified molecules and a combined modified product of 20/31/51 sites respectively coupled with 5kDa-PEG modified molecules, and carries out pSTAT5 phosphorylation detection and receptor affinity detection on the combined products. The results show that the combination product of PEG modified at position 31/51 has a trend of significantly decreasing affinity for IL-2R alpha and beta as the molecular weight of PEG increases. The combined modification at position 31/51 significantly reduced the binding of the modifier to IL-2R β and affected its binding to IL-2R α to some extent, compared to WT-IL-2; while the coupling of 5kDa-PEG modification at position 20/31/51, respectively, less affected its binding to IL-2R α, but masked its binding to IL-2R β. The result of the detection of pSTAT5 phosphorylation activity on the modified product shows that the effective concentration of the targeted Treg is remarkably increased by carrying out combined modification on IL-2: performing 10 kDa-20 kDa PEG modification on 31/51 locus of IL-2, wherein the effective concentrations of the modification product for activating Treg reach 1 μ g/ml and 5 μ g/ml respectively; the 5kDa-PEG modification of the 20/31/51 site of IL-2 resulted in targeted activation of Tregs at a concentration of 1. mu.g/ml without significant activation of CD8+ T cells, as shown in FIG. 4 and Table 2.

TABLE 2 affinity assay for IL-2 derivatives modified by multi-site combinations with IL-2R alpha and beta

Example 5 detection of Treg and CD8+ T activation marker by modification

The detection of the Treg and CD8+ T cell activation markers can confirm the targeting activation effect of the modifier more clearly, and complement phosphorylation detection. Different postcomplement products and PBMC are stimulated together for 48 hours, and then cells after stimulation are fixed, membranes are broken, and multicolor antibody flow detection is carried out. The specific labeled antibodies were as follows: anti-CD3-APC/Cy7, anti-CD4-PE/Cy7, anti-CD8-FITC, anti-CD25-APC, anti-Foxp3-Pacific blue, anti-CD26-PE, anti-CD49d-BV605, CD25 and Foxp3 of Treg cells after activation, and CD25 and CD49d of CD8+ T cells, respectively. The experimental results are consistent with the results of pSTAT5 phosphorylation: the IL-2 modified by PEG can obviously improve the Treg activation marker in a targeting way. More specifically, IL-2 derivatives of PEG modified at 20K at positions 31,51 and 79, PEG derivatives modified at

positions

10K and 20K at positions 20, and derivatives modified at positions 31/51 and 20/31/51 in combination can achieve more effective targeted activation of Treg within the detection concentration range (0.04-1 μ g/ml), and the results are shown in FIG. 5.

Example 6 detection of activation of isolated pure subpopulations of immune cells by modified PEGylated IL-2 derivatives

In order to more clearly verify the targeted activation effect of the modified PEG IL-2 derivative on Treg, the inventor separates human primary Treg, NK, memory CD4+ T (MP-CD4), memory CD8+ T (MP-CD8), juvenile CD4+ T (B/C) by a magnetic bead sorting method

CD4), naive CD8+ T cells (

CD8) and the activation of pSTAT5 by different posttonics on these subpopulations of immune cells was examined separately. The results show that the modification of the selected site or the combination of sites can obviously improve the activation of Tregs in the detection concentration range compared with WT-IL-2, and lower level, even no activation, including NK, MP-CD4, MP-CD8,

CD4，

other effector cells including CD 8. More specifically, IL-2 derivatives of 20K-PEG modified at

position

20 and 5K-PEG modified simultaneously at position 20/31/51The IL-2 derivative can obviously block other effector cells, and can activate Tregs with low activity and targeting; the IL-2 derivative of the 20K-PEG is modified at the 31 site, so that Tregs can be activated at a higher level in a biased manner, and a moderate blocking effect on other effector cells is achieved; the IL-2 derivative of the 20K-PEG is modified at the 31 and 51 double sites simultaneously, and other effector cells are blocked to a higher degree while Tregs are activated with high-level bias. The results are shown in FIG. 6.

Example 7 pharmacokinetic assay of modified PEGylated IL-2 derivatives

Modification of PEG can improve the molecular volume of protein, reduce glomerular filtration, and shield antigenic sites, thereby improving the stability of the modified IL-2 in vivo and prolonging the half-life period. On the other hand, the IL-2 derivative with prolonged half-life period can continuously activate Tregs in a safe concentration range due to longer in vivo exposure time, and has a safer and more effective effect. The inventor evaluates the influence of PEG modification at different sites and in different molecular ranges on the in vivo half-life of IL-2. The results show that the stability in vivo is obviously improved along with the increase of the molecular weight of PEG modification at different sites. More specifically, compared with WT-IL-2, the half-life can be improved by 1.6 times, 7.8 times and 17 times by modifying 5-, 10-and 20kDa-PEG at the 20-site; the half-life period can be respectively improved by 3.2 times, 6.7 times and 8.3 times by modifying 5-, 10-and 20kDa-PEG at the 31 site; the half-life period can be improved by 7.8, 7.2 and 16 times by modifying 20kDa-PEG at the sites of 51, 73 and 79 respectively; on the other hand, the 20kDa-PEG is modified at the 31 and 51 sites simultaneously, so that the half-life period of the modifier can be greatly improved and is improved by about 41 times compared with that of WT; the simultaneous modification of 5kDa-PEG at

positions

20, 31 and 51 can reach the level of single-point modification of 20kDa-PEG, which is increased by about 8 times, and the results are shown in tables 3-5.

TABLE 3 pharmacokinetic analysis of the 20 th site modified 5-, 10-, 20kDa-PEG of IL-2

TABLE 4 pharmacokinetic analysis of the 31 st site modified 5-, 10-, 20kDa-PEG of IL-2

TABLE 5 pharmacokinetic analysis of different site-modified 20kDa-PEG and site combination modification

Example 8 Treg Targeted detection of IL-2 modifications in mice

DBA/1 mice were selected and injected with 1. mu.g and 5. mu.g of WT-hIL-2, h31-20K, 31/51-20K, m103-20K modified IL-2 derivatives, respectively. Wherein WT-hIL-2 is injected once a day for 5 times; PEG modified IL-2 in every other day injection of 3 times. On the third day after injection, splenic lymphocytes of mice were isolated for multicolor flow detection of cell activation markers, and labeled antibodies were as follows: anti-mouse-CD3-APC/Cy7, anti-mouse-CD4-PE/Cy7, anti-mouse-CD8-FITC, anti-mouse-CD25-APC, anti-mouse-Foxp3-Pacific blue, anti-mouse-CD103-PerCP/Cy5.5, and anti-mouse-CTLA4-BV 605. The results show that the modification product of the 31-site modified 20kDa-PEG and the modification product of the 31/51 two-site modified 20kDa-PEG can obviously activate Tregs, activate CD8+ T and Tconv to a lesser extent and improve Treg/CD8+ T and Treg/Tconv under the injection dosage of low dose (1 mu g) and high dose (5 mu g); 31-20K under the condition of low-dose injection, the Treg/CD8+ T and Treg/Tconv are respectively improved by 2.4 times and 2 times compared with WT; 1.8 times and 2.2 times respectively under high-dose injection; 31/51-20K under low-dose injection condition, the Treg/CD8+ T and Treg/Tconv are respectively improved by 6.8 times and 5.4 times compared with WT; increases were 5.8-fold and 4.6-fold, respectively, under high dose injection. The sample of the mouse source 103 site modified 20K has no obvious targeting on activating Treg cells compared with WT under low dose, but Treg/CD8+ T and Treg/Tconv are respectively improved by 5.3 times and 5 times compared with WT under high concentration; the results of the detection of the cell surface activation marker were consistent with the results of the cell count detection, and the PEG-modified IL-2 derivatives were able to effectively activate CD25, CD103 and CTLA4 on the surface of Treg compared to WT-IL-2 at the effective target activation concentration, but activated CD25 of CD8+ T cells at the same level as WT-IL-2, and the results are shown in fig. 7.

Example 9 Treg targeting assay of IL-2 modified at sites H16 and D20 in mice

According to the research result, the reduction of the combination of IL-2 to IL-2R beta is beneficial to the reduction of the dependence of IL-2R beta receptor by IL-2, the reduction of the activation to effector cells and the improvement of the targeting of Treg. The H16 and D20 sites are two key sites for IL-2 binding to the IL-2R β receptor. The modified product of human IL-2 at the 20 th site has obvious Treg targeting property, but the in vitro retention activity is lower. The inventors performed in vivo evaluations of this site at various doses. The results show that 20-20K under the injection conditions of 1ug and 5ug, Treg is not only not obviously increased but also obviously inhibited compared with WT, and the percentage of Treg in CD4 cells is 66% and 56% of WT respectively; the percentage of Treg/CD8+ T over WT was 84% and 77% under 1 μ g and 5 μ g injection conditions, respectively; the percentages of Treg/Tconv compared to WT were 63% and 53%, respectively; when the injection dose is increased to a higher concentration of 50 mug, obvious Treg activation targeting is realized: Treg/CD8+ T and Treg/Tconv increased 1.2-fold and 1.4-fold respectively compared with WT. The results of the activation marker molecules on the tregs and CD8+ T cells are consistent with the results of the cell proportion analysis: injection of 50 μ g could significantly activate CD25 activity on tregs but not CD8+ T, with the results shown in figure 8.

The inventors subsequently performed an evaluation of Treg targeting in vivo by IL-2 modified with PEG at the H16 site. The results show that H16-20K (5. mu.g) injected mice expanded Treg significantly compared to WT-IL-2 and PBS injected mice, while CD8+ T cells remained essentially unchanged. The Treg/CD8 ratio was greatly increased, and the results are shown in FIG. 9.

Example 10 use of IL-2 modifications in models of graft versus host disease

Allogeneic hematopoietic stem cell transplantation is one of the main clinical means for radically treating various blood diseases, genetic diseases and solid tumors. The main mechanism is to help the recipient to rebuild the immune and hematopoietic system in vivo by donor-derived stem cells, producing a graft-versus-leukemia effect to kill tumor cells. However, in clinical treatment, the donor and the recipient are the main reasonsThe difference between histocompatibility complexes or minor histocompatibility complexes is that many patients develop graft versus host disease to varying degrees after blood cell transplantation. Clinically, graft-versus-host disease is a syndrome manifested as an autoimmune disease, resulting in inflammatory reactions of multiple organs and multiple tissue cells and fibrous lesion damage, and is a main reason and a complicated sequelae of treatment failure. As one of the immune diseases, the treatment of graft-versus-host disease is mainly based on the extensive immunosuppression and the non-selective elimination of immune cells, and the harm caused by the problems can be improved for a long time through the targeted activation of tregs. The inventors evaluated the therapeutic effect of modified IL-2 derivatives on graft-versus-host disease by examining the targeted activation of the postcomplement products on tregs in peripheral blood mononuclear cells from human sources activated by mouse transplantation. Human PBMC were isolated and stimulated overnight with anti-human-CD3/CD28 magnetic beads. Selecting NOD immunodeficient mice, injecting 3 × 10 intravenous injection to each mouse tail the next day ⁷ PBMC, and injecting 0.4. mu.g, 2. mu.g, 10. mu.g-WT-IL-2, 20-20K, 31-20K, 31/51-20K samples; wherein WT-hIL-2 is injected once a day for 5 times; PEG modified IL-2 was injected 3 times a day after each injection. On the third day after injection, splenic lymphocytes of mice were isolated for multicolor flow detection of cell activation markers, and labeled antibodies were as follows: anti-human-CD3-APC/Cy7, anti-human-CD4-PE/Cy7, anti-human-CD8-FITC, anti-human-CD25-BV605, anti-human-Foxp 3-PE. The result shows that the modification product of the 20kDa-PEG modified by the 31 site and the modification product of the 31/51 two sites modified by the 20kDa-PEG can obviously activate Tregs, activate CD8+ T and Tconv to a lesser extent and improve Treg/CD8+ T and Treg/Tconv within the range of the detected dose; 31-20K under the condition of low dose (0.4 mu g) injection, the Treg/CD8+ T and Treg/Tconv are respectively improved by 2.5 times and 3.6 times compared with the WT; under the condition of medium dose (2 mu g) injection, the Treg/CD8+ T and Treg/Tconv are respectively improved by 4.6 times and 4.2 times compared with the WT; under the condition of high-dose (10 mu g) injection, the Treg/CD8+ T and Treg/Tconv are respectively improved by 1.5 times and 2.5 times compared with the WT; 31/51-20K under the condition of low dose (0.4 mu g) injection, the Treg/CD8+ T and Treg/Tconv are respectively improved by 4.1 times and 1.2 times compared with WT; Treg/CD8+ T and Treg/Tconv vs W at moderate dose (2 μ g) injection conditionsT is respectively improved by 6 times and 1.13 times; under the condition of high-dose (10 mu g) injection, the Treg/CD8+ T and Treg/Tconv are respectively improved by 2.4 times and 1.4 times compared with the WT; the results of the activation marker molecules on the tregs and CD8+ T cells are consistent with the results of the cell proportion analysis: the modification product of the 31-site modified 20kDa-PEG and the modification product of the 31/51 two-site modified 20kDa-PEG can more effectively activate CD25 and Foxp3 on Tregs in a detection range than WT, but have no obvious difference on CD8+ T (FIG. 10 ABCD). Furthermore, we evaluated 31/51-20K for treatment of graft versus host disease. The results show that the mice injected with 31/51-20K can obviously resist the death of the mice from graft-versus-host disease, and the effect is better than that of the wild type IL-2 and PBS control group, and the results are shown in FIG. 10E.

Example 11 evaluation of the effectiveness of IL-2 modifications in a collagen-induced arthritis model

A Collagen Induced Arthritis (CIA) model is reported by Trentham in 1977 for the first time and is widely used for researching pathogenesis and drug screening of rheumatoid arthritis at home and abroad. The inventor evaluates the therapeutic action of the modified IL-2 derivative on rheumatoid arthritis by detecting the activation and therapeutic action of a postcomplement product on immune cell subsets of a CIA model mouse. Firstly, mixing bovine type II Collagen (COII) with Complete Freund's Adjuvant (CFA), selecting DBA/1 mice, and injecting 100 mu g of COII into the tail root of each mouse; on day 21 after injection, each mouse was reinjected with 100. mu.g of COII mixed Incomplete Freund's Adjuvant (IFA) as an activation to induce arthritis. After the second COII injection, PBS, 1 mug-WT-IL-2, 1 mug-31-20K, 1 mug-m 103-20K and 0.2 mug-31/51-20K samples are respectively injected into each group of mice, wherein WT-hIL-2 is injected once a day for 5 times; PEG modified IL-2 in every other day injection of 3 times. Mice were scored every other day for severity of arthritis: 0 is no red swelling; 1, minor swollen joint; 2 ═ toe joint and toe swelling; swelling of the paw below the ankle joint; swelling of all paws including ankle. On the third day after injection, 3 mice per group were taken and splenic lymphocytes were isolated for multi-color flow assay, labeled antibodies as follows: anti-mouse-CD3-APC/Cy7, anti-mouse-CD4-PE/Cy7, anti-mouse-CD8-FITC, anti-mouse-CD25-APC, anti-mouse-Foxp3-Pacific blue, anti-mouse-CD103-PerCP/Cy5.5, anti-mouse-CTLA4-BV 605; among them, after half spleen lymphocytes are stimulated overnight, TH17 cell detection is carried out, and the labeled antibodies are as follows: anti-mouse-CD3-APC/Cy7, anti-mouse-CD4-PE/Cy7, anti-mouse-IL-17-PE. The results show that the Treg cells of the experimental group injected with 31-20K, 31/51-20K and m103-20K are significantly higher than those of the WT and PBS treated group, the TH17 cells are lower than those of the WT and PBS treated group, the levels of Treg/CD8+ T and Treg/TH17 are both significantly higher than those of the WT and PBS treated group, and the generation and development of arthritis can be effectively prevented, and the results are shown in FIG. 11 ABCD. Further, the inventors evaluated the prophylactic and therapeutic effects of 31/51-20K on CIA by injecting PBS and different doses of WT-IL-2 and 31/51-20K to each group of mice one week before the second collagen injection. The results showed that 31/51-20K was effective in delaying the onset time and severity of arthritis and had significant prophylactic and therapeutic effects compared to WT and PBS groups (FIG. 11E).

Example 12 evaluation of effectiveness and immunogenicity of IL-2 modified substance in systemic lupus erythematosus model mice

The MRL/lpr model mouse is generated by the complex mating of LG/J, AKR/J, C3H/Di and C57BL/6 strain mice, and has the symptoms of systemic lupus erythematosus of humans such as systemic lymphadenectasis, erosive arthritis, anti-DNA, anti-Sm, anti-Su, anti-nucleoside P antibodies, high-titer ANA and the like due to apoptosis-related mutation of a Fas gene, so that the MRL/lpr model mouse is a widely applied lupus drug evaluation model with high acceptance at present. The inventor gives PBS to an impending MRL/lpr mouse and 31/51-20K and WT-IL-2 with different doses, periodically draws blood, and detects the influence of different administration groups on lupus indexes of the MRL/lpr mouse. Experimental results show that 31/51-20K mice have obvious decrease in lupus index, specifically antinuclear antibody reduction, double-chain DNA antibody reduction and proteinuria reduction, compared with WT-IL-2 and PBS mice, suggesting that 31/51-20K has better treatment effect on systemic lupus erythematosus (FIG. 12A).

Treatment involved long-term dosing, with WT administered 3 times a week for 3 weeks; 31/51-20K is administered once a week for 3 weeks. Immunogenicity is one of the key factors affecting the efficacy of protein drugs administered over a long period of time, and therefore the inventors have also evaluated the immunogenicity of WT-IL-2 and 31/51-20K in long-term administration. The results show that 31/51-20K has lower level immunogenicity than WT-IL-2, which induces mice to produce total IgG against IL-2, IgG1, IgGa2 are significantly lower than WT group, demonstrating that 31/51-20K has lower immunogenicity in long term administration.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Beijing coordination hospital of Chinese academy of medical sciences

Peking University

<120> long-acting interleukin-2 targeting regulatory T cells and its use in the treatment of autoimmune diseases

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Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

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Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

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Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

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Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

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Claims

1. Site-directed mutagenesis of human interleukin-2, characterized in that the mutated human interleukin-2 targets the activation of tregs, modified at a specific site of the human interleukin-2 shown in SEQ ID No.1, said specific site being selected from the group consisting of (1): shown at position A73 of SEQ ID No. 1; (2): the combination of any one or more of the amino acid sites of A73 th site and H16 th site, D20 th site, Y31 th site, T51 th site, K76 th site and H79 th site shown in SEQ ID No.1, wherein the modification is that the amino acid at the specific site is mutated into an unnatural amino acid

As shown.

2. The PEG-modified site-directed mutagenesis human interleukin-2 is characterized in that: the site-directed mutagenesis of human interleukin-2 as described in claim 1 is subjected to PEG modification by site-directed coupling with PEG containing cyclooctyne functional group after substituting unnatural amino acid containing azide group in claim 1 for amino acid at specific site of human interleukin-2 shown in SEQ ID No.1, and the introduced PEG has molecular weight of 5kDa, 10kDa, 20kDa, 30kDa, or 40 kDa.

3. The PEG-modified site-directed mutagenesis human interleukin-2 of claim 2, wherein when there is more than one specific site, the PEG coupled at different sites may be the same or different.

4. The PEG-modified site-directed mutagenesis of human interleukin-2 as claimed in claim 2, in particular a modification of PEG with a molecular weight of 5kDa, 10kDa or 20kDa on the mutated unnatural amino acid at position H16; PEG with the molecular weight of 5kDa, 10kDa or 20kDa is modified on the mutated unnatural amino acid at position D20; modifying the mutated unnatural amino acid at position Y31 with PEG of 5kDa, 10kDa or 20kDa in molecular weight; modifying the mutated unnatural amino acid at position T51 with PEG with molecular weight of 5kDa, 10kDa or 20 kDa; modifying the mutated unnatural amino acid at position A73 with PEG with molecular weight of 5kDa, 10kDa or 20 kDa; modifying the mutated unnatural amino acid at position K76 with PEG with molecular weight of 5kDa, 10kDa or 20 kDa; the mutated unnatural amino acid at position H79 is modified with a PEG of 5kDa, 10kDa or 20kDa molecular weight.

5. A pharmaceutical composition comprising an effective amount of the site-directed mutagenesis human interleukin-2 of claim 1 or the PEG-modified site-directed mutagenesis human interleukin-2 of any one of claims 2 to 4, and a pharmaceutically acceptable carrier.

6. Use of the site-directed mutant human interleukin-2 of claim 1 or the PEG-modified site-directed mutant human interleukin-2 of any one of claims 2 to 4 in the preparation of a medicament for immunomodulation of a variety of autoimmune diseases, such as graft versus host disease, rheumatoid arthritis and systemic lupus erythematosus.