CN112279906B - Human interleukin 2-polyethylene glycol conjugate, preparation method and application thereof - Google Patents

Abstract

The invention discloses a human interleukin 2-polyethylene glycol conjugate, a preparation method and application thereof. The invention discloses a human interleukin 2-polyethylene glycol conjugate, which comprises recombinant human interleukin2 containing at least one unnatural amino acid and PEG coupled on the at least one unnatural amino acid, wherein the position of the at least one unnatural amino acid is selected from one or more sites of L36, M39, L40, M46, P47, L63, L66, E67, L70 and A73 corresponding to SEQ ID NO. 2, and the recombinant human interleukin2 is a protein shown in SEQ ID NO. 3 or a functional active fragment thereof. The human interleukin 2-polyethylene glycol conjugate provided by the invention can be used for treating solid tumors or blood tumors by single medicine or combined use with anti-tumor medicines.

Description

Human interleukin 2-polyethylene glycol conjugate, preparation method and application thereof

Technical Field

The invention belongs to the field of biological pharmacy, and relates to a human interleukin 2-polyethylene glycol conjugate, and a preparation method and application thereof.

Background

Interleukin-2 (Interleukin2, IL-2, often abbreviated as Interleukin 2) is an important immunomodulatory factor produced by activated T helper T lymphocytes of type I (Th1), and has been called T cell growth factor, and its major biological function promotes the growth, proliferation and differentiation of T cells (including CD4+ and CD8+ T cells) and further secretion of cytokines in a dual manner of stimulation and anti-apoptosis. Besides, interleukin2 also stimulates the proliferation of NK cells, enhances the killing activity of NK cells, generates cytokines and induces the generation of LAK cells; promoting B cell proliferation and secretion of antibodies; therefore, interleukin2 plays an important role in immune response of the body and in resisting viral infection, etc. (Gaffena S.L., Cytokine 28: 109e123, 2004).

Since the first discovery of IL-2 by Morgan et al in 1976, it has been widely used clinically. In 1991, rhIL-2 (product name: Aldesukin) produced by Cetus in the United states was approved by FDA to be on the market, widely applied to malignant tumors such as renal cell carcinoma, malignant melanoma, and malignant lymphoma (Proleukin Specification), and also has potential effect in adjuvant therapy of hepatitis B and hepatitis C infections (Tomova R. et al, Anticancer Research, 29: 5241-. The recombinant human interleukin2 biological products of more than 10 recombinant human interleukin2 are produced and marketed in China so far, and are widely applied to the treatment of malignant tumors such as renal cell carcinoma, melanoma, breast cancer, bladder cancer, liver cancer, rectal cancer, lymph cancer, lung cancer and the like, the control of cancerous hydrothorax and ascites, the enhancement of body immunity of tumor patients after operations, radiotherapy and chemotherapy, the improvement of cellular immunity and anti-infection capacity of patients with congenital or acquired immunodeficiency, the treatment of various autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, sjogren syndrome and the like, and certain treatment effects on certain viral, bacillary diseases, intracellular parasitic bacteria infectious diseases such as hepatitis B, leprosy, tuberculosis, candida albicans infection and the like.

The human IL-2 precursor consists of 153 amino acid residues, and upon secretion from the cell, the signal peptide (containing 20 amino acid residues) is cleaved off, yielding 133 amino acids of mature IL-2 with a relative molecular weight of 15.4 kD. Activation of effector cells by IL-2 is achieved by binding to the IL-2 receptor (IL-2R) on the cell surface. It has now been found that the IL-2 receptor includes three of IL-2 ra, IL-2R β and IL-2R γ, which form a high affinity heteromultimeric glycoprotein functional complex IL-2R α β γ (Kd ═ 10) ^-11 mol/L). IL-2R beta and IL-2R gamma form a moderate affinity receptor complex IL-2R beta gamma (Kd 10 ═ 10) ^{- 9} mol/L) has biological activity after being activated by IL-2; the IL-2R alpha subunit is a low affinity receptor form (Kd 10) ^-8 mol/L), its binding to IL-2 is unable to transmit intracellular proliferation signals; although IL-2R alpha and IL-2R beta can also form high affinity receptor complexes, no biological function can be activated by IL-2. The IL-2 receptor species are expressed to different extents in different cells, different developmental stages of the same cell type, and different states of the disease, thereby forming different receptor complexes. For example, LAK cell precursors express high levels of the IL-2R β 0 γ complex, which can attack and lyse cancer cells upon activation by IL-2; macrophages also express the IL-2R β 3 γ complex, and may also be activated by IL-2; monocytes express large amounts of IL-2R gamma and small amounts of IL-2R beta, while NK cells express large amounts of IL-2R beta and small amounts of IL-2R gamma, which form IL-2R beta gamma receptors of moderate affinity, respectively, which activate monocytes or NK cells after binding to high concentrations of IL-2 to form trimers; the surface of the activated T cell expresses IL-2R beta 2, IL-2R beta and IL-2R gamma, the excessive IL-2R alpha is beneficial to polymerizing with the IL-2R beta, and the IL-2R beta 1 is combined with the IL-2R gamma after being combined with the IL-2 to form a high-affinity receptor-IL-2 compound, so as to transmit signals and cause cell proliferation reaction. After the cell reaction, IL-2R alpha, IL-2R beta and IL-2R gamma are dissociated, and the cell is no longer sensitive to IL-2; human tumor cells also have IL-2 receptor expression, IL-2 can inhibit the proliferation of tumor cells after being combined with receptor complexes on the tumor cells, and different cancer cells express respective special IL-2 receptor complexes, so the IL-2 structure is improved to only act on corresponding receptors on specific tumor surfaces, and the purpose of realizing the effect of the IL-2 receptor on the specific tumor surfacesOnly attack cancer cells and reduce the damage to normal cells.

Based on the research theory, a plurality of researchers modify IL-2 in different directions, enhance the combination of the IL-2 with a specific receptor compound (such as an IL-2R beta gamma compound) on the surface of an anti-tumor related effector cell, activate cell types related to tumor killing, and simultaneously reduce the combination with an IL-2R alpha beta gamma compound highly expressed on the surface of a negative immunoregulation T cell (such as a Treg cell) as much as possible, thereby not only enhancing the drug effect, but also reducing the side effect of the drug. Existing modifications to IL-2 include: designing specific IL-2 variant protein, changing the amino acid sequence of the binding site of IL-2R alpha, IL-2R beta or IL-2R gamma to make the space structure unfavorable for the interaction with IL-2R alpha or strengthen the interaction with IL-2R beta or IL-2R gamma; designing an IL-2/anti-IL-2 antibody compound, and specifically covering a binding site with IL-2R by using an anti-IL-2 antibody to realize the change of IL-2 function and the extension of in vivo half-life; performing fusion expression of IL-2 with Fc or Human Serum Albumin (HSA) to realize half-life extension of IL-2 in vivo; non-site-specific coupling PEGylation of IL-2 prolongs the half-life of IL-2.

The existing research on the modification of IL-2 has the following defects:

1. the simple amino acid site-directed mutation can weaken the binding capacity with IL-2R alpha or strengthen the binding capacity with IL-2R beta or IL-2R gamma, but cannot effectively prolong the half-life of molecules, and the mutation product is easy to generate immunogenic reaction in vivo, easily reduces the biological activity of the product and generates larger toxicity risk.

2. Pure fusion expression (e.g., with Fc or HSA) or IL-2 modification, while able to extend the half-life of the molecule, does not yield significant advantages over unmodified IL-2 in practical use.

3. The site-directed mutagenesis in combination with the fusion expressed IL-2 showed no particular advantage in practical applications.

4. The conventional non-site coupling PEGylation IL-2 does not show special advantages in practical application, and the PEGylation IL-2 comprehensively weakening the binding capacity of IL-2R alpha is successful in stages, but the defects of difficult control of production process and quality, complex molecular structure, complex action mechanism and the like exist due to the characteristics of the non-site coupling process.

Genetic code expansion technique

All proteins in natural organisms are composed of 20 natural amino acids encoded by triplet codons. In 1986, a team led by England scientists I.Chambers and German scientists F.Zinoni respectively finds selenocysteine in selenase contained in prokaryotic and eukaryotic organisms, and is called as '21 st amino acid'; in 2002, the scientists Srinivasan and Hao written that pyrrolysine was found in Methanosarcina pasteurii and was called "amino acid 22". The discovery of the two amino acids breaks through the cognition of people on the amino acid coded by organisms, and attempts are made to develop scientific research by using the unnatural amino acid.

It was found by study that selenocysteine is encoded with the stop code UGA and pyrrolysine is encoded with the stop code UAG. In organisms containing the two amino acids, corresponding tRNA and aminoacyl-tRNA synthetases are respectively arranged, and the introduction of corresponding amino acid residues into protein sequences is completed together. While the corresponding trnas and aminoacyl-tRNA synthetases do not interact with other trnas and aminoacyl-tRNA synthetases in the organism, a strict match between an unnatural amino acid, the corresponding trnas and aminoacyl-tRNA synthetases is also referred to as an orthogonal reaction.

The 20 natural amino acids contain only a limited number of functional groups, such as hydroxyl, carboxyl, amino, alkyl, and aromatic groups. In order to make proteins produce more abundant or specialized functional groups, scientists have attempted to encode other classes of unnatural amino acids into proteins using the translation machinery of selenocysteine or pyrrolysine. With the intensive and extensive research and application, dozens of tRNA/aminoacyl-tRNA synthetase orthogonal systems are developed to realize the expression of hundreds of unnatural amino acids in recombinant protein (Dufang Chuan, et al, proceedings of Hangzhou university (Nature science edition), 12:437-445, 2013).

Click reaction

The concept of Click Chemistry was first proposed in 2001 by nobel chemical awards, professor k.barry sharp, us Scripps research, and colleagues, to attempt to modularly, regioselectively, and rapidly construct new libraries of chemical molecules using reactions such as Click Chemistry. Click chemistry has the following characteristics: the method has the advantages of easily obtained reaction raw materials, very reliable reaction, insensitivity to oxygen and water, good product stereoselectivity, high yield, simple and convenient post-reaction treatment and product separation, no need of column chromatography generally, and environment-friendly reaction byproducts.

One of the most important and widely used types of reactions in click chemistry is the Huisgen l, 3-dipolar cycloaddition of azido (azide) and alkynyl (alkyne) under the catalysis of cu (i) to form a stable l, 2, 3-triazole compound, known as the "CUAAC" reaction, which was discovered by Sharpless et al. The Huisgen 1, 3-dipolar cycloaddition reaction is a reaction scheme that combines two reaction substrates with different unsaturated bonds into a new class and nature of five-membered heterocyclic compounds that are distinct. Cycloaddition of azide ligands to alkynyl ligands is one of the most typical examples.

However, for in vivo applications, the presence of transition metals (copper salts) can be toxic to both bacteria and human cells. In order to avoid the addition of copper salts, a subject group has developed a new click chemistry method, i.e., a cyclooctyne derivative is prepared, which undergoes a strain-promoted [3+2] cycloaddition reaction (i.e., SPAAC reaction) with an azide group. This novel copper-free click chemistry approach does not produce significant toxicity to living cells and can be used in many aspects of glycobiology. For example, glycan trafficking imaging may be performed in cells, tissues, or even whole organisms under conditions of cellular stimulation or drug intervention.

Disclosure of Invention

Aiming at the defects existing in the modification aspect of the prior IL-2, the invention provides a human interleukin 2-polyethylene glycol conjugate, which is prepared by recombining one or more natural amino acids in an amino acid sequence of the human interleukin2 by site-specific mutagenesis of the unnatural amino acids through codon expansion or a chemical synthesis technology, and then coupling PEG with a certain molecular weight on the side chain of the unnatural amino acids through a connecting molecule by a Click reaction site-specific coupling.

In one aspect, the present invention provides a human interleukin 2-polyethylene glycol conjugate comprising recombinant human interleukin2 comprising at least one unnatural amino acid and PEG conjugated to the at least one unnatural amino acid;

wherein the position of the at least one unnatural amino acid is selected from one or more of positions corresponding to L36, M39, L40, M46, P47, L63, L66, E67, L70, and A73 of SEQ ID NO 2;

the recombinant human interleukin2 is protein shown as SEQ ID NO. 3 or a functional active fragment thereof;

compared with wild type IL-2 (such as, for example, Quanqi (recombinant human IL-2 of a commercial wild type)), the conjugate has reduced binding with IL-2R alpha and retains the binding activity with IL-2R beta gamma, the activation of CD8+ T cells through an IL-2R beta gamma complex retains the capacity of activating and expanding CD8+ T cells, can inhibit the expansion of Treg cells, has a remarkably prolonged half-life period in vivo, and can effectively promote immunity and inhibit tumors.

In some embodiments, in the human interleukin-2-polyethylene glycol conjugate described above, the unnatural amino acid is an azide-containing lysine analog.

In some embodiments, in any of the human interleukin-2-polyethylene glycol conjugates described above, the azido-containing lysine analog is N ∈ -2-azidoethoxycarbonyl-L-lysine (NAEK) having the formula:

in some embodiments, the human interleukin-2-polyethylene glycol conjugate of any one of the above, wherein the PEG has a molecular weight of 20KD to 50KD, e.g., 20KD, 30KD, 40KD, and 50 KD.

In some embodiments, in the human interleukin-2-polyethylene glycol conjugate of any preceding claim, the PEG is conjugated to the side chain of the at least one unnatural amino acid via a linker, and the linker is BCN or a derivative thereof.

In a second aspect, the present invention provides a method of preparing a human interleukin 2-polyethylene glycol conjugate as described in any one of the above, comprising the steps of: preparing the recombinant human interleukin2 containing at least one unnatural amino acid by a codon expansion technology or a chemical synthesis mode, and coupling the PEG on the at least one unnatural amino acid;

wherein the position of the at least one unnatural amino acid is selected from one or more of positions corresponding to L36, M39, L40, M46, P47, L63, L66, E67, L70 and A73 of SEQ ID NO 2;

the recombinant human interleukin2 is protein shown in SEQ ID NO. 3 or a functional active fragment thereof.

In some embodiments, in the above method, the recombinant human interleukin2 containing at least one unnatural amino acid is prepared by codon expansion techniques.

In some embodiments, in any of the methods described above, the codon expansion technique is effected in E.coli.

In some embodiments, the method of any one of the above, wherein the unnatural amino acid is an azido-containing lysine analog, preferably N ∈ -2-azidoethoxycarbonyl-L-lysine.

In some embodiments, in any of the methods described above, the codon expansion technique specifically comprises the steps of: mutating a nucleic acid molecule of recombinant human interleukin2, the mutated nucleic acid molecule differing from a nucleic acid molecule encoding recombinant human interleukin2 in that: the codon corresponding to amino acids at least one of positions L36, M39, L40, M46, P47, L63, L66, E67, L70 and A73 of SEQ ID NO 2 was mutated to the amber codon UAG; expressing the mutated nucleic acid molecule in E.coli, and simultaneously incorporating an azido-containing lysine analog (e.g., N epsilon-2-azidoethoxycarbonyl-L-lysine) into the expressed recombinant human interleukin-2 via an orthogonal tRNA synthetase/tRNA pair;

the working principle of the codon expansion system is as follows: tRNA ^Pyl Lysyl tRNA enzymes which cannot utilize the host cell, can only be tRNA ^Pyl RS acylation, tRNA ^Pyl RS can only acylate tRNA ^Pyl Not acylating other tRNA's, i.e. tRNA' s ^Pyl And tRNA ^Pyl Orthogonality among RSs, only tRNA ^Pyl The RS can acylate a corresponding unnatural amino acid (e.g., a lysine analog containing an azido group) to the orthogonal tRNA and can acylate only the tRNA and not other tRNA. The codon expansion system allows the azido-containing lysine analog to correspond to the amber codon UAG (i.e., tRNA) ^Pyl The corresponding codon is UAG) to introduce an azido-containing lysine analog into IL-2 at a site.

In some embodiments, the method further comprises the steps of protein denaturation, renaturation and ultrafiltration after expressing the mutant recombinant human interleukin2 in escherichia coli.

In some embodiments, the method of any one of the above, wherein the PEG has a molecular weight of 20KD to 50KD, e.g., 20KD, 30KD, 40KD, and 50 KD.

In some embodiments, the method of any one of the above, wherein the PEG is coupled to the side chain of the at least one unnatural amino acid by a Click reaction with a linker that is BCN or a derivative thereof; for example, the pH of the mutated recombinant human interleukin2 solution is adjusted to 3 by using dilute hydrochloric acid, and the BCN-PEG solid is put into the mutated recombinant human interleukin2 solution, fully dissolved, sealed and reacted in a constant temperature shaking table by shaking.

In some embodiments, in any of the above methods, after the coupling, the reaction solution comprises a portion of unreacted IL-2, a hetero-protein, and unreacted PEG, and is further purified by cation exchange chromatography; for example, chromatographic media: CM Sepharose FF; and (3) an equilibrium buffer: 20mM sodium acetate buffer (pH3.0), elution buffer: 20mM sodium acetate buffer solution-1M NaCl (pH4.5), specifically comprises adjusting pH of the coupling reaction solution to 3.0 + -0.2 with an equilibrium buffer solution, the conductivity is less than or equal to 2.5ms/CM, loading to CM Sepharose FF, performing linear elution (0-30% elution buffer solution, 15CV) with an elution buffer solution, and collecting target protein components.

In a third aspect, the present invention provides the use of a human interleukin 2-polyethylene glycol conjugate as described in any one of the above in the manufacture of a medicament for promoting immunity, treating malignant solid and hematological tumors, and/or expanding CD8+ T cells.

In some embodiments, in the above use, the solid tumor is bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer.

In some embodiments, in the above-mentioned uses, the hematologic tumor is Chronic Lymphocytic Leukemia (CLL), Small Lymphocytic Lymphoma (SLL), Follicular Lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), Mantle Cell Lymphoma (MCL), waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, burkitt's lymphoma, non-burkitt's high malignancy B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

In a fourth aspect, the present invention provides a kit comprising any one of the human interleukin 2-polyethylene glycol conjugates described above.

In a fifth aspect, the present invention provides a method of treating a malignant solid tumour or a haematological tumour comprising the step of administering to a patient in need thereof a therapeutically effective amount of a human interleukin 2-polyethylene glycol conjugate as described in any of the above.

In some embodiments, in the above methods, the solid tumor is bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer.

In some embodiments, the method of any one of the above, wherein the hematologic tumor is Chronic Lymphocytic Leukemia (CLL), Small Lymphocytic Lymphoma (SLL), Follicular Lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), Mantle Cell Lymphoma (MCL), waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, burkitt's lymphoma, non-burkitt's highly malignant B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large-cell lymphoma, precursor B-lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, lymphoblastic leukemia, lymphoplasmacytoma, lymphoblastic lymphoma, lymphoblastic large B-cell lymphoma, lymphoblastic lymphoma, peripheral zone lymphoma, splenic zone lymphoma, plasma cell myeloma, and plasmacytoma, Primary effusion lymphoma or lymphomatoid granulomatosis.

In some embodiments, in any of the methods described above, the human interleukin-2-polyethylene glycol conjugate can be administered alone or in combination with an anti-tumor agent.

Analysis of the binding activity of the conjugates of the invention to IL-2R by SPR showed that the binding activity of the conjugates of the invention to IL-2 ra was reduced to a different extent compared to wild-type IL-2, but the binding activity to IL-2R β was substantially retained; CTLL-2 cells and YT cells are respectively used, and the JAK-STAT signal channel is activated by analyzing the binding of a sample and IL-2R on the cell surface, and the comprehensive experiment result shows that the binding activity of the conjugate obtained by the invention and IL-2R alpha is reduced to different degrees relative to the binding activity of IL-2R beta; in addition, pharmacokinetic and pharmacodynamic studies show that compared with wild type IL-2, the conjugate provided by the invention has a remarkably prolonged half-life, can remarkably inhibit tumors, expand CD8+ T cells, inhibit Treg cells and promote immunity.

The most important improvement of the present invention is that,

(1) the non-natural amino acid is introduced into the appointed site by the codon expansion technology, thereby realizing the precise fixed-point coupling of PEG and interleukin-2, overcoming the defect that the traditional random coupling mode can not be precisely coupled, and having high product uniformity.

(2) Unlike traditional mutation site selection of surface amino acid residues of the IL-2 molecular domain that interact with the receptor, the present invention selectively mutates amino acid residues of the internal folded and surface exposed border regions of the IL-2 molecular domain that interact with the alpha receptor. Through the design and screening of mutation sites, interleukin-2 mutation sites capable of reducing the binding activity of IL-2R alpha and keeping the binding activity of IL-2R beta and IL-2R gamma relatively unchanged are obtained, so that the site-specific modified human interleukin-2-polyethylene glycol conjugate specifically promotes the proliferation of CD8+ T cells in a tumor microenvironment without obvious effect on the proliferation of CD4+ T cells, and the immunotherapy of tumors is facilitated. And the mutated amino acid sites are not on the prominent surface, reducing possible immunogenicity. The amino acid sites do not participate in the overall structure maintenance of the interleukin-2, but have a maintenance function on the local tertiary structure of the protein, and the NAEK replacement reduces the stability of the local protein structure and reduces the affinity with alpha receptors.

(3) Coupling of PEG further alters local tertiary structure, reducing affinity to the alpha receptor.

(4) PEG coupling achieves an increase in the half-life of IL-2 in vivo, reducing the frequency of administration to the patient.

Drawings

FIG. 1 is a schematic representation of expression plasmid NB1S 3-WT.

FIG. 2 is a schematic representation of the helper plasmid NB 1W.

FIG. 3 is an SDS-PAGE electrophoresis of each strain. Wherein, A: crushing rhIL2-L36-BL21 thallus, centrifuging and precipitating; b: crushing rhIL2-M39-BL21 thalli, centrifuging and precipitating; c: crushing rhIL2-L40-BL21 thallus, centrifuging and precipitating; d: crushing rhIL2-M46-BL21 thalli, centrifuging and precipitating; e: crushing rhIL2-L63-BL21 thallus, centrifuging and precipitating; f: crushing rhIL2-A73-BL21 thalli, centrifuging and precipitating; g: rhIL2-L63-BL21 inclusion body; arrows indicate the expressed mutant rhIL-2.

FIG. 4 shows SEC-HPLC profiles before and after coupling of mutant rhIL-2 with PEG. Wherein, A: each mutant rhIL-2 prior to conjugation; b: after coupling, the PEG of each mutant rhIL-2 is 30KD PEG.

FIG. 5 shows the tumor suppression effect of 30kD PEG-rhIL2 in the B16-F10 allograft tumor model.

FIG. 6 is a photograph of the tumor suppression effect of 30KD PEG-rhIL2 in B16-F10 allograft tumor model.

FIG. 7 is a graph showing the change in the ratio of each immune cell population in a mouse tumor tissue sample after administration.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

Human YT cells are disclosed in the literature "Yodoi, J.et al. (1985),. TCGF (IL 2) -receptor inducing factor(s),. I.Regulation of IL 2receptor on a natural killer-like cell line (YT cells),. Journal of Immunology,134(3),. 1623-.

Example 1: construction of expression Strain expressing recombinant human IL-2(rhIL-2) with site-directed insertion of unnatural amino acids

1. Obtaining of expression plasmid NB1S3-WT for wild-type recombinant human IL-2

The precursor protein sequence of homo sapiens IL-2 (GenBank ID: CAA25292.1) was obtained from the National Center for Biotechnology Information (NCBI) as shown in SEQ ID NO: 1. The N-terminus of the precursor sequence contains a 20 amino acid signal peptide sequence which is cleaved during processing and maturation of the IL-2 protein molecule, thus removing the signal peptide sequence to yield the mature homo sapiens IL-2 protein sequence (SEQ ID NO: 2). According to literature reports (Liang S.M et al. Journal of Biological Chemistry,261(1):334-337, 1986) mature homo sapiens IL-2 protein sequences contain 3 cysteine Cys, of which two Cys at positions 58 and 105 form disulfide bonds, which are important for the Biological activity of homo sapiens IL-2. The Cys at the position 125 does not participate in the formation of disulfide bonds, but interferes with the formation of normal disulfide bonds in the renaturation process of the protein inclusion body of the recombinant homo sapiens IL-2, so that the Cys at the position 125 can be mutated into serine Ser, and the efficiency of renaturation is improved without obviously influencing the activity of the serine Ser. Meanwhile, in order to express the recombinant protein in E.coli, methionine Met is added to the N-terminus of the protein sequence for initiating the translation of the protein, thereby obtaining the mature protein sequence of recombinant human IL-2 (SEQ ID NO: 3). The gene sequence (SEQ ID NO:4) of the coding recombinant human IL-2 is obtained through the reverse translation process of amino acid and codon optimization, and the coding gene of the recombinant human IL-2 is obtained through whole-gene synthesis. Then the wild type recombinant human IL-2 expression plasmid NB1S3-WT (see FIG. 1) is obtained by connecting the wild type recombinant human IL-2 expression plasmid into an NB1S3 expression vector (the expression vector is modified from a commercial vector pET-21a, and an ampicillin resistance gene screening marker of the expression vector is replaced by a spectinomycin resistance gene amplified from a commercial vector pCDF-duet1 by a PCR mode), and the sequence is shown as SEQ ID NO: 5.

SEQ ID NO:1

MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

SEQ ID NO:2

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

SEQ ID NO:3

MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT

SEQ ID NO:4

ATGGCGCCTACATCCAGCTCGACCAAAAAGACGCAGCTGCAACTGGAACACCTGCTCCTGGATCTGCAAATGATTCTTAACGGTATCAATAACTACAAAAATCCGAAACTGACCCGTATGCTGACGTTTAAATTCTATATGCCAAAGAAAGCGACCGAGCTGAAACATCTGCAGTGCCTGGAAGAGGAACTGAAACCGCTGGAGGAAGTTTTGAACCTGGCTCAGTCTAAAAACTTTCACCTGCGCCCTCGTGACCTGATTTCCAATATCAACGTGATTGTTCTGGAACTGAAAGGCTCTGAAACCACGTTTATGTGCGAGTACGCCGATGAAACCGCCACGATTGTGGAATTTCTGAATCGCTGGATCACCTTCTCCCAGAGCATTATTAGCACGCTGACCTAA

2. Site-directed mutagenesis site selection

According to the crystal structure of IL-2, the binding site of IL-2 and its receptor, the conserved sequences of different IL-2 subtypes and the amino acid exposure range, and comprehensively considering the information of antigen epitope, enzymolysis site and the like, several suitable sites are selected for modification, which are mainly based on the following factors: (1) the amino acid sites are relatively close to the alpha receptors and far from the beta and gamma receptors in the crystal structure of the interleukin-2 and receptor complex; (2) the amino acid sites do not protrude on the surface, reducing possible immunogenicity; (3) the amino acid sites do not influence the overall structure of interleukin-2, but have certain influence on the local tertiary structure of the protein, and the stability of the local protein structure is reduced after NAEK (N epsilon-2-azidoethoxycarbonyl-L-lysine) is replaced, so that the affinity with alpha receptor is reduced; (4) after PEG coupling, the local tertiary structure can be further changed, and the affinity with alpha receptor is reduced. L36, M39, L40, M46, P47, L63, L66, E67, L70 and A73 of SEQ ID NO. 2 were selected for site-directed mutagenesis.

3. Primer design for site-directed mutagenesis and construction of mutagenesis vector

Mutation primers capable of mutating the codon encoding the amino acid into an amber codon are designed aiming at L36, M39, L40, M46, P47, L63, L66, E67, L70 and A73 of SEQ ID NO. 2, and specific primers are shown in Table 1.

TABLE 1 mutant primer List

The method comprises the steps of carrying out double digestion on a plasmid NB1S3-WT by using restriction endonucleases XbaI and XhoI to obtain a linearized DNA plasmid, using the linearized DNA plasmid as a template, using a high fidelity DNA polymerase (purchased from Takara, Cat. No. R045A), using primers XbaI-F in Table 1 to pair primers R at various sites, using primers XhoI-R in Table 1 to pair primers F at various sites, carrying out PCR amplification and overlapping PCR to obtain a mutant gene in which amino acid codons of IL-2 at several sites such as L36, M39, L40, M46, P47, L63, L66, E67, L70 and A73 are mutated to form an amber stop codon (for example, using the linearized plasmid NB1S3-WT as a template, using XbaI-F and L36-R as primers, carrying out PCR amplification to obtain an upstream fragment of L36 site mutation, using the linearized plasmid NB1S3-WT as a primer, using XhoI-WT as an L356-R as a primer, carrying out PCR amplification to obtain an upstream fragment of the L6327 and an upstream fragment of the aforementioned L36, and carrying out PCR amplification to obtain an upstream fragment of the aforementioned fragment of IL-2 The mutant downstream fragment is used as a template, XbaI-F and XhoI-R are used as primer pairs for carrying out overlapping PCR amplification to obtain a full-length gene with a mutation at the L36 locus, and then the obtained mutant gene is respectively replaced by the fragment between the XbaI and the XhoI restriction sites of the NB1S3-WT plasmid by using a high-fidelity DNA assembly cloning kit (purchased from NEB and with the product number of E5520S) according to the operation of instructions to construct 10 expression plasmids NB1S3-L36, NB1S3-M39, NB1S3-L40, NB1S3-M46, NB1S3-P47, NB1S3-L63, NB1S3-L66, NB1S3-E67, NB1S3-L70 and NB1S3-A73, and the mutation is verified by sequencing.

4. Construction of site-directed mutant rhIL-2 expression strains

The plasmid pUltra construct described in the reference (Chatterjee, A. et al, Biochemistry,52(10),1828-1837,2013) was constructed by obtaining a DNA fragment containing tRNA specifically recognizing the unnatural amino acid Lys-azido and a tRNA synthetase encoding gene (wild-type Archimedes aeoglobus pyrrilysin synthetase and a gene encoding the corresponding tRNA, Lys-azido is a pyrrilysine analog) and a chloramphenicol resistance gene (SEQ ID NO:28) by total gene synthesis, amplifying a DNA fragment (SEQ ID NO:29) containing the replication initiation site of CloDF13 from a commercial vector pCDF-duet1 by PCR amplification, and further subcloning and ligating the two DNA fragments by using a high fidelity DNA assembly cloning kit to obtain an accessory plasmid NB1W (see FIG. 2, which is hereinafter referred to as an accessory plasmid), which was selected for chloramphenicol resistance. Respectively transforming the auxiliary plasmid and the expression plasmid (spectinomycin resistance) obtained in the step 3 into escherichia coli BL21(DE3) together, screening double-positive strains (the double-positive strains represent strains which simultaneously obtain spectinomycin resistance and chloramphenicol resistance) by spectinomycin resistance and chloramphenicol resistance plates, wherein the double-positive strains comprise rhIL2-L36-BL21, rhIL2-M39-BL21, rhIL2-L40-BL21, rhIL2-M46-BL21, rhIL2-P47-BL21, rhIL2-L63-BL21, rhIL2-L66-BL21, rhIL2-E67-BL21, rhIL2-L70-BL21 and rhIL2-A73-BL 21.

The DNA fragment comprising the wild-type Gum methane coccus pyrrilysin synthetase encoding gene, the corresponding tRNA encoding gene and the chloramphenicol resistance gene (SEQ ID NO:28) is as follows:

CCTTATGCGACTCCCTGCATTAGGGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACAAAGGAGGTCATATGGATAAAAAGCCTCTGAACACTCTGATTTCTGCGACCGGTCTGTGGATGTCCCGCACCGGCACCATCCACAAAATCAAACACCATGAAGTTAGCCGTTCCAAAATCTACATTGAAATGGCTTGCGGCGATCACCTGGTTGTCAACAACTCCCGTTCTTCTCGTACCGCTCGCGCACTGCGCCACCACAAATATCGCAAAACCTGCAAACGTTGCCGTGTTAGCGATGAAGATCTGAACAAATTCCTGACCAAAGCTAACGAGGATCAGACCTCCGTAAAAGTGAAGGTAGTAAGCGCTCCGACCCGTACTAAAAAGGCTATGCCAAAAAGCGTGGCCCGTGCCCCGAAACCTCTGGAAAACACCGAGGCGGCTCAGGCTCAACCATCCGGTTCTAAATTTTCTCCGGCGATCCCAGTGTCCACCCAAGAATCTGTTTCCGTACCAGCAAGCGTGTCTACCAGCATTAGCAGCATTTCTACCGGTGCTACCGCTTCTGCGCTGGTAAAAGGTAACACTAACCCGATTACTAGCATGTCTGCACCGGTACAGGCAAGCGCCCCAGCTCTGACTAAATCCCAGACGGACCGTCTGGAGGTGCTGCTGAACCCAAAGGATGAAATCTCTCTGAACAGCGGCAAGCCTTTCCGTGAGCTGGAAAGCGAGCTGCTGTCTCGTCGTAAAAAGGATCTGCAACAGATCTACGCTGAGGAACGCGAGAACTATCTGGGTAAGCTGGAGCGCGAAATTACTCGCTTCTTCGTGGATCGCGGTTTCCTGGAGATCAAATCTCCGATTCTGATTCCGCTGGAATACATTGAACGTATGGGCATCGATAATGATACCGAACTGTCTAAACAGATCTTCCGTGTGGATAAAAACTTCTGTCTGCGTCCGATGCTGGCCCCGAACCTGTACAACTATCTGCGTAAACTGGACCGTGCCCTGCCGGACCCGATCAAAATTTTCGAGATCGGTCCTTGCTACCGTAAAGAGTCCGACGGTAAAGAGCACCTGGAAGAATTCACCATGCTGAACTTTTGCCAGATGGGTAGCGGTTGCACGCGTGAAAACCTGGAATCCATTATCACCGACTTCCTGAATCACCTGGGTATCGATTTCAAAATTGTTGGTGACAGCTGTATGGTGTACGGCGATACGCTGGATGTTATGCACGGCGATCTGGAGCTGTCTTCCGCAGTAGTGGGCCCAATCCCGCTGGATCGTGAGTGGGGTATCGACAAACCTTGGATCGGTGCGGGTTTTGGTCTGGAGCGTCTGCTGAAAGTAAAACACGACTTCAAGAACATCAAACGTGCTGCACGTTCCGAGTCCTATTACAATGGTATTTCTACTAACCTGTAACTAGTGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAGCACTGCAGCGGCTAACTAAGCGGCCTGCTGACTTTCTCGCCGATCAAAAGGCATTTTGCTATTAAGGGATTGACGAGGGCGTATCTGCGCAGTAAGATGCGCCCCGCATTGGAAACCTGATCATGTAGATCGAATGGACTCTAAATCCGTTCAGCCGGGTTAGATTCCCGGGGTTTCCGCCAAATTCGAAAAGCCTGCTCAACGAGCAGGCTTTTTTGCATCTCGAGCAGCTCAGGGTCGAATTTGCTTTCGAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCAACCAGGCGTTTAAGGGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTGTCCCTCCTGTTCAGCTACTGACGGGGTGGTGCGTAACGGCAAAAGCACCGCCGGACATCAGCGCGCGCTGCGGACAC

the DNA fragment of the replication initiation site of CloDF13 (SEQ ID NO:29) is as follows: CGGACATCAGCGCGCGCTGCGGACACATACAAAGTTACCCACAGATTCCGTGGATAAGCAGGGGACTAACATGTGAGGCAAAACAGCAGGGCCGCGCCGGTGGCGTTTTTCCATAGGCTCCGCCCTCCTGCCAGAGTTCACATAAACAGACGCTTTTCCGGTGCATCTGTGGGAGCCGTGAGGCTCAACCATGAATCTGACAGTACGGGCGAAACCCGACAGGACTTAAAGATCCCCACCGTTTCCGGCGGGTCGCTCCCTCTTGCGCTCTCCTGTTCCGACCCTGCCGTTTACCGGATACCTGTTCCGCCTTTCTCCCTTACGGGAAGTGTGGCGCTTTCTCATAGCTCACACACTGGTATCTCGGCTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTAAGCAAGAACTCCCCGTTCAGCCCGACTGCTGCGCCTTATCCGGTAACTGTTCACTTGAGTCCAACCCGGAAAAGCACGGTAAAACGCCACTGGCAGCAGCCATTGGTAACTGGGAGTTCGCAGAGGATTTGTTTAGCTAAACACGCGGTTGCTCTTGAAGTGTGCGCCAAAGTCCGGCTACACTGGAAGGACAGATTTGGTTGCTGTGCTCTGCGAAAGCCAGTTACCACGGTTAAGCAGTTCCCCAACTGACTTAACCTTCGATCAAACCACCTCCCCAGGTGGTTTTTTCGTTTACAGGGCAAAAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACTGAACCGCTCTAGATTTCAGTGCAATTTATCTCTTCAAATGTAGCACCTGAAGTCAGCCCCATACGATATAAGTTGTAATTCTCATGTTAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATGTCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTC

Example 2: expression and purification of rhIL-2 by site-directed mutagenesis of inserted unnatural amino acids

1. Expression of unnatural amino acid incorporation of mutant rhIL-2

The 10 expression strains rhIL2-L36-BL21 obtained in example 1 were subjected to,rhIL2-M39-BL21, rhIL2-L40-BL21, rhIL2-M46-BL21, rhIL2-P47-BL21, rhIL2-L63-BL21, rhIL2-L66-BL21, rhIL2-E67-BL21, rhIL2-L70-BL21 and rhIL2-A73-BL21 were inoculated into LB medium (yeast extract 5g/L, tryptone 10g/L, NaCl 10g/L, containing 100mg/L spectinomycin and 37.5mg/L chloramphenicol), and after 5-8 hours of culture at 37 ℃, secondary seed expansion (same medium composition as before) was carried out to OD bacterial liquid ₆₀₀ The concentration is 2.0 +/-0.2, and secondary seed liquid is obtained.

The secondary seed liquid was inoculated into a fermentation medium for fermentation culture in a 5L fermentor in a culture volume of 2L in a2 XYT medium (16 g/L yeast extract, 10g/L tryptone, 5g/L NaCl), and the inoculum size: 5% (v/v); the culture temperature is as follows: 37 ℃; and (3) pH control: 6.90 +/-0.05, and automatically adding ammonia water or H when necessary ₃ PO ₄ (ii) a And (4) DO control: 30%, DO-related speed; bacterial liquid OD ₆₀₀ When the concentration is 20.0 +/-2.0, IPTG and the unnatural amino acid NAEK are added to the mixture, the final concentration is 1mM, and simultaneously 50% glycerol is fed at the feeding speed of 0.6 +/-0.1 mL/min; after induction expression for 5-6 hours, the cells were collected. The SDS-PAGE electrophoresis of each strain is shown in FIG. 3.

2. Separation and extraction of mutant rhIL-2

Resuspending the collected thallus with buffer solution (25mM Tris, 6mM EDTA, 1mM DTT, pH8.0), adding 1% DNase (1mg/mL) and 0.5% PMSF, mixing, and homogenizing at 50-80MPa for 3 times with ultra-high pressure homogenizer; centrifuging the homogeneous solution at 10000rpm for 20min, and collecting the lower layer inclusion body crude body.

The obtained inclusion body crude body was washed twice with a washing buffer (20mM Tris-HCl, 100mM NaCl, 2% TritonX-100, pH8.0), and then washed once with ultrapure water to obtain a purified inclusion body.

The purified inclusion bodies were dissolved in a denaturation buffer (20mM Tris-HCl, 100mM NaCl, 6M guanidine hydrochloride, 1mM DTT, pH8.0), centrifuged at 10000rpm after 30min, and the supernatant was collected as a denatured protein solution. Adding renaturation buffer solution (20mM Tris-HCl, 100mM NaCl, pH8.0) with 4 times volume into the collected denatured protein solution, fully stirring, standing for 12h, centrifuging at 10000rpm, and collecting supernatant to obtain the renaturation protein solution.

Concentrating renaturated protein solution to the original volume 1/4 by using an ultrafiltration membrane package (Millipore, Biomax-5) with the molecular weight cutoff of 5kDa, changing the solution by using a replacement buffer solution (20mM Tris-HCl, pH8.0) until the conductance is about 2ms/cm, further concentrating until the protein concentration is about 0.5-1mg/mL, centrifuging at 10000rpm, and collecting supernatant, namely mutant rhIL-2 crude protein rhIL2-L36, rhIL2-M39, rhIL2-L40, rhIL2-M46, rhIL2-P47, rhIL2-L63, rhIL2-L66, rhIL2-E67, rhIL2-L70 and rhIL2-A73, wherein the supernatant can be directly used for subsequent PEG coupling.

Example 3: preparation of PEG-linker

As shown in formula 1, a certain amount of BCN-PEG with a general formula _N Mixing cycloalkyne of-NHS (succinimide activation-polyethylene glycol-octacyclo olefin) and 30KD of PEG according to the proportion of 1:1.2 equivalent, dissolving the mixture to be clear by using dichloromethane, adding TEA (TEA acid) with 10 times equivalent of cycloalkyne as an acid-binding agent, stirring the mixture at 25 ℃ for reaction for 18-24h, carrying out TLC (thin layer chromatography) detection on the raw materials after complete reaction, concentrating the reaction liquid under reduced pressure, adding methyl tert-butyl ether, precipitating a product BCN-PEG in the form of white solid, filtering and drying the product for later use. The reaction yield is more than 95%.

Example 4: site-directed conjugation of PEG to rhIL-2 with site-directed mutagenesis of inserted unnatural amino acids

As shown in formula 2, BCN-PEG was site-specifically coupled to NAEK of site-specifically mutated rhIL-2 by Click reaction.

Taking 30KD PEG Click reaction coupling rhIL2-M39 as an example, the reaction system is as follows:

rhIL2-M39 1eq

30KD BCN-PEG 15eq

Tris-HCl 20mM(pH≈3)

the pH of the rhIL2-M39 solution was adjusted to 3 by using dilute hydrochloric acid, BCN-PEG solid was put into the rhIL2-M39 solution and sufficiently shaken to dissolve it, thereby obtaining a clear and transparent solution, and then the reaction solution was sealed and shaken in a constant temperature shaker (25 ℃, 70rpm) to react. Samples were taken at intervals and the reaction results were checked by SDS-PAGE, and after 48h the reaction was stopped with a conversion of about 50% -80%.

After a Click reaction, 30KD PEG-rhIL2-L36, 30KD PEG-rhIL2-M39, 30KD PEG-rhIL2-L40, 30KD PEG-rhIL2-M46, 30KD PEG-rhIL2-P47, 30KD PEG-rhIL2-L63, 30KD PEG-rhIL2-L66, 30KD PEG-rhIL2-E67, 30KD PEG-rhIL2-L70 and 30KD PEG-rhIL2-A73 coupling reaction liquid are obtained.

Example 5: purification of site-directed modified 30KD PEG-rhIL2

The chromatography medium was CM Sepharose FF (available from GE Co.), the equilibration buffer was 20mM sodium acetate buffer (pH3.0), and the elution buffer was 20mM sodium acetate buffer-1M NaCl (pH 4.5). Adjusting the pH of each coupling reaction solution obtained in the embodiment 4 to 3.0 +/-0.2 by using an equilibrium buffer solution, enabling the conductivity to be less than or equal to 2.5ms/CM, loading the coupling reaction solution to a CM Sepharose FF chromatographic column, performing linear elution (0-30% of elution buffer solution and 15CV) by using the elution buffer solution, and collecting target protein components to obtain a target protein sample with the purity of more than 95%. The SEC-HPLC profiles of the purified conjugates 30KD PEG-rhIL2-L36, 30KD PEG-rhIL2-M39, 30KD PEG-rhIL2-L40, 30KD PEG-rhIL2-M46, 30KD PEG-rhIL2-L63 and 30KD PEG-rhIL2-A73 and the conjugates before rhIL2-L36, rhIL2-M39, rhIL2-L40, rhIL2-M46, rhIL2-L63 and rhIL2-A73 are shown in figure 4.

In FIG. 4, rhIL2-V91 is a mutant rhIL-2 which is obtained by constructing rhIL2-V91-BL21 according to the method of example 1, and then expressing and purifying the mutant rhIL-2 according to example 2; the primers used in the construction process were as follows:

V91-F：5’-GATTTCCAATATCAACTAGATTGTTCTGGAACTGA-3’(SEQ ID NO:30)

V91-R：5’-TCAGTTCCAGAACAATCTAGTTGATATTGGAAATC-3’(SEQ ID NO:31)

30KD PEG-rhIL2-V91 is the conjugate after coupling rhIL2-V91 with 30KD PEG Click reaction and purifying according to the above steps according to the method of example 4.

Example 6: in vitro activity evaluation of site-directed modified 30KD PEG-rhIL2

1. Binding Activity

The SPR method is adopted to detect the binding activity of each fixed-point modified 30KD PEG-rhIL2 with rhIL-2R alpha and rhIL-2R beta, and the reference substance is recombinant human IL-2(Solarbio, P00020-1mg) of Solarbio company. The method comprises the following steps: first, the surface of a CM5 chip (available from GE, cat # BR-1008-30) was activated for 420 seconds with 400mM 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 100mM N-hydroxysuccinimide at a flow rate of 10. mu.L/min. Next, the murine anti-His antibody (purchased from GE, cat. No. 28-9950-56) was diluted to 50. mu.g/mL with a fixative reagent (10mM sodium acetate, pH4.5) and injected into the experimental channel (FC4) at a flow rate of 10. mu.L/min for about 420s in a fixed amount of about 9000 to 14000 RU. Finally, the chip was blocked with 1M ethanolamine at 10. mu.L/min for 420 s. Two channels were prepared in total, and the reference channel (FC3) and the experimental channel (FC4) were subjected to the same operation as described above to prepare a CM5 chip. Samples of stock solutions of rhIL-2R α (from Acro, Cat. ILA-H52H9) and rhIL-2R β (from Acro, Cat. CD2-H5221) were diluted to 4 μ g/mL with running reagent (containing 10mM HEPES, 150mM NaCl, 3mM EDTA, 0.005% Tween-20, pH adjusted to 7.4) and injected into the experimental channel (FC4) at a flow rate of 10 μ L/min to approximately 500 RU. The reference channel (FC3) does not require capture of ligand and serves as a control channel to monitor binding of analyte to the chip and allows subtraction upon fitting. Different 30KD PEG-rhIL2 samples with corresponding running reagent for 2 times of dilution, and set the sample zero concentration negative control, can monitor the running reagent and chip binding conditions, and can be fitted when deducting. The diluted sample was injected into the experimental channel and the reference channel sequentially at a flow rate of 30. mu.L/min for 60s binding time and 90s dissociation time. After each concentration analysis, the chip was regenerated for 60s with glycine HCl pH 1.5 at a flow rate of 30 μ L/min, washing off the ligand and undissociated analyte. For the next concentration analysis, the experimental channel needs to recapture the same amount of ligand. KD values for each protein molecule were calculated using Biacore 8K (GE Healthcare Life Sciences) analysis software, and zero concentration of reference channel (FC3) and analyte was used for background subtraction, and the results are shown in table 2. The binding affinities of each sample to rhIL-2R α are ranked as: the reference substance is more than 30KD PEG-rhIL2-V91 more than 30KD PEG-rhIL2-L40 more than 30KD PEG-rhIL 2-M46; the binding affinities of each sample to rhIL-2R β are ranked as: the reference substance is more than 30KD PEG-rhIL2-L40 more than 30KD PEG-rhIL2-M46 more than 30KD PEG-rhIL 2-V91. The ratio of β/α binding affinities for each sample was: reference product: 0.064, 30kD PEG-rhIL 2-L40: 1.137,30kD PEG-rhIL 2-M46: 5.231, control 30KD PEG-rhIL 2-V91: 0.068.

TABLE 2 detection of binding activity of reference substance, site-directed modified 30K PEG-rhIL2 with rhIL-2R alpha and rhIL-2R beta

2. Cell activity (STAT5 phosphorylation assay)

The method adopts two cell strains, wherein a mouse CTLL-2 cell is a cell strain containing IL-2R alpha beta gamma, a human YT cell is a cell strain containing IL-2R beta gamma, and rhIL-2 is combined with the IL-2R on the cell surface to activate a JAK-STAT signal channel. The modification sites of the samples are different, the relative activity to the two cells is different, the higher the relative activity value of YT cells/CTLL-2 cells is, the better the effect of the samples on promoting the immune function is; on the contrary, the better the effect of suppressing the immune function.

The specific process is as follows: mouse CTLL-2 cells (purchased from American Type Culture Collection) and human YT cells were cultured in respective media (CTLL-2 cell Culture medium: RPMI 1640+ 10% FBS +400IU/mL rhIL-2, 2mM L-glutamine, 1mM sodium pyruvate; YT cell Culture medium: RPMI 1640+ 10% FBS +1mM Non-Essential Amino Acids Solution (purchased from Gibco, Cat. No. 11140050)) at 37 ℃ in 5% carbon dioxide to a sufficient amount, starved for 4h before detection, and then adjusted to a cell density of 1 × 10 ⁶ cells/mL are ready for use. Each site-directed modified 30KD PEG-rhIL2 sample was diluted in gradient at a total concentration of 6, cells were stimulated at 37 ℃ for 10 minutes, and then lysed for western blot experiments using pSTAT5 antibody (purchased from CST under Cat. No. 9359L) and β -actin (purchased from CST under N.C.; produced byCatalog No. 8457S), detecting the protein amount of pSTAT5 and beta-actin in cell lysate, and calculating EC50 according to the gray scale result of pSTAT 5/beta-actin and the concentration of the sample. The results are shown in Table 3. The results show that rhIL2 with unnatural amino acid inserted into sites M39, L40, M46 and L63 and coupled with 30KD PEG achieves the original design requirements, while the effects of sites L36 and A73 are not expected.

TABLE 3 results of STAT5 phosphorylation experiments on CTLL-2 and YT cells by mutant rhIL-2 and 30KD PEG-rhIL2

Example 7: pharmacokinetic Studies in mice

Female C57 mice (SPF grade, Zhejiang Weitongli laboratory animal technology Co., Ltd.) were used for the experiment, and after random grouping, the positive control drugs of quanji rhIL-2 (purchased from Shandong quanghong pharmaceutical Co., Ltd.) and 30KD PEG-rhIL2-M46 were administered by single intravenous injection at 1mg/kg, respectively, and quanji was collected at 0.0833h, 0.5h, 1h, 4h, 8h, 16h and 24h after administration (n is 5); 30KD PEG-rhIL2-M46 was administered at 0.0833h, 0.5h, 1h, 4h, 8h, 16h, 24h, 48h, 72h, 96h, 120h, 144h (n ═ 5) after administration. The blood sample is placed at room temperature for 15min, and centrifuged at 6800g/min for 6min to obtain serum. Blood serum concentration was analyzed as follows:

50 μ L of 1 μ g/mL Anti-IL-2 antibody (purchased from abcam, cat. No. ab9618) working solution was added to each well of a high adsorption 96-well plate using a multi-channel pipette and incubated overnight at 2-8 ℃. Discard the well, wash 3 times with 300. mu.L/well of 1 XPBST (0.05% Tween-20), blot clean on absorbent paper and blot. Casein blocking solution (available from Thermo under catalog No. 37528) was added at 200. mu.L/well using a multi-channel pipette and allowed to stand at room temperature for 90 min. Discard the well liquid, wash 3 times with 300. mu.L/well of 1 XPBST, blot on absorbent paper and blot. The quanji rhIL-2, 30KD PEG-rhIL2-M46 and the serum sample to be detected are diluted by mouse serum gradient and transferred into a micropore plate according to 50 mu L/hole. Standing at room temperature for 120 min. Discard the well, wash 3 times with 300. mu.L/well of 1 XPBST, and blot on absorbent paper. Multipurpose use50 μ L of a 0.25 μ g/mL IL-2Monoclonal Antibody (BG5), Biotin (ex Invitrogen, Cat. No. M600B) working solution was added to each well of the pipette and allowed to stand at room temperature for 60 min. Discard the well liquid, wash 3 times with 300. mu.L/well 1 XPBST, pat clean on absorbent paper and suck dry. Will Pierce ^TM High Sensitivity Streptavidin-HRP (available from Thermo, catalog No. 21130) was diluted 4000-fold with casein blocking solution, distributed into microwell plates at 50. mu.L/well, and allowed to stand at room temperature for 60 min. Discard the well liquid, wash 4 times with 300. mu.L/well 1 XPBST, pat clean on absorbent paper and suck dry. Add 50. mu.L of 1-Step to each well using a multichannel pipettor ^TM Turbo TMB-ELISA Substrate Solution (available from Thermo, catalog No. 34022), 25min later 2M sulfuric acid stop Solution was added and absorbance at 450nm and 650nm was read on a microplate reader (available from Perkin Elmer, model EnSight). Four-parameter fitting is carried out through Dazdaq Ltd.WorkOut 1.5 analysis software, the concentrations and units of the corresponding quanzhi rhIL-2 and 30K PEG-rhIL2-M46 are input, a curve is fitted for each of the concentrations and the units, and the blood concentration of the serum to be detected is calculated. According to the non-atrioventricular model (statistical moment parameter) of the DAS software, the average half-life t of the fudge rhIL-2 and the 30KD PEG-rhIL2-M46 is calculated _1/2 Respectively 0.796h and 17.7 h. The pharmacokinetic parameters of SpongrIL-2 and 30KD PEG-rhIL2-M46 are shown in Table 4.

TABLE 4 pharmacokinetic parameters of quackie rhIL-2 and 30KD PEG-rhIL2-M46 (n ═ 5)

Example 8: pharmacodynamic study in mice-antitumor Activity

The experiment adopts female C57BL/6 mouse (SPF grade, Zhejiang Weitonglihua laboratory animal technology Co., Ltd.) of 8 × 10 ⁴ 0.1 mL/mouse B16-F10 (purchased from ATCC, Cat. No. CRL-6475) cell suspension was inoculated subcutaneously into the right dorsal part of the mouse to reach a tumor volume of 50mm ³ On the left and right, 10 mice were randomized in each group and given vehicle (1 × PBS), control drugs of quanzhi rhIL-2, 30KD PEG-rhIL2-L40, 30KD PEG-rhIL2-M46 and 30KD PEG-rhIL2-L63 (administration volume 10mL/kg), respectively.Animal body weights and tumor volumes were measured 3 times a week during the experiment, and the administration mode and the experimental results are shown in table 5 and fig. 5. The Relative Tumor Volume (RTV) is calculated as: v _t /V ₀ In which V is ₀ Tumor volume in groups, V _t For the tumor volume at each measurement. The relative tumor proliferation rate T/C (%) is calculated as: t is _RTV /C _RTV X 100% where T _RTV To the treatment group RTV, C _RTV Negative control group RTV.

The results show that the Sponger rhIL-2, 30KD PEG-rhIL2-L40, 30KD PEG-rhIL2-M46 and 30KD PEG-rhIL2-L63 have obvious inhibiting effect on mouse melanoma B16-F10 allograft tumors compared with a solvent group. Compared with Quanqi rhIL-2, the mean volume values of 30KD PEG-rhIL2-L40, 30KD PEG-rhIL2-M46 and 30KD PEG-rhIL2-L63 are all smaller than that of Quanqi rhIL-2, and have significant difference.

TABLE 5 mode of administration of test substances in the B16-F10 model of allograft tumors and Effect on tumor volume in animals

Note: *: p < 0.05; **: p < 0.01; ***: p <0.001, compared to vehicle group.

When the average tumor volume of each group of mice tested exceeds 2000mm ³ Mice were sacrificed at time and tumors dissected and photographed as shown in figure 6.

Example 9: pharmacodynamic study in mice-expression of immune cell populations

The experiment adopts female C57BL/6 mouse (SPF grade, Zhejiang Weitonglihua laboratory animal technology Co., Ltd.) of 8 × 10 ⁴ 0.1 mL/mouse B16-F10 cell suspension was inoculated subcutaneously into the right dorsal part of the mouse to achieve a tumor volume of 100mm ³ On the left and right, the mice were randomly divided into groups of 6 mice, and each test substance was administered (administration volume: 10mL/kg) as shown in Table 6. At Day5 and Day7, 3 animals per group were selected and samples of blood and tumor tissue from each group were collected and subjected to flow-testing for changes in the proportion of each immune cell population, Day5 resultsAs shown in fig. 7.

TABLE 6 modes of administration of test substances in the B16-F10 model of allograft tumors

Compared with Quanqi rhIL-2, the proportion of CD8+ T cells of 30KD PEG-rhIL2-L40, 30KD PEG-rhIL2-M46 and 30KD PEG-rhIL2-L63 is obviously increased, the proportion of Treg cells is obviously reduced, the proportion of CD8+ T/Treg is obviously increased, and the drug effect of well enhancing immunity is shown.

Sequence listing

<110> Zhejiang New code biomedical Co Ltd

<120> human interleukin 2-polyethylene glycol conjugate, preparation method and application thereof

<130> DSP1F202708JW

<160> 31

<170> SIPOSequenceListing 1.0

<210> 1

<211> 153

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 1

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu

20 25 30

Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile

35 40 45

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

50 55 60

Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu

65 70 75 80

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

85 90 95

Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile

100 105 110

Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala

115 120 125

Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe

130 135 140

Cys Gln Ser Ile Ile Ser Thr Leu Thr

145 150

<210> 2

<211> 133

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 2

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 3

<211> 134

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu

1 5 10 15

His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser

115 120 125

Ile Ile Ser Thr Leu Thr

130

<210> 4

<211> 405

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atggcgccta catccagctc gaccaaaaag acgcagctgc aactggaaca cctgctcctg 60

gatctgcaaa tgattcttaa cggtatcaat aactacaaaa atccgaaact gacccgtatg 120

ctgacgttta aattctatat gccaaagaaa gcgaccgagc tgaaacatct gcagtgcctg 180

gaagaggaac tgaaaccgct ggaggaagtt ttgaacctgg ctcagtctaa aaactttcac 240

ctgcgccctc gtgacctgat ttccaatatc aacgtgattg ttctggaact gaaaggctct 300

gaaaccacgt ttatgtgcga gtacgccgat gaaaccgcca cgattgtgga atttctgaat 360

cgctggatca ccttctccca gagcattatt agcacgctga cctaa 405

<210> 5

<211> 5398

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc 60

ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc 120

ccattcgcca atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta 180

gaggccccaa ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc 240

tttcgggctt tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagttaggtc 300

agcgtgctaa taatgctctg ggagaaggtg atccagcgat tcagaaattc cacaatcgtg 360

gcggtttcat cggcgtactc gcacataaac gtggtttcag agcctttcag ttccagaaca 420

atcacgttga tattggaaat caggtcacga gggcgcaggt gaaagttttt agactgagcc 480

aggttcaaaa cttcctccag cggtttcagt tcctcttcca ggcactgcag atgtttcagc 540

tcggtcgctt tctttggcat atagaattta aacgtcagca tacgggtcag tttcggattt 600

ttgtagttat tgataccgtt aagaatcatt tgcagatcca ggagcaggtg ttccagttgc 660

agctgcgtct ttttcgtgga gctagaggtc ggggccatat gtatatctcc ttcttaaagt 720

taaacaaaat tatttctaga ggggaattgt tatccgctca caattcccct atagtgagtc 780

gtattaattt cgcgggatcg agatctcgat cctctacgcc ggacgcatcg tggccggcat 840

caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg atggggaaga 900

tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc 960

cgtggccggg ggactgttgg gcgccatctc cttgcatgca ccattccttg cggcggcggt 1020

gctcaacggc ctcaacctac tactgggctg cttcctaatg caggagtcgc ataagggaga 1080

gcgtcgagat cccggacacc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc 1140

gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg 1200

cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg 1260

tttctgcgaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa 1320

ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag 1380

tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact 1440

gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc 1500

ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga 1560

ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt 1620

ctctgaccag acacccatca acagtattat tttctcccat gaagacggta cgcgactggg 1680

cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag 1740

ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat 1800

tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat 1860

gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc 1920

gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt 1980

agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa 2040

acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg 2100

ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct 2160

ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 2220

acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc 2280

tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac ccagtcagct 2340

ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgaagctt ctatcgtcgc 2400

cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctg 2460

ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc 2520

ggtattcgga atcttgcacg ccctcgctca agccttcgtc actggtcccg ccaccaaacg 2580

tttcggcgag aagcaggcca ttatcgccgg catggcggcc ccacgggtgc gcatgatcgt 2640

gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga 2700

atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc 2760

aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc 2820

gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac 2880

acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg 2940

ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc 3000

atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa 3060

cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat 3120

ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc 3180

ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag 3240

ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac 3300

ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc 3360

gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta 3420

tactggctta actatgcggc atcagagcag attgtactga gagtgcacca tatatgcggt 3480

gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct 3540

cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 3600

aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 3660

aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 3720

tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 3780

caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 3840

cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 3900

ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 3960

gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 4020

agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 4080

gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 4140

acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 4200

gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 4260

gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 4320

cggggtctga cgctcagtgg aacgaaaact cacgttaagg gactgcagct atttgtttat 4380

ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 4440

aataatattg aaaaaggaag agtatgaggg aagcggtgat cgccgaagta tcgactcaac 4500

tatcagaggt agttggcgtc atcgagcgcc atctcgaacc gacgttgctg gccgtacatt 4560

tgtacggctc cgcagtggat ggcggcctga agccacacag tgatattgat ttgctggtta 4620

cggtgaccgt aaggcttgat gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa 4680

cttcggcttc ccctggagag agcgagattc tccgcgctgt agaagtcacc attgttgtgc 4740

acgacgacat cattccgtgg cgttatccag ctaagcgcga actgcaattt ggagaatggc 4800

agcgcaatga cattcttgca ggtatcttcg agccagccac gatcgacatt gatctggcta 4860

tcttgctgac aaaagcaaga gaacatagcg ttgccttggt aggtccagcg gcggaggaac 4920

tctttgatcc ggttcctgaa caggatctat ttgaggcgct aaatgaaacc ttaacgctat 4980

ggaactcgcc gcccgactgg gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca 5040

tttggtacag cgcagtaacc ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa 5100

tggagcgcct gccggcccag tatcagcccg tcatacttga agctagacag gcttatcttg 5160

gacaagaaga agatcgcttg gcctcgcgcg cagatcagtt ggaagaattt gtccactacg 5220

tgaaaggcga gatcaccaag gtagtcggca aataatgtct aacaattcgt tcaagccgag 5280

gggccgcaag atccggccac gatgacccgg tcgtcggttc agggcagggt cgttaaatag 5340

ccgcttatgt ctattgctgg tttaccggtt tattgactac cggaagcagt gtgaccgt 5398

<210> 6

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ctacaaaaat ccgaaataga cccgtatgct gacgt 35

<210> 7

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

acgtcagcat acgggtctat ttcggatttt tgtag 35

<210> 8

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tccgaaactg acccgttagc tgacgtttaa attct 35

<210> 9

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

agaatttaaa cgtcagctaa cgggtcagtt tcgga 35

<210> 10

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gaaactgacc cgtatgtaga cgtttaaatt ctata 35

<210> 11

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tatagaattt aaacgtctac atacgggtca gtttc 35

<210> 12

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gacgtttaaa ttctattagc caaagaaagc gaccg 35

<210> 13

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cggtcgcttt ctttggctaa tagaatttaa acgtc 35

<210> 14

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gtttaaattc tatatgtaga agaaagcgac cgagc 35

<210> 15

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gctcggtcgc tttcttctac atatagaatt taaac 35

<210> 16

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gtgcctggaa gaggaataga aaccgctgga ggaag 35

<210> 17

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

cttcctccag cggtttctat tcctcttcca ggcac 35

<210> 18

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

agaggaactg aaaccgtagg aggaagtttt gaacc 35

<210> 19

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ggttcaaaac ttcctcctac ggtttcagtt cctct 35

<210> 20

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ggaactgaaa ccgctgtagg aagttttgaa cctgg 35

<210> 21

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ccaggttcaa aacttcctac agcggtttca gttcc 35

<210> 22

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

accgctggag gaagtttaga acctggctca gtcta 35

<210> 23

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

tagactgagc caggttctaa acttcctcca gcggt 35

<210> 24

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

ggaagttttg aacctgtagc agtctaaaaa ctttc 35

<210> 25

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

gaaagttttt agactgctac aggttcaaaa cttcc 35

<210> 26

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

tgagcggata acaattcccc tctagaaata attttgttta ac 42

<210> 27

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

gatctcagtg gtggtggtgg tggtgctcga g 31

<210> 28

<211> 2997

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

ccttatgcga ctccctgcat tagggagctg ttgacaatta atcatcggct cgtataatgt 60

gtggaattgt gagcggataa caatttcaca aaggaggtca tatggataaa aagcctctga 120

acactctgat ttctgcgacc ggtctgtgga tgtcccgcac cggcaccatc cacaaaatca 180

aacaccatga agttagccgt tccaaaatct acattgaaat ggcttgcggc gatcacctgg 240

ttgtcaacaa ctcccgttct tctcgtaccg ctcgcgcact gcgccaccac aaatatcgca 300

aaacctgcaa acgttgccgt gttagcgatg aagatctgaa caaattcctg accaaagcta 360

acgaggatca gacctccgta aaagtgaagg tagtaagcgc tccgacccgt actaaaaagg 420

ctatgccaaa aagcgtggcc cgtgccccga aacctctgga aaacaccgag gcggctcagg 480

ctcaaccatc cggttctaaa ttttctccgg cgatcccagt gtccacccaa gaatctgttt 540

ccgtaccagc aagcgtgtct accagcatta gcagcatttc taccggtgct accgcttctg 600

cgctggtaaa aggtaacact aacccgatta ctagcatgtc tgcaccggta caggcaagcg 660

ccccagctct gactaaatcc cagacggacc gtctggaggt gctgctgaac ccaaaggatg 720

aaatctctct gaacagcggc aagcctttcc gtgagctgga aagcgagctg ctgtctcgtc 780

gtaaaaagga tctgcaacag atctacgctg aggaacgcga gaactatctg ggtaagctgg 840

agcgcgaaat tactcgcttc ttcgtggatc gcggtttcct ggagatcaaa tctccgattc 900

tgattccgct ggaatacatt gaacgtatgg gcatcgataa tgataccgaa ctgtctaaac 960

agatcttccg tgtggataaa aacttctgtc tgcgtccgat gctggccccg aacctgtaca 1020

actatctgcg taaactggac cgtgccctgc cggacccgat caaaattttc gagatcggtc 1080

cttgctaccg taaagagtcc gacggtaaag agcacctgga agaattcacc atgctgaact 1140

tttgccagat gggtagcggt tgcacgcgtg aaaacctgga atccattatc accgacttcc 1200

tgaatcacct gggtatcgat ttcaaaattg ttggtgacag ctgtatggtg tacggcgata 1260

cgctggatgt tatgcacggc gatctggagc tgtcttccgc agtagtgggc ccaatcccgc 1320

tggatcgtga gtggggtatc gacaaacctt ggatcggtgc gggttttggt ctggagcgtc 1380

tgctgaaagt aaaacacgac ttcaagaaca tcaaacgtgc tgcacgttcc gagtcctatt 1440

acaatggtat ttctactaac ctgtaactag tgtctccagc ttggctgttt tggcggatga 1500

gagaagattt tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag 1560

aatttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg 1620

agcactgcag cggctaacta agcggcctgc tgactttctc gccgatcaaa aggcattttg 1680

ctattaaggg attgacgagg gcgtatctgc gcagtaagat gcgccccgca ttggaaacct 1740

gatcatgtag atcgaatgga ctctaaatcc gttcagccgg gttagattcc cggggtttcc 1800

gccaaattcg aaaagcctgc tcaacgagca ggcttttttg catctcgagc agctcagggt 1860

cgaatttgct ttcgaatttc tgccattcat ccgcttatta tcacttattc aggcgtagca 1920

accaggcgtt taagggcacc aataactgcc ttaaaaaaat tacgccccgc cctgccactc 1980

atcgcagtac tgttgtaatt cattaagcat tctgccgaca tggaagccat cacaaacggc 2040

atgatgaacc tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat aatatttgcc 2100

catggtgaaa acgggggcga agaagttgtc catattggcc acgtttaaat caaaactggt 2160

gaaactcacc cagggattgg ctgagacgaa aaacatattc tcaataaacc ctttagggaa 2220

ataggccagg ttttcaccgt aacacgccac atcttgcgaa tatatgtgta gaaactgccg 2280

gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt tcagtttgct catggaaaac 2340

ggtgtaacaa gggtgaacac tatcccatat caccagctca ccgtctttca ttgccatacg 2400

gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt 2460

gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt 2520

ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga 2580

tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga 2640

aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt 2700

ggaacctctt acgtgccgat caacgtctca ttttcgccaa aagttggccc agggcttccc 2760

ggtatcaaca gggacaccag gatttattta ttctgcgaag tgatcttccg tcacaggtat 2820

ttattcggcg caaagtgcgt cgggtgatgc tgccaactta ctgatttagt gtatgatggt 2880

gtttttgagg tgctccagtg gcttctgttt ctatcagctg tccctcctgt tcagctactg 2940

acggggtggt gcgtaacggc aaaagcaccg ccggacatca gcgcgcgctg cggacac 2997

<210> 29

<211> 2153

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

cggacatcag cgcgcgctgc ggacacatac aaagttaccc acagattccg tggataagca 60

ggggactaac atgtgaggca aaacagcagg gccgcgccgg tggcgttttt ccataggctc 120

cgccctcctg ccagagttca cataaacaga cgcttttccg gtgcatctgt gggagccgtg 180

aggctcaacc atgaatctga cagtacgggc gaaacccgac aggacttaaa gatccccacc 240

gtttccggcg ggtcgctccc tcttgcgctc tcctgttccg accctgccgt ttaccggata 300

cctgttccgc ctttctccct tacgggaagt gtggcgcttt ctcatagctc acacactggt 360

atctcggctc ggtgtaggtc gttcgctcca agctgggctg taagcaagaa ctccccgttc 420

agcccgactg ctgcgcctta tccggtaact gttcacttga gtccaacccg gaaaagcacg 480

gtaaaacgcc actggcagca gccattggta actgggagtt cgcagaggat ttgtttagct 540

aaacacgcgg ttgctcttga agtgtgcgcc aaagtccggc tacactggaa ggacagattt 600

ggttgctgtg ctctgcgaaa gccagttacc acggttaagc agttccccaa ctgacttaac 660

cttcgatcaa accacctccc caggtggttt tttcgtttac agggcaaaag attacgcgca 720

gaaaaaaagg atctcaagaa gatcctttga tcttttctac tgaaccgctc tagatttcag 780

tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat aagttgtaat 840

tctcatgtta gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa 900

gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg 960

ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 1020

acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg 1080

agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt 1140

ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat 1200

aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca 1260

gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca 1320

tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg 1380

cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat 1440

gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga 1500

tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg 1560

agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat 1620

tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca 1680

gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc 1740

ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg 1800

ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca 1860

acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca 1920

tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc 1980

gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt 2040

tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg 2100

ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctc 2153

<210> 30

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

gatttccaat atcaactaga ttgttctgga actga 35

<210> 31

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

tcagttccag aacaatctag ttgatattgg aaatc 35

Claims (8)

1. A human interleukin 2-polyethylene glycol conjugate comprising recombinant human interleukin2 comprising one unnatural amino acid and PEG conjugated to the unnatural amino acid;

wherein the position of the unnatural amino acid is selected from the group consisting of positions corresponding to L40, M46, and L63 of SEQ ID NO. 2;

the recombinant human interleukin2 is protein shown as SEQ ID NO. 3;

the unnatural amino acid is N epsilon-2-azido ethoxycarbonyl-L-lysine, and the structural formula is as follows:

2. the human interleukin 2-polyethylene glycol conjugate of claim 1, wherein: the molecular weight of the PEG is 20KD to 50 KD.

3. The human interleukin 2-polyethylene glycol conjugate according to claim 1 or 2, wherein: the PEG is coupled to the unnatural amino acid via a linker, which is BCN.

4. The preparation process of human interleukin-2-polyglycol conjugate includes the following steps: preparing recombinant human interleukin2 containing an unnatural amino acid by a codon expansion technology or a chemical synthesis mode, and coupling PEG on the unnatural amino acid;

wherein the position of the unnatural amino acid is selected from the group consisting of a position corresponding to one of L40, M46, and L63 of SEQ ID NO 2;

the recombinant human interleukin2 is protein shown as SEQ ID NO. 3;

the unnatural amino acid is N epsilon-2-azidoethoxycarbonyl-L-lysine, and the structural formula is as follows:

5. the method of claim 4, wherein: the codon expansion technique was carried out in E.coli.

6. The method according to claim 4 or 5, characterized in that: the molecular weight of the PEG is 20KD to 50 KD.

7. The method according to claim 4 or 5, characterized in that: the PEG is coupled on the side chain of the unnatural amino acid through a Click reaction by a linker, and the linker is BCN.

8. Use of the human interleukin 2-polyethylene glycol conjugate of any one of claims 1 to 3 for the preparation of a medicament for promoting immunity, treating malignant solid tumors and hematological tumors, and/or expanding CD8+ T cells.

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