Conjugate of human interleukin-2 and polyethylene glycol and use thereof

Abstract

The present invention provides a conjugate of human interleukin-2 and polyethylene glycol and its use. The conjugate of human interleukin-2 and polyethylene glycol comprises recombinant human interleukin-2 containing at least one unnatural amino acid and PEG coupled to the at least one unnatural amino acid, the unnatural amino acid being a carbonyl-terminated compound having a structure represented by formula (I) or an enantiomer thereof, and the PEG is coupled to the at least one unnatural amino acid by forming an oxime bond between the carbonyl-terminated group and the hydroxylamine-terminated PEG. The conjugate of human interleukin-2 and polyethylene glycol may be used alone or in combination with other antitumor drugs for the treatment of diseases such as solid tumors and blood tumors. [Formula 1] TIFF2024530031000053.tif26170

Description

[Technical Field]

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to a conjugate of human interleukin-2 and polyethylene glycol and uses thereof. [Background technology]

[0002] Interleukin-2 (IL-2) is an important immunoregulatory factor produced by activated type I helper T lymphocytes (Th1), formerly known as T cell growth factor. Its primary biological function is to stimulate and inhibit apoptosis in a dual manner in T cells (CD4 + and CD8 + Interleukin-2 promotes the growth, proliferation, and differentiation of immune cells (including T cells) and promotes the further secretion of cellular factors. In addition, interleukin-2 stimulates the proliferation of NK cells, enhances NK cell killing activity, produces cellular factors, induces the production of LAK cells, and promotes the proliferation and antibody secretion of B cells. Therefore, interleukin-2 plays an important role in the body's immune response and antiviral infection (Gaffena SL, Cytokine 28:109e123, 2004).

[0003] IL-2 has been widely used clinically since its first discovery by Morgan et al. in 1976. In 1991, rhIL-2 (product name: Aldesleukin) manufactured by Cetus, USA, was approved for sale by the FDA and is widely used in the treatment of malignant tumors such as renal cell carcinoma, malignant melanoma, and malignant lymphoma (Proleukin instruction manual), and is also potentially effective as an adjuvant treatment for hepatitis B and hepatitis C infections (Tomova R. et al., Anticancer Research, 29:5241-5244, 2009). To date, more than 10 companies in China have produced and sold biological products based on recombinant human interleukin-2. These products are widely used in the treatment of malignant tumors, including renal cell carcinoma, melanoma, breast cancer, bladder cancer, liver cancer, rectal cancer, lymphatic cancer, and lung cancer; for controlling cancerous pleural and ascites effusion; for enhancing the biological immune function of tumor patients after surgery, radiotherapy, and chemotherapy; for improving the cellular immune function and anti-infective ability of patients with congenital or acquired immunodeficiency syndromes; and for treating various autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, and sicca syndrome. In addition, they also have a certain therapeutic effect on certain viral, bacillary, and intracellular parasitic bacterial infections, such as hepatitis B, leprosy, pulmonary tuberculosis, and Monilia albicans infection.

[0004] The human IL-2 precursor consists of 153 amino acid residues. Upon secretion from cells, its signal peptide (containing 20 amino acid residues) is cleaved to produce the mature IL-2 of 133 amino acids, with a relative molecular weight of 15.4 kD. IL-2 activates effector cells by binding to the IL-2 receptor (IL-2R) on the cell surface. It has now been discovered that there are three types of IL-2 receptors: IL-2Rα, IL-2Rβ, and IL-2Rγ. According to these three receptors, the functional complex of IL-2Rαβγ, a heteromeric glycoprotein with high affinity, binds to the IL-2Rαβγ (Kd=10 -11 IL-2Rβ and IL-2Rγ can form a receptor complex with intermediate affinity, IL-2Rβγ (Kd=10 -9mol / L), and this receptor complex is biologically active after activation by IL-2, whereas the IL-2Rα subunit is a receptor form with low affinity (Kd=10 -8 mol / L), and cannot transmit intracellular proliferation signals upon binding to IL-2. Although IL-2Rα and IL-2Rβ can form a high-affinity receptor complex, this receptor complex has no biological function and cannot be activated by IL-2. Different cells, allogeneic cells at different developmental stages, and disease states express different types of IL-2 receptors to different degrees, resulting in the formation of different receptor complexes. For example, LAK cell precursors express high levels of IL-2Rβγ complexes, which can attack and degrade cancer cells after activation by IL-2. Macrophages also express IL-2Rβγ complexes and can be activated by IL-2. Monocytes express large amounts of IL-2Rγ and small amounts of IL-2Rβ. NK cells express large amounts of IL-2Rβ and small amounts of IL-2Rγ, which can form intermediate-affinity IL-2Rβγ receptors, which bind to high concentrations of IL-2 to form trimers and activate monocytes or NK cells. Activated T cells express IL-2Rα, IL-2Rβ, and IL-2Rγ on their surface. Excessive IL-2Rα favors polymerization with IL-2Rβ, which then binds to IL-2Rγ, forming a high-affinity receptor-IL-2 complex, further transmitting signals and inducing a cell proliferation response. After the cell response, IL-2Rα, IL-2Rβ, and IL-2Rγ dissociate, rendering the cells insensitive to IL-2. Human tumor cells also express IL-2 receptors, and IL-2 binding to the receptor complex on tumor cells can inhibit tumor cell proliferation. Because different cancer cells express unique IL-2 receptor complexes, modifying the structure of IL-2 so that it interacts only with the corresponding receptor on the specific tumor surface can target cancer cells and reduce damage to normal cells.

[0005] Based on this research theory, many researchers have made various modifications to IL-2, enhancing its binding to specific receptor complexes (e.g., IL-2Rβγ complexes) on the surface of anti-tumor-related effector cells, activating tumor-killing-related cell types, and at the same time minimizing its binding to the IL-2Rαβγ complexes highly expressed on the surface of negative immune regulatory T cells (e.g., Treg cells). This can improve efficacy while reducing drug side effects. Improvements to existing IL-2 include designing specific IL-2 mutant proteins (e.g., Aron ML et al., Nature, 484(7395):529-33, 2012), changing the amino acid sequence of the binding site with IL-2Rα, IL-2Rβ, or IL-2Rγ so that the spatial structure is unfavorable for interaction with IL-2Rα or strengthens the interaction with IL-2Rβ or IL-2Rγ; designing IL-2 / anti-IL-2 antibody (or IL-2 receptor) complexes (e.g., Jared EL et al., J Immunother Cancer, 8(1):e000673, 2020), utilizing the specificity of anti-IL-2 antibodies to mask the binding site with IL-2R, thereby changing the function of IL-2 and extending its half-life in vivo; and expressing IL-2 in a fusion with Fc or human serum albumin (HSA) to extend the half-life of IL-2 in vivo (e.g., Jianyong Lei et al., Protein Expression and Purification, 84(1):154-160, 2012), combining site-specific mutagenesis with HAS / Fc fusion to simultaneously alter function and extend half-life (e.g., CN112724259A), and non-site-specific PEGylation of IL-2 to extend its half-life (e.g., Deborah HC et al., Clin Cancer Res, 22(3):680-90, 2016).

[0006] The above-mentioned studies on improving IL-2 have the following drawbacks.

[0007] 1. Simple site-specific mutation of amino acids can weaken the binding ability to IL-2Rα or strengthen the binding ability to IL-2Rβ or IL-2Rγ, but it cannot effectively extend the half-life of the molecule. In addition, the mutation products are likely to cause immunogenic reactions in the body, reduce the biological activity of the products, and have a high risk of toxicity.

[0008] 2. Simple fusion expression (e.g., fusion with Fc or HSA) or modification of IL-2 can extend the half-life of the molecule, but no clear advantage has been found compared to unmodified IL-2 in actual use. Fusion expression can only modify the fusion molecule at the N-terminus or C-terminus of the target protein, and it is not possible to optimize the modification site.

[0009] 3. IL-2 that combines some site-specific mutations and fusion expression has not shown any particular advantages in practical applications (e.g., Rodrigo Vazquez-Lombardi et al., Nat Commun, 8:15373, 2017).

[0010] 4. PEGylated IL-2 obtained by conventional non-site-specific coupling has not shown any particular advantages in practical applications. Although the combination of PEGylated IL-2 with weak IL-2Rα binding ability has achieved some success, the characteristics of non-site-specific coupling technology have drawbacks such as difficulty in controlling production technology and quality, complex molecular structure, and complex mechanism of action.

[0011] In response to the limitations of the above-mentioned IL-2 improvements, some researchers have developed IL-2 to which PEG is site-specifically coupled using codon extension technology (e.g., WO2019028419A1). The unnatural amino acid used was Lys-azido, and its structural formula is as follows:

[0012] [ka] The azide structure (-N3) at the end of Lys-azido can be used to form alkyne-containing structures (e.g., BCN, i.e. [ka] (See, for example, Chinese Patent CN103153927B) and can be chemically linked to carrier drugs (e.g., PEG) modified with PEG to form conjugates with high specificity. However, these coupling and chemical modification methods require the introduction of costly alkyne structures, and acceptable drug-antibody coupling rates can only be achieved when a large equivalent amount is used, which increases the corresponding production costs, complicates the production process, and imposes strict production conditions.

[0013] Considering the above, there are still many shortcomings in the improvement of IL-2 at present, and further research is required. Summary of the Invention

[0014] In order to overcome the drawbacks in the improvement of IL-2 existing in the prior art, one object of the present invention is to provide a conjugate of human interleukin-2 and polyethylene glycol, specifically, using one or more natural amino acids in the amino acid sequence of recombinant human interleukin-2 by site-directed mutagenesis of a series of novel unnatural amino acids, polyethylene glycol (PEG) is site-specifically coupled to the unnatural amino acid through an oximation reaction to form the conjugate of the present invention.

[0015] Another object of the present invention is to provide uses of the conjugate of human interleukin-2 and polyethylene glycol, which can be used to treat diseases such as malignant solid tumors and hematological tumors.

[0016] In a first aspect of the present invention, there is provided a conjugate of human interleukin-2 and polyethylene glycol, comprising recombinant human interleukin-2 comprising at least one unnatural amino acid, and PEG coupled to the at least one unnatural amino acid, the unnatural amino acid is a carbonyl-terminated compound having a structure represented by Formula (I) or an enantiomer thereof, and PEG is coupled to the at least one unnatural amino acid by forming an oxime bond between the carbonyl-terminated group and a hydroxylamine-terminated (i.e., aminooxy) PEG; [ka] In the formula, X and Z each independently represent a substituted or unsubstituted C0 to C20 linear or branched alkylene group, in which one or more -CH2- groups can be optionally replaced by one or more selected from -O-, -S-, -NH-, -C(O)-, and -S(O)-; Y represents -C(O)-, -S(O)-, or -CH2-; A represents a substituted or unsubstituted C6 to C20 aryl group; When X, Z, and A each independently represent a substituted group, the substituent is one or more selected from a hydroxyl group, a mercapto group, a halogen, a nitro group, a cyano group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an acyl group, an amido group, a carboxyl group, an ester group, an amino group, a sulfonyl group, a sulfinyl group, a cycloalkyl group, a heterocyclo group, an aryl group, and a heteroaryl group.

[0017] As shown in Example 9, the inventors of the present invention found that in addition to the skyrocketing cost and the complicated production process, the azide structure (-N3) at the end of Lys-azido is easily reduced to an amino structure (-NH2) when inserted into recombinant human interleukin-2 (as shown in formula (1)), which makes the coupling activity easily lost, and therefore the yield of the coupling complex during the production process is reduced due to this reduction reaction.

[0018] [ka] The unnatural amino acids of the present invention have a terminal carbonyl group introduced as an active reactive group, which not only provides a novel structure and is easy to produce, but also allows for mild coupling conditions, reduces production costs, and is less likely to undergo structural changes when inserted into proteins, resulting in less loss of reactivity. The unnatural amino acids of the present invention also contain an aryl group bonded to the carbonyl group, which increases the stability of the resulting conjugates and makes them less susceptible to decomposition even under low pH conditions. Furthermore, the unnatural amino acids of the present invention also contain an alkylene group of a predetermined chain length, which provides good compound flexibility and makes it easier to form a variety of conjugates.

[0019] In the conjugates provided herein, the unnatural amino acid contained in the recombinant human interleukin-2 contains a carbonyl terminal group, while the PEG used contains a hydroxylamine terminal group and has the structure represented by formula (II).

[0020] [ka] The carbonyl group in the unnatural amino acid can undergo an oximation reaction with the hydroxylamine group in PEG to form an oxime bond, the structure of which is shown in formula (III), thereby coupling PEG to the unnatural amino acid.

[0021] [ka] In formula (III), D' represents a residue in which the carbonyl group of the unnatural amino acid has been removed from the recombinant human interleukin-2 of the present invention, and D" represents PEG in which the "NH2-O-" terminal group has been removed.

[0022] Compared with wild-type IL-2 or commercially available recombinant human IL-2, the conjugates provided by the present invention have reduced binding ability to IL-2Rα, maintained binding activity to IL-2Rβγ, and inhibited CD8 activation by the IL-2Rβγ complex. + Activation of T cells into CD8 + The conjugates provided by the present invention can maintain the activation and proliferation ability of T cells while suppressing the proliferation of Treg cells, have a significantly extended half-life in the body, and can effectively promote immunity and suppress tumors. In addition, the conjugates provided by the present invention have a higher coupling rate and better stability.

[0023] In some preferred embodiments according to the present invention, the recombinant human interleukin-2 is the protein set forth in SEQ ID NO:3 or a functionally active fragment thereof.

[0024] In some preferred embodiments according to the present invention, the position of the at least one unnatural amino acid in the recombinant human interleukin-2 containing at least one unnatural amino acid is one or more of: P34, K35, T37, R38, L40, T41, F42, K43, F44, Y45, E61, E62, K64, P65, E67, E68, N71, L72, and Y107, relative to SEQ ID NO:2. In some more preferred embodiments according to the present invention, the position of the at least one unnatural amino acid in the recombinant human interleukin-2 containing at least one unnatural amino acid is one or more of: K35, T41, K43, Y45, E61, K64, and P65, relative to SEQ ID NO:2.

[0025] In the unnatural amino acids of the present invention, "C0-Cn" includes C0-C1, C0-C2, ...C0-Cn, and when it refers to C0, it means that this group is not present, and the C atoms at both ends are directly linked to form a bond. For example, the "C0-C6" group refers to a group containing 0 to 6 carbon atoms in the corresponding moiety, i.e., no group is present, or the group contains 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms. The "C6-C10" group refers to a group containing 6 to 10 carbon atoms in the corresponding moiety, i.e., 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, or 10 carbon atoms.

[0026] In the context of unnatural amino acids of the invention, an "aryl group" refers to a carbocyclic aromatic system containing one or two rings, where such rings may be linked together in a fused fashion. An "aryl group" includes monocyclic or bicyclic aryl groups, such as phenyl, naphthyl, and tetrahydronaphthyl aromatic groups. The aryl group is preferably a C6-C10 aryl group, more preferably a phenyl group or a naphthyl group, and most preferably a phenyl group.

[0027] In some preferred embodiments according to the present invention, the substituent is one or more selected from a hydroxyl group, a mercapto group, a halogen, a nitro group, a cyano group, a C1 to C6 alkyl group, a C1 to C6 alkoxy group, an acyl group, an amido group, a carboxyl group, an ester group, an amino group, a sulfonyl group, a sulfinyl group, a C3 to C8 cycloalkyl group, a C3 to C8 heterocyclo group, a C6 to C20 aryl group, and a C4 to C10 heteroaryl group.

[0028] In some preferred embodiments according to the present invention, X and Z each independently represent a C0-C10 linear or branched alkylene group, in which one or more -CH2- groups may be optionally substituted with one or more selected from -O-, -S-, and -NH-. In some more preferred embodiments according to the present invention, X and Z each independently represent a C0-C6 linear alkylene group, in which one or more -CH2- groups may be optionally substituted with one or more selected from -O-, -S-, and -NH-. In some more preferred embodiments according to the present invention, X and Z are not simultaneously C0 alkylene groups; in other words, it is not permitted for both an X group and a Z group to be absent.

[0029] In some preferred embodiments according to the present invention, A represents a substituted or unsubstituted C6 to C10 aryl group, and more preferably A represents a substituted or unsubstituted phenyl group or naphthyl group.

[0030] In some preferred embodiments according to the present invention, the unnatural amino acid is a compound having a structure represented by formula (I-1): [ka] In the formula, X, Z and A are each independently defined in any one of the above technical solutions.

[0031] In some preferred embodiments according to the present invention, the unnatural amino acid is a compound having a structure represented by formula (I-2): [ka] In the formula, X is defined as in any one of the above technical solutions; R1 and R2 each independently represent hydrogen, a hydroxyl group, a mercapto group, halogen, a nitro group, a cyano group, a C1 to C6 alkyl group, a C1 to C6 alkoxy group, an acyl group, an amido group, a carboxyl group, an ester group, an amino group, a sulfonyl group, a sulfinyl group, a C3 to C8 cycloalkyl group, a C3 to C8 heterocyclo group, a C6 to C20 aryl group, or a C4 to C10 heteroaryl group.

[0032] In some relatively preferred embodiments according to the present invention, the unnatural amino acid is a compound having a structure represented by Formula (I-3), Formula (I-4), Formula (I-5), or Formula (I-6): [ka] [ka] [ka] [ka] In the formula, X' represents a C0-C6 linear alkylene group, more preferably a C0-C4 linear alkylene group, in which one or more -CH2- groups can be optionally substituted with -O- and / or -NH-; The R1 and R2 are each independently defined in any one of the above technical solutions.

[0033] The unnatural amino acids of the present invention include optically pure enantiomers and racemates.

[0034] In some more preferred embodiments according to the present invention, an unnatural amino acid according to the present invention is a compound having a structure represented by any one of the following: [ka]

[0035] In some preferred embodiments according to the present invention, the molecular weight of the hydroxylamine ("NH-O-") terminated PEG of the present invention is 10-100 KD, including, but not limited to, about 10 KD, about 20 KD, about 30 KD, about 40 KD, about 50 KD, about 60 KD, about 70 KD, about 80 KD, about 90 KD, about 100 KD, or any combination of molecular weight ranges. In some more preferred embodiments according to the present invention, the molecular weight of the "NH-O-" terminated PEG is 20-50 KD.

[0036] In some preferred embodiments provided herein, recombinant human interleukin-2 containing at least one unnatural amino acid of the present invention is produced by codon extension technology or chemical synthesis. In some more preferred embodiments provided herein, recombinant human interleukin-2 containing at least one unnatural amino acid of the present invention is produced by codon extension technology, where the codon extension technology is achieved in E. coli.

[0037] The codon extension technology of the present invention specifically includes the following steps: comparing a nucleic acid molecule of a mutant recombinant human interleukin-2 with a nucleic acid molecule encoding recombinant human interleukin-2, the difference between the mutated nucleic acid molecule is that the codon for an amino acid at at least one of positions P34, K35, T37, R38, L40, T41, F42, K43, F44, Y45, E61, E62, K64, P65, E67, E68, N71, L72, and Y107 corresponding to SEQ ID NO: 2 is mutated to an amber codon UAG; and expressing the mutated nucleic acid molecule in E. coli, the carbonyl-containing lysine analog of the present invention (e.g., NBOK) is incorporated into the expressed recombinant human interleukin-2 by an orthogonal tRNA synthetase / tRNA pair. The operating principle of the above codon extension system is as follows: tRNA Pyl cannot utilize the host cell lysyl-tRNA enzyme and Pyl can only be acylated by RS, and tRNA PylRS is tRNA Pyl It can only acylate one tRNA and cannot acylate other tRNAs. Pyl and tRNA Pyl The orthogonal tRNAs are capable of acylating the corresponding unnatural amino acids with RSs. Pyl There is only RS, and tRNA Pyl RS can only acylate such tRNAs and cannot acylate other tRNAs. The codon extension system allows the carbonyl-containing lysine analog to correspond to the amber codon UAG (i.e., tRNA Pyl The corresponding codon is UAG), thereby allowing the site-specific introduction of carbonyl-group-containing lysine analogs into IL-2.

[0038] In some preferred embodiments of the present invention, the step of expressing the mutated recombinant human interleukin-2 in E. coli further comprises a protein denaturation step, a protein refolding step, and an ultrafiltration step.

[0039] In some preferred embodiments of the present invention, the oximation reaction between an unnatural amino acid in recombinant human interleukin-2 and a hydroxylamine-containing PEG involves the following steps: Prior to the oximation reaction, the pH of the mutated recombinant human interleukin-2 solution is adjusted to about 3.5-4.5 (e.g., about 4.0 using 2 M acetic acid), the protein concentration is adjusted to 0.5-1.5 mg / ml (e.g., using 20 mM sodium acetate buffer, pH 4.0), and the protein is added at a predetermined molar ratio (e.g., protein:PEG = 1:15), dissolved thoroughly, sealed, and reacted on a thermostatic shaker for 30-60 hours. After the reaction, the coupling status can be analyzed by standard analytical methods (e.g., RP-HPLC).

[0040] In some preferred embodiments of the present invention, the reaction solution after the oximation reaction (i.e., coupling reaction) contains some unreacted IL-2, impurity proteins, and unreacted PEG. Therefore, further purification can be performed using cation exchange chromatography. For example, the purification process was as follows: using Capto MMC as the chromatography medium, 20 mM sodium citrate buffer (pH = 3.0) as the equilibration buffer, and 20 mM sodium citrate buffer-1 M NaCl (pH = 7.8) as the elution buffer. The coupling reaction solution was adjusted to pH 3.0 ± 0.2 with the equilibration buffer and adjusted to a conductivity of 5 mS / cm or less. The sample was then loaded onto the Capto MMC and linearly eluted with the elution buffer (0-100% elution, 20 CV) to collect the target protein component. This purification process yielded a target protein sample with a purity of approximately 95%.

[0041] In a second aspect of the present invention, the conjugate of human interleukin-2 and polyethylene glycol according to any one of the above technical solutions is used as an immunostimulatory drug, a drug for the prevention and / or treatment of solid tumors (especially malignant solid tumors) and hematological tumors, and / or a drug for the treatment of CD8 + There is provided a use in the manufacture of a medicament for T cell proliferation.

[0042] In some preferred embodiments according to the present invention, the solid tumor is bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, renal cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer.

[0043] In some preferred embodiments according to the present invention, the hematological malignancies are selected from the group consisting of 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, Hodgkin's lymphoma, hyperplasia, and leukemia. Malignant B-cell non-Hodgkin's lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-cell lymphoblastic lymphoma, B-cell juvenile lymphocytic 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 lymphomatous granuloma.

[0044] In a third aspect of the present invention, there is provided a reagent kit comprising the conjugate of human interleukin-2 and polyethylene glycol according to any one of the above technical solutions.

[0045] In a fourth aspect of the present invention, there is provided a method for preventing and / or treating solid tumors (particularly malignant solid tumors) or hematological tumors, comprising the step of administering to a patient in need thereof a therapeutically effective amount of a conjugate of human interleukin-2 and polyethylene glycol described in any one of the above technical solutions.

[0046] In some preferred embodiments according to the present invention, the solid tumor is bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, renal cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer.

[0047] In some preferred embodiments according to the present invention, the hematological malignancies are selected from the group consisting of 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, Hodgkin's lymphoma, hyperplasia, and leukemia. Malignant B-cell non-Hodgkin's lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-cell lymphoblastic lymphoma, B-cell juvenile lymphocytic 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 lymphomatous granuloma.

[0048] In some preferred embodiments according to the present invention, the conjugate of human interleukin-2 and polyethylene glycol may be administered alone or in combination with one or more other anti-tumor agents.

[0049] In a fifth aspect of the present invention, there is provided a method for promoting immune function, comprising administering an effective amount of a conjugate of human interleukin-2 and polyethylene glycol described in any one of the above technical solutions to a patient in need thereof.

[0050] In some preferred embodiments according to the present invention, the promotion of immune function is promotion of the immune function of human interleukin-2.

[0051] In a sixth aspect of the present invention, there is provided a method for treating CD8, which comprises administering an effective amount of a conjugate of human interleukin-2 and polyethylene glycol according to any one of the above technical solutions to a patient in need thereof. + A method for expanding T cells is provided.

[0052] The technical solution provided in the present invention has the following advantages:

[0053] (1) The novel unnatural amino acids of the present invention can be introduced into specific sites using codon extension technology, thereby enabling precise site-specific coupling of PEG and interleukin-2, overcoming the drawback of conventional random coupling, which is unable to achieve precise coupling, and also resulting in highly uniform products.

[0054] (2) The unnatural amino acids of the present invention are lysine analogs containing a terminal carbonyl group and an aryl group in their structure. Compared with common azide-containing lysine analogs (e.g., Lys-azido), they are easier to prepare, safer, less likely to be inactivated when inserted into proteins, have a higher conjugation rate with PEG, and the resulting conjugates have better stability. They also have a coupling efficiency of over 95% even after denaturation and renaturation of recombinant protein inclusion bodies.

[0055] (3) By designing and screening the mutation site, the present invention provides a mutation site of interleukin-2 that can reduce the IL-2Rα binding activity while maintaining the IL-2Rβ and IL-2Rγ binding activities relatively unchanged, thereby enabling the site-specifically modified human interleukin-2 / polyethylene glycol conjugate to inhibit CD8 in the tumor microenvironment. + It specifically promotes T cell proliferation, while CD4 + It has no significant effect on T cell proliferation, making it advantageous for tumor immunotherapy.

[0056] (4) The conjugate of the present invention achieves an extension of the half-life of IL-2 in the body by coupling with PEG, thereby reducing the frequency of administration to patients. [Brief explanation of the drawings]

[0057] [Figure 1] FIG. 1 is a schematic diagram of the expression plasmid NB1S3-WT. [Figure 2] FIG. 1 is a schematic diagram of the helper plasmid NB1W. [Figure 3]FIG. 1 shows an SDS-PAGE electrophoresis diagram of the fermentation products obtained by adding the unnatural amino acid NBOK to the rhIL-2-expressing strains with different mutation sites obtained in Example 2 (arrows indicate the position of the target product strip). Each lane in the diagram represents the following: Lane 1: rhIL2-K35-BL21 cells administered with NBOK were disrupted and precipitated after centrifugation; Lane 2: rhIL2-T41-BL21 cells administered with NBOK were disrupted and precipitated after centrifugation; Lane 3: rhIL2-K43-BL21 cells administered with NBOK were disrupted. Lane 4: rhIL2-Y45-BL21 cells administered with NBOK were disrupted and the precipitate was obtained after centrifugation. Lane 5: rhIL2-E61-BL21 cells administered with NBOK were disrupted and the precipitate was obtained after centrifugation. Lane 6: rhIL2-K64-BL21 cells administered with NBOK were disrupted and the precipitate was obtained after centrifugation. Lane 7: rhIL2-P65-BL21 cells administered with NBOK were disrupted and the precipitate was obtained after centrifugation. Lane 8: Inclusion bodies obtained by washing the precipitate of rhIL2-Y45-BL21 cells administered with NBOK. [Figure 4A] Figure 4 shows the RP-HPLC spectra before and after the coupling reaction of mutant rhIL-2 with PEG in Example 3. Figure 4A shows each mutant rhIL-2 before the coupling reaction, and the main peak of the target protein is located at approximately 22.5 minutes in the figure. [Figure 4B] FIG. 4B shows each mutant rhIL-2 after the coupling reaction (both PEGs are 30 KD PEG), and the main peak of the target protein is at about 21 minutes in the figure. [Figure 5] 1 shows RP-HPLC spectra of the conjugate 30KD PEG-rhIL2-Y45 in Example 4 before and after column chromatography. [Figure 6] FIG. 10 shows the mass spectrum of rhGH-V91 in Example 9. [Figure 7] FIG. 1 shows SDS-PAGE electrophoresis of recombinant IL-2 cells containing different unnatural amino acids obtained in Example 10. [Figure 8]1 shows SDS-PAGE electrophoresis of mutant IL-2 crude proteins containing different unnatural amino acids obtained in Example 10. FIG. [Figure 9A] Figure 9 shows the RP-HPLC spectra of mutant rhIL-2 (rhIL2-T41NPAK) in Example 11 before and after the coupling reaction with PEG. Figure 9A shows mutant rhIL-2 before the coupling reaction, and the main peak of the target protein is located at approximately 22,380 minutes in the figure. [Figure 9B] FIG. 9B shows the mutant rhIL-2 after the coupling reaction (PEG is 30KD PEG), with the main peak of the target protein at approximately 21.175 minutes. [Figure 10] 1 is an RP-HPLC spectrum of the purified conjugate in Example 12. [Figure 11A] FIG. 13 shows the results of Western blot experiments of pSTAT5 and β-actin proteins in CTLL2 cells stimulated with different concentrations of reference rhIL2 at 37°C for 10 minutes in Example 13, and an EC50 graph fitted based on the grayscale results. [Figure 11B] FIG. 13 shows the results of a Western blot experiment of pSTAT5 and β-actin proteins in CTLL2 cells stimulated with different concentrations of T41NPAK mutant IL-2 (i.e., rhIL2-T41 NPAK) and 30KD PEG-rhIL2-T41NPAK at 37°C for 10 minutes in Example 13, and an EC50 graph fitted based on the grayscale results. [Figure 11C] FIG. 13 shows the results of Western blot experiments of pSTAT5 and β-actin proteins in YT cells stimulated with different concentrations of reference rhIL2 at 37°C for 10 minutes in Example 13, and an EC50 graph fitted based on the grayscale results. [Figure 11D]FIG. 13 shows the results of a Western blot experiment of pSTAT5 and β-actin proteins in YT cells stimulated with different concentrations of T41NPAK mutant IL-2 (i.e., rhIL2-T41NPAK) and 30KD PEG-rhIL2-T41NPAK at 37°C for 10 minutes in Example 13, and an EC50 graph fitted based on the grayscale results. DETAILED DESCRIPTION OF THE INVENTION

[0058] The technical solution of the present invention will be further described in detail below with reference to specific examples.

[0059] The reagents or raw materials used in the preparations and examples of the present invention are commercially available products unless otherwise specified, and the experimental methods used in the present invention are conventional methods in this field unless otherwise specified.

[0060] Human YT cells are those disclosed in the literature "Yodoi, J. et al. (1985). TCGF (IL2)-receptor inducing factor(s). I. Regulation of IL2 receptor on a natural killer-like cell line (YT cells). Journal of Immunology, 134(3), 1623-1630" and are publicly available from Zhejiang Xinma Biopharmaceutical Co., Ltd.

[0061] Production Example 1: Production of unnatural amino acid NBOK The structural formula of NBOK is shown below. [ka]

[0062] The reaction process was as follows: [ka]

[0063] The manufacturing process includes the following steps:

[0064] a) A reaction flask was charged with p-methylacetophenone (4.0 mL, 30.0 mmol), DCM (50.0 mL), NBS (6.41 g, 36.0 mmol), and BPO (0.05 g, 0.3 mmol). The resulting mixture was refluxed at 80°C for 24 hours, and then the vessel was cooled in ice water to precipitate a solid. The solid was filtered off, washed three times with saturated Na2CO3, and extracted three times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude product 1-1 (5.46 g, 85% yield). The crude product 1-1 was used directly in the next step without further purification.

[0065] b) Product 1-1 (2.73 g, 12.80 mmol) was added to a reaction flask, and the solvents dioxane (40 mL) and water (40 mL) were added. Calcium carbonate (7.68 g, 76.8 mmol) was then added. The resulting mixture was refluxed at 105°C for 24 hours, then cooled to room temperature, filtered off the solid, extracted three times with DCM, and the organic phases were combined and concentrated under reduced pressure. After purification by column chromatography (eluent: PE:EA = 3:1), product 1-2 (1.80 g, yield 94%) was obtained.

[0066] c) A two-necked reaction flask was charged with p-nitrophenyl chloroformate (2.90 g, 14.4 mmol) and DCM (10.0 mL). The mixture was cooled to 0°C, and product 1-2 (1.80 g, 12.0 mmol) and pyridine (1.2 mL, 14.4 mmol) were added. After stirring at room temperature for 18 hours, saturated sodium carbonate solution (10 mL) was added to the reaction mixture, which was extracted three times with DCM (50 mL). The organic phases were combined, washed twice with water, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent: PE:EA = 5:1) to obtain product 1-3 (3.14 g, 83% yield).

[0067] d) Product 1-3 (1.26 g, 4.0 mmol) and Fmoc-Lys-OH hydrochloride (1.40 g, 3.33 mmol) were added to a reaction flask, and the solvent dioxane (15 mL) and water (5 mL) were added, followed by triethylamine (1.2 mL, 8.3 mmol). The resulting mixture was reacted at room temperature for 24 hours, and then an appropriate amount of 1 M HCl solution was added. The mixture was extracted with DCM and concentrated under reduced pressure to obtain crude product 1-4, which was used directly in the next step without further purification.

[0068] e) In a reaction flask, product 1-4 (1.10 g, 0.19 mmol) was dissolved in DCM (10 mL), diethylamine (5.0 mL) was added, and the mixture was allowed to react at room temperature for 6 hours. The product was precipitated, filtered, and triturated three times with DCM to obtain the target product 1-5 (817 mg, 63% yield for two steps).

[0069] 1 H-NMR (400MHz, heavy water) δ8.04(d,J=8.4Hz,2H),7.55(d,J=8.0Hz,2H),5.21(s,2H),3.74(t,J=6.0Hz, 1H),3.17(t,J=6.4Hz,2H),2.70(s,3H),1.95-1.83(m,2H),1.62-1.52(m,2H),1.47-1.35(m,2H).

[0070] Production Example 2: Production of unnatural amino acid NPAK The structural formula of NPAK is shown below. [ka]

[0071] The reaction process was as follows: [ka]

[0072] The manufacturing process includes the following steps:

[0073] a) p-Chloroacetophenone (1.00 g, 6.47 mmol) was added to a reaction flask, and under a nitrogen atmosphere, diethyl malonate (6.84 g, 47.70 mmol), KHCO3 (0.97 g, 9.70 mmol), and K2CO3 (1.34 g, 9.70 mmol) were added, followed by Pd(dba)2 (0.019 g, 0.030 mmol) and P(t-Bu)3HBF4 (0.021 g, 0.071 mmol). After the addition was complete, the nitrogen protection was replaced, and the temperature was raised to 160°C and the reaction was carried out for 40 hours. When the reaction was complete as determined by TLC, water (30 mL) was added to the reaction mixture, which was then extracted with EA three times. The combined organic phases were washed twice with water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at 0-5°C to obtain a colorless, transparent liquid. This was purified by column chromatography (eluent: PE:EA = 10:1) to obtain product 2-1 (0.80 g, yield 60%).

[0074] b) A reaction flask was charged with LiOH (0.30 g, 11.64 mmol) and water (5.0 mL), ethanol (10 mL), and product 2-1 (0.80 g, 3.88 mmol). The mixture was stirred at room temperature for 2 hours. When the reaction was complete as determined by TLC, the reaction mixture was adjusted to pH 1-2 by adding 2M HCl solution, extracted three times with EA, and the organic phases were combined, washed twice with water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain product 2-2 (0.5 g, 72% yield).

[0075] c) Product 2-2 (0.20 g, 1.12 mmol) was added to a reaction flask, followed by N-hydroxysuccinimide (NHS, 0.19 g, 1.68 mmol), DIPEA (0.07 g, 0.56 mmol), and DCM (2.0 mL) in that order. After cooling to 0-5 °C, a solution of DCC (0.23 g, 1.12 mmol) and DCM (2.0 mL) was added and the mixture was allowed to react for 2 hours. The mixture was then warmed to room temperature and stirred overnight. When the reaction was complete as determined by TLC, the mixture was filtered and washed with DCM. The mother liquor was concentrated under reduced pressure and purified by column chromatography (eluent: PE:EA = 5:1) to obtain product 2-3 (0.19 g, 62% yield).

[0076] d) Product 2-3 (0.10 g, 0.36 mmol) was added to a reaction flask, followed by triethylamine (0.04 g, 0.36 mmol), Fmoc-Lys-OH hydrochloride (0.13 g, 0.36 mmol), dioxane (2.0 mL), and water (2.0 mL) in that order, and the mixture was stirred at room temperature for 18 hours. When the reaction was complete as determined by TLC, the mixture was concentrated under reduced pressure, extracted three times with EA, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: DCM:MeOH = 15:1) to obtain oily liquid product 2-4 (0.03 g, 61% yield).

[0077] e) Product 2-4 (0.08 g, 0.15 mmol), DCM (1.0 mL), and piperidine (0.04 g, 0.47 mmol) were added to a reaction flask and stirred at room temperature for 3 hours. After the reaction was complete as determined by TLC, the mixture was concentrated under reduced pressure, triturated with petroleum ether (5 mL) for 1 hour, filtered, and the resulting filter cake was triturated with petroleum ether (5 mL) for 1 hour, filtered, and triturated with ethanol four times to remove residual piperidine, finally obtaining an off-white solid 2-5 (0.02 g, 43% yield).

[0078] 1 H-NMR (400MHz, heavy water) δ7.85(d,J=8.2Hz,2H),7.33(d,J=8.2Hz,2H),3.94(t,J=6.3Hz,1H),3.56(s,2 H),3.12(t,J=6.8Hz,2H),2.54(s,3H),1.80-1.70(m,2H),1.54-1.45(m,2H),1.40-1.224(m,2H).

[0079] Production Example 3: Production of unnatural amino acid NBPK The structural formula of NBPK is shown below. [ka]

[0080] The reaction process was as follows: [ka]

[0081] The manufacturing process includes the following steps:

[0082] a) A reaction flask was charged with p-methylacetophenone (4.0 mL, 30.0 mmol), DCM (50.0 mL), NBS (6.41 g, 36.0 mmol), and BPO (0.05 g, 0.3 mmol). The resulting mixture was refluxed at 80 °C for 24 h. When the reaction was complete as determined by TLC, the vessel was cooled in ice water to precipitate a solid. The solid was filtered off, washed three times with saturated Na2CO3, and extracted three times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude product 3-1 (5.46 g, 85% yield). The crude product 3-1 was used directly in the next step without further purification.

[0083] b) To a reaction flask, NaH (0.58 g, 14.64 mmol, 60%) and dry THF (20 mL) were added. Under ice bath cooling, ethylene glycol (6.7 mL, 122.0 mmol) was slowly added and stirred at room temperature for 1 hour. Then, product 3-1 (2.60 g, 12.2 mmol) was added and heated to reflux at 70 °C for 48 hours to complete the reaction. Under ice bath cooling, saturated NH4Cl was slowly added dropwise to quench the NaH, washed with water, and extracted three times with EtOAc. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent: PE:EA = 2:1) to obtain product 3-2 (1.39 g, 59% yield).

[0084] c) Product 3-2 (1.39 g, 7.2 mmol) was added to a reaction flask, and DCM (10 mL) was added. Under ice bath cooling, p-nitrophenyl chloroformate (1.74 g, 8.64 mmol) and pyridine (0.7 mL, 8.64 mmol) were added, and the mixture was stirred at room temperature for 18 hours. When the reaction was complete as determined by TLC, the mixture was washed with water and extracted three times with EtOAc. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent: PE:EA = 3:1) to obtain product 3-3 (2.27 g, 88% yield).

[0085] d) Product 3-3 (2.27 g, 6.32 mmol) was added to a reaction flask, followed by the solvents dioxane (16 mL) and water (4 mL). Fmoc-Lys-OH hydrochloride (2.13 g, 5.27 mmol) and triethylamine (1.85 mL, 13.2 mmol) were added, and the mixture was stirred at room temperature for 18 hours to complete the reaction. The pH was adjusted to approximately 2 with 1 M HCl, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude product 3-4, which was used directly in the next step.

[0086] e) In a reaction flask, the product 3-4 obtained in the previous step was dissolved in DCM (10 mL), and diethylamine (5 mL) was added and reacted at room temperature for 6 hours. When the reaction was complete as determined by TLC, the mixture was concentrated under reduced pressure and purified by column chromatography (eluent: DCM:MeOH:HO = 40:10:1) to obtain the white solid product 3-5 (0.95 g, 49% yield for two steps).

[0087] 1 H-NMR (400MHz, heavy water) δ8.04(d,J=8.0Hz,2H),7.56(d,J=8.0Hz,2H),4.72(s,2H),4.25(s,2H),3.81(s,2H),3.7 4(t,J=6.0Hz,1H),3.16-3.08(m,2H),2.70(s,3H),1.97-1.79(m,2H),1.60-1.48(m,2H),1.48-1.35(m,2H).

[0088] Production Example 4: Production of unnatural amino acid NPOK The structural formula of NPOK is shown below. [ka]

[0089] The reaction process was as follows: [ka]

[0090] The manufacturing process includes the following steps:

[0091] a) Triphosgene (BTC, 2.18 g, 7.35 mmol) was added to a reaction flask, followed by the addition of THF (10.0 mL). Under ice bath cooling, p-hydroxyacetophenone (2.0 g, 14.7 mmol) and pyridine (1.5 mL, 17.64 mmol) were added, and the resulting mixture was reacted at room temperature for 24 hours. When the reaction was complete as determined by TLC, an appropriate amount of water was added, followed by extraction with EtOAc three times, and the organic phases were combined. After drying with anhydrous sodium sulfate, filtration, and concentration under reduced pressure, crude product 4-1 (1.20 g) was obtained, which was used directly in the next step.

[0092] b) Boc-lysine (1.1 g, 5.0 mmol) was added to a reaction flask, followed by DCM (10.0 mL), product 4-1 (1.20 g), and triethylamine (2 mL, 15 mmol). After stirring at room temperature for 24 hours, the reaction was confirmed by TLC. The pH was adjusted to slightly acidic by adding 1 M HCl in small amounts, and the mixture was extracted three times with DCM. The organic phases were combined. The mixture was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent: DCM:MeOH = 5:1) to obtain product 4-2 (1.70 g, 84% yield).

[0093] c) Product 4-2 (1.70 g, 4.2 mmol) was added to a reaction flask, followed by the addition of DCM (5 mL) and trifluoroacetic acid (5 mL). The resulting mixture was allowed to react at room temperature for 1 hour. When the reaction was complete as determined by TLC, the mixture was concentrated under reduced pressure and purified by column chromatography (eluent: DCM:MeOH:HO = 40:10:1) to obtain product 4-3 (1.19 g, 92% yield).

[0094] 1 H-NMR (400MHz, heavy water) δ8.09(d,J=8.6Hz,2H),7.31(d,J=8.6Hz,2H),3.78(t,J=6.0Hz,1H),3. 27(t,J=6.8Hz,2H),2.70(s,3H),1.98-1.87(m,2H),1.72-1.60(m,2H),1.55-1.43(m,2H).

[0095] Production Example 5: Production of unnatural amino acid NBGK The structural formula of NBGK is shown below. [ka]

[0096] The reaction process was as follows: [ka]

[0097] The manufacturing process includes the following steps:

[0098] a) A reaction flask was charged with p-methylacetophenone (8.0 mL, 60.0 mmol), DCM (80.0 mL), NBS (12.82 g, 72.0 mmol), and BPO (145 mg, 0.6 mmol). The resulting mixture was refluxed at 90 °C for 24 h. When the reaction was complete as determined by TLC, the vessel was cooled in ice water to precipitate a solid. The solid was filtered off, washed three times with saturated Na2CO3, extracted three times with DCM, and the organic phases were combined. After drying with anhydrous sodium sulfate, filtration, and concentration under reduced pressure, crude product 5-1 (11.12 g, 87% yield) was obtained. The crude product 5-1 was used directly in the next step without further purification.

[0099] b) In a reaction flask, 4 Å MS (14 g) and LiOH (1.45 g, 34.54 mmol) were dissolved in DMF (70 mL) and stirred at room temperature for 20 minutes. Glycine methyl ester hydrochloride (2.0 g, 15.7 mmol) was added, and the mixture was stirred for another 45 minutes. Product 5-1 (4.0 g, 18.8 mmol) was added, and the mixture was stirred at room temperature for 18 hours. When the reaction was complete as determined by TLC, the solid was filtered off, and the resulting filter cake was washed with EA. The resulting filtrate was washed twice with water. After drying over anhydrous sodium sulfate and concentration under reduced pressure, crude product 5-2 was obtained and used directly in the next step.

[0100] c) In a reaction flask, the product 5-2 obtained in the previous step was dissolved in dioxane (20 mL), and 1M NaOH was slowly added dropwise. After reacting for 2 hours, the hydrolysis reaction was completed as determined by TLC to give product 5-3. 20 mL of saturated NaHCO3 was added, followed by the slow addition of Fmoc-OSu dissolved in dioxane (10 mL). The mixture was stirred at room temperature overnight. When the reaction was complete as determined by TLC, the mixture was adjusted to a weak acidity with 1M HCl, extracted with EA, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: DCM:MeOH = 10:1) to give product 5-4 (3.60 g, 89% yield).

[0101] d) Product 5-3 (3.60 g, 8.0 mmol), NBS (1.10 g, 9.6 mmol), EDCI (1.85 g, 9.6 mmol) were added to a reaction flask, and DCM (50 mL) was added. The resulting mixture was reacted at room temperature for 18 hours. When the reaction was complete as determined by TLC, the mixture was washed with water three times, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain product 5-5 (3.20 g, 75% yield).

[0102] e) Product 5-5 (3.20 g, 6.0 mmol) was added to a reaction flask, followed by dioxane (40 mL) and water (10 mL). Fmoc-Lys-OH hydrochloride (3.0 g, 7.2 mmol) and triethylamine (2.0 mL, 15.0 mmol) were added, and the mixture was stirred at room temperature for 18 hours to complete the reaction. The pH was adjusted to approximately 2 with 1 M HCl, extracted with EA, and the combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent: DCM:MeOH:AcOH = 20:1:0.5) to obtain product 5-5 (3.50 g, 75% yield).

[0103] f) In a reaction flask, product 5-5 was dissolved in DCM (20 mL), and diethylamine (20 mL) was added and reacted at room temperature for 6 hours. After the reaction was complete as determined by TLC, the mixture was concentrated under reduced pressure and purified by column chromatography (eluent: DCM:MeOH:HO = 30:10:1) to obtain the final product 5-7 (0.55 g, 37% yield) as a white powder.

[0104] 1 H-NMR (400MHz, heavy water) δ7.98(d,J=8.2Hz,2H),7.50(d,J=8.2Hz,2H),3.84(s,2H),3.71(s,1H),3.31(s ,2H),3.17(t,J=6.9Hz,2H),2.67(s,3H),1.97-1.73(m,2H),1.58-1.45(m,2H),1.44-1.27(m,2H).

[0105] Production Example 6: Production of unnatural amino acid NPOK-2 The structural formula of NPOK-2 is shown below. [ka]

[0106] The reaction process was as follows: [ka]

[0107] The manufacturing process includes the following steps:

[0108] a) p-Acetylphenol (2.05 g, 15.0 mmol) and bromoacetic acid (2.50 g, 18.0 mmol) were added to a reaction flask, followed by an aqueous solution (6 mL) of NaOH (1.20 g, 30 mmol). The resulting mixture was refluxed at 100 °C for 24 hours to complete the reaction. The reaction mixture was cooled to room temperature and acidified with 1 M hydrochloric acid to precipitate a solid. After filtration, a white crude product 6-1 (3.32 g, 113% yield) was obtained. The crude product 6-1 was used directly in the next step without further purification.

[0109] b) In a reaction flask, the crude product 6-1 (3.32 g, 17.0 mmol) obtained in the previous step was dissolved in DCM (50 mL), and NHS (2.35 g, 20.4 mmol) and EDCI (3.90 g, 20.4 mmol) were added. The resulting mixture was stirred at room temperature for 18 hours to allow the reaction to complete. After extraction with DCM, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. After further purification by column chromatography (eluent: DCM:MeOH:AcOH = 20:1:0.5), product 6-2 (1.67 g, 38% yield) was obtained.

[0110] c) Product 6-2 (1.67 g, 5.7 mmol) was added to a reaction flask, followed by dioxane (20 mL) and water (50 mL). Fmoc-Lys-OH hydrochloride (1.9 g, 4.8 mmol) and triethylamine (1.7 mL, 12.0 mmol) were added and stirred at room temperature for 18 hours to complete the reaction. The pH was adjusted to approximately 2 with 1 M HCl, extracted with EA, and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting crude product 6-3 was directly dissolved in DCM (10 mL) and diethylamine (5 mL) was added. The resulting mixture was stirred at room temperature for 18 hours to complete the reaction. After concentration under reduced pressure and purification by column chromatography (eluent: DCM:MeOH:HO = 40:10:1), the final product 6-4 (709 mg, 39% yield for two steps) was obtained.

[0111] 1 H-NMR (400MHz, heavy water) δ7.91(d,J=8.8Hz,2H),6.98(d,J=8.8Hz,2H),4.60(s,2H),3.59(t,J=6.4Hz, 1H),3.19(t,J=6.8Hz,2H),2.52(s,3H),1.87-1.65(m,2H),1.56-1.40(m,2H),1.35-1.15(m,2H).

[0112] Production Example 7: Production of unnatural amino acid NBGK-2 The structural formula of NBGK-2 is shown below. [ka]

[0113] The reaction process was as follows: [ka]

[0114] The manufacturing process includes the following steps:

[0115] a) A solution of bromoacetic acid (2.10 g, 15.0 mmol) and NaOH (0.80 g, 20 mmol) in water (10 mL) was added to a reaction flask and stirred for 10 minutes. p-Acetanilide (1.40 g, 10.0 mmol) was then added, and the resulting mixture was refluxed at 100 °C for 18 hours to complete the reaction. The reaction mixture was cooled to room temperature, filtered, and washed with water to obtain the white crude product 7-1 (1.30 g, 67% yield), which was used directly in the next step without further purification.

[0116] b) A reaction flask was charged with a solution of product 7-1 (1.30 g, 6.7 mmol) and NaHCO3 (1.70 g, 20.1 mmol) in water (20 mL). Fmoc-OSu (2.80 g, 8.1 mmol) and DMF (20 mL) were then added. The resulting mixture was stirred at 60 °C for 18 hours to allow the reaction to complete. The mixture was cooled to room temperature and extracted with EA. The pH of the remaining aqueous phase was adjusted to about 2 with 1 M hydrochloric acid, and then extracted with EA to obtain an organic phase. Anhydrous sodium sulfate was added for drying, filtered, and concentrated under reduced pressure. Product 7-2 was obtained, and the crude product 7-2 was used in the next step without further purification.

[0117] c) In a reaction flask, the product 7-2 (approximately 6.7 mmol) obtained in the previous step, NHS (0.90 g, 8.0 mmol), and EDCI (1.50 g, 8.0 mmol) were dissolved in DMF (50 mL). The reaction mixture was stirred at room temperature for 24 hours to allow for a complete reaction. Water was added, and the mixture was extracted with DCM to obtain the organic phase. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the product 7-3. The crude product 7-3 was used in the next step without further purification.

[0118] d) To a reaction flask, the product 7-3 (approximately 6.7 mmol) obtained in the previous step, Fmoc-Lys-OH hydrochloride (2.30 g, 5.6 mmol), and triethylamine (2.0 mL, 14.0 mmol) were added. The reaction mixture was stirred at room temperature for 3 hours to allow for a complete reaction. The pH was then adjusted to approximately 2 with 1 M hydrochloric acid and extracted with EA. The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain product 7-4, which was used directly in the next step without further purification.

[0119] e) The product 7-4 obtained in the previous step was added to a reaction flask. The solvents DCM (20 mL) and diethylamine (10 mL) were added. The reaction mixture was stirred at room temperature for 12 hours to allow for a complete reaction. The mixture was first concentrated under reduced pressure, redissolved in acetonitrile (50 mL), and then concentrated under reduced pressure three times to remove excess diethylamine. After triturating twice with DCM, the final product 7-5 (1.65 g, 51% overall yield) was obtained.

[0120] 1 H-NMR (400MHz, heavy water) δ7.71(d,J=8.8Hz,2H),6.51(d,J=8.8Hz,2H),3.80(s,2H),3.53(t,J=6.8Hz, 1H),3.09(t,J=6.8Hz,2H),2.39(s,3H),1.73-1.60(m,2H),1.42-1.33(m,2H),1.25-1.14(m,2H).

[0121] Example 1: Construction of an expression strain expressing recombinant human IL-2 (rhIL-2) with site-specific insertion of unnatural amino acids 1. Obtaining wild-type recombinant human IL-2 expression plasmid NB1S3-WT The precursor protein sequence of Homo sapiens IL-2 (GenBank ID: CAA25292.1), shown as SEQ ID NO:1, was obtained from the National Center for Biotechnology Information (NCBI). This precursor sequence contains a 20-amino acid signal peptide sequence at its N-terminus, which is removed during the processing and maturation of the IL-2 protein molecule. After removal of this signal peptide sequence, the mature Homo sapiens IL-2 protein sequence (SEQ ID NO:2) was obtained. According to a literature report (Liang SM et al., Journal of Biological Chemistry, 261(1):334-337, 1986), the mature Homo sapiens IL-2 protein sequence contains three cysteines, two of which, at positions 58 and 105, form disulfide bonds that are crucial for the biological activity of Homo sapiens IL-2. Cys at position 125 not only does not participate in disulfide bond formation but also interferes with normal disulfide bond formation during the refolding of recombinant Homo sapiens IL-2 protein inclusion bodies. Therefore, mutating Cys at position 125 to serine (Ser) improves refolding efficiency without significantly affecting activity. At the same time, for recombinant protein expression in E. coli, a methionine (Met) must be added to the N-terminus of the protein sequence to initiate translation of the protein, resulting in the mature recombinant human IL-2 protein sequence (SEQ ID NO: 3). The gene encoding recombinant human IL-2 (SEQ ID NO: 4) was obtained through amino acid and codon back-translation and codon optimization, and the recombinant human IL-2 coding gene was then obtained by total gene synthesis. It was then subcloned into the NB1S3 expression vector (this expression vector was adapted from the commercially available vector pET-21a, and its ampicillin resistance genetic screening marker was replaced with the spectinomycin resistance gene amplified by PCR from the commercially available vector pCDF-duet1), to obtain the wild-type recombinant human IL-2 expression plasmid NB1S3-WT (see Figure 1), the sequence of which is shown in SEQ ID NO:5.

[0122] [ka] [ka] [ka]

[0123] 2. Selection of site-specific mutation sites Specific sites were selected as positions P34, K35, T37, R38, L40, T41, F42, K43, F44, Y45, E61, E62, K64, P65, E67, E68, N71, L72, and Y107 of SEQ ID NO:2 for point mutation, and site-specific modification was carried out using this mutant IL-2 as a starting material.

[0124] 3. Primer design for site-directed mutagenesis and construction of mutation vector Primers capable of mutating the codons encoding the amino acids at the following positions in SEQ ID NO: 2 to amber codons were designed, and specific primers are shown in Table 1.

[0125] [Table 1(1)] [Table 1(2)]

[0126] The linearized DNA plasmid obtained by cleaving the NB1S3-WT plasmid with both the DNA restriction endonucleases XbaI and XhoI was used as a template. High-fidelity DNA polymerase (purchased from Takara, product number R045A) was used to amplify the IL-2 genes by PCR amplification and overlap PCR using primers XbaI-F (Table 1) paired with primer R for each site, and primer XhoI-R (Table 1) paired with primer F for each site. Mutant genes in which the amino acid codons at the following sites in IL-2, K35, T41, K43, Y45, E61, K64, and P65, were mutated to amber stop codons were obtained. (For example, PCR amplification was performed using the linearized plasmid NB1S3-WT as a template and XbaI-F and T41-R as a primer pair to obtain an upstream fragment in which the T41 site was mutated.) The downstream fragment mutated at the T41 site was obtained by PCR amplification using the upstream fragment mutated at the T41 site as a primer pair. Subsequently, the upstream fragment mutated at the T41 site and the downstream fragment mutated at the T41 site obtained above were used as templates for overlapping PCR amplification using XbaI-F and XhoI-R as a primer pair to obtain the full-length gene mutated at the T41 site. Furthermore, using a High-Fidelity DNA Integrative Cloning Reagent Kit (purchased from NEB, product number E5520S) and following the manufacturer's instructions, each of the fragments between the XbaI and XhoI enzyme cleavage sites of the NB1S3-WT plasmid was replaced with the obtained mutant gene. Seven expression plasmids, NB1S3-K35, NB1S3-T41, NB1S3-K43, NB1S3-Y45, NB1S3-E61, NB1S3-K64, and NB1S3-P65, were constructed and sequenced, demonstrating successful mutation.

[0127] 4. Construction of site-specifically mutated rhIL-2 expressing strain Using the structure of the pUltra plasmid described in a literature reference (Chatterjee, A. et al., Biochemistry, 52(10), 1828-1837, 2013), we synthesized a gene encoding a tRNA and tRNA synthetase that specifically recognizes a carbonyl-terminated lysine analogue, such as the structure represented by formula (I) of the present invention (the gene encoding the wild-type methanarchaeal pyrrolysine synthase and its corresponding tRNA), as well as a chloramphenicol resistance gene (SEQ ID NO: 46). A DNA fragment (SEQ ID NO: 47) containing the CloDF13 replication origin was amplified from the commercially available vector pCDF-duet1 by PCR amplification, and the two DNA fragments were ligated by subcloning using a high-fidelity DNA integration cloning reagent kit to obtain the helper plasmid NB1W (see FIG. 2; hereinafter, this plasmid will be referred to as the helper plasmid). The screening marker of this plasmid was chloramphenicol resistance. The helper plasmid and the expression plasmid (spectinomycin resistance) obtained in step 3 were co-transformed into E. coli BL21(DE3), and then passaged on spectinomycin-resistance and chloramphenicol-resistance plates to obtain double-positive strains (double-positive strains indicate strains that simultaneously acquired spectinomycin resistance and chloramphenicol resistance): rhIL2-K35-BL21, rhIL2-T41-BL21, rhIL2-K43-BL21, rhIL2-Y45-BL21, rhIL2-E61-BL21, rhIL2-K64-BL21, and rhIL2-P65-BL21.

[0128] The DNA fragment (SEQ ID NO: 46) containing the wild-type methanarchaeal pyrrolysine synthase-encoding gene, the corresponding tRNA-encoding gene, and the chloramphenicol resistance gene was as follows:

[0129] [ka] [ka]

[0130] The DNA fragment (SEQ ID NO: 47) containing the CloDF13 replication origin was as follows:

[0131] [ka]

[0132] Example 2: Expression and purification of rhIL-2 with unnatural amino acids inserted by site-directed mutagenesis 1. Expression of mutant rhIL-2 incorporating unnatural amino acids Each of the seven expression strains obtained in Example 1, rhIL2-K35-BL21, rhIL2-T41-BL21, rhIL2-K43-BL21, rhIL2-Y45-BL21, rhIL2-E61-BL21, rhIL2-K64-BL21, and rhIL2-P65-BL21, was inoculated into LB medium (containing 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, 100 mg / L spectinomycin, and 37.5 mg / L chloramphenicol) and cultured at 37°C for 5 to 8 hours. The OD of the bacterial solution was then measured. 600 Secondary propagation was carried out until the pH reached 2.0±0.2 (the composition of the medium was the same as above) to obtain secondary seed solution.

[0133] The secondary seed solution was inoculated into a fermentation medium and cultured in a 5 L fermentation tank. The culture volume was 2 L, the medium was 2xYT medium (16 g / L yeast extract, 10 g / L tryptone, 5 g / L NaCl), the inoculum amount was 5% (v / v), the culture temperature was 37°C, the pH was controlled at 6.90±0.05, ammonia water or H3PO4 was automatically added as needed, and the DO was controlled at 30% by setting the DO-related rotation speed. 600 When the pH reached 20.0±2.0, IPTG and the unnatural amino acid NBOK obtained in Preparation 1 were added to a final concentration of 1 mM. Simultaneously, 50% glycerol was added at a rate of 0.6±0.1 mL / min. After 5-6 hours of induced expression, the cells were harvested. SDS-PAGE electrophoresis of each strain is shown in Figure 3.

[0134] 2. Separation and extraction of mutant rhIL-2 Each of the collected bacterial cells was resuspended in a buffer solution (25 mM Tris, 6 mM EDTA, 1 mM DTT, pH 8.0), 1% DNA enzyme (1 mg / mL) and 0.5% PMSF were added, and the mixture was homogenized three times at 50-80 MPa using an ultra-high pressure homogenizer. The homogenate was centrifuged at 10,000 rpm for 20 minutes, and the crude inclusion bodies in the lower layer were collected.

[0135] The obtained crude inclusion bodies were washed twice with washing buffer (20 mM Tris-HCl, 100 mM NaCl, 2% Triton X-100, pH 8.0) and then once with ultrapure water to obtain purified inclusion bodies.

[0136] The purified inclusion bodies were dissolved in a denaturing buffer (20 mM Tris-HCl, 100 mM NaCl, 6 M guanidine hydrochloride, 1 mM DTT, pH 8.0) for 30 minutes, and the supernatant was collected at 10,000 rpm. A four-fold volume of renaturing buffer (20 mM Tris-HCl, 100 mM NaCl, pH 8.0) was added to the denatured protein solution, and the solution was thoroughly stirred and allowed to stand for 12 hours. The supernatant was collected at 10,000 rpm and the renatured protein solution was collected.

[0137] The refolded protein solution was concentrated to 1 / 4 of its original volume using an ultrafiltration membrane bag (Millipore, Biomax-5) with a molecular weight cutoff of 5 kDa, and then subjected to liquid exchange with a replacement buffer (20 mM Tris-HCl, pH 8.0) until the electrical conductivity reached approximately 2 mS / cm. The solution was then further concentrated to a protein concentration of approximately 0.5-1 mg / mL. The supernatant was centrifuged at 10,000 rpm and collected to obtain the mutant rhIL-2 crude proteins: rhIL2-K35, rhIL2-T41, rhIL2-K43, rhIL2-Y45, rhIL2-E61, rhIL2-K64, and rhIL2-P65. These were ready for subsequent PEG coupling.

[0138] Example 3: Site-specific coupling of PEG to rhIL-2 into which the unnatural amino acid NBOK has been inserted by site-directed mutagenesis [ka] The synthetic route for the site-specific coupling of PEG with rhIL-2 into which an unnatural amino acid had been inserted by site-directed mutagenesis was as shown in Scheme 2 (where the direction from P1 to P2 is from the N-terminus to the C-terminus of the amino acid sequence).

[0139] For example, to prepare rhIL-2 coupled with 30KD aminooxy PEG (i.e., hydroxylamine PEG) via oximation, the coupling procedure was as follows: Before coupling, the target protein obtained as described above was adjusted to pH 4.0 with 2M acetic acid solution, and the protein concentration was adjusted to approximately 1mg / ml with 20mM sodium acetate buffer (pH 4.0). 30KD aminooxy PEG solid (purchased from Beijing Keykai Technology Co., Ltd.) was added at a molar ratio of 1:15 (protein to aminooxy PEG), and the mixture was thoroughly dissolved by shaking to obtain a clear, transparent solution. The reaction mixture was then sealed and allowed to react while shaking on a thermostatic shaker (25°C, 100 rpm). After 48 hours, the coupling status was analyzed by RP-HPLC; see Figures 4A and 4B. As is clear from the results, only a small rhIL-2 peak was observed in Figure 4B, indicating that PEG was coupled to all seven target proteins. Furthermore, the coupling rates (100% - concentration of rhIL-2 remaining after completion of coupling / concentration of rhIL-2 at time zero of coupling reaction × 100%) were all greater than 95%, further indicating that the unnatural amino acid NBOK was inserted into the target proteins.

[0140] The PEG-coupled mutant rhIL-2 proteins are designated as 30KD PEG-rhIL2-K35, 30KD PEG-rhIL2-T41, 30KD PEG-rhIL2-K43, 30KD PEG-rhIL2-Y45, 30KD PEG-rhIL2-E61, 30KD PEG-rhIL2-K64, and 30KD PEG-rhIL2-P65.

[0141] The analytical conditions for RP-HPLC were as follows:

[0142] Mobile phase A (0.1% TFA-H2O), Mobile phase B (0.1% TFA-ACN).

[0143] [Table 2]

[0144] Example 4: Purification of site-specifically modified 30KD PEG-rhIL2 The chromatographic medium was Capto MMC, the equilibration buffer was 20 mM sodium citrate buffer (pH=3.0), and the elution buffer was 20 mM sodium citrate buffer-1 M NaCl (pH=7.8).

[0145] The purification process specifically included the following: the coupling reaction mixtures of 30KD PEG-rhIL2-K35, 30KD PEG-rhIL2-T41, 30KD PEG-rhIL2-K43, 30KD PEG-rhIL2-Y45, 30KD PEG-rhIL2-E61, 30KD PEG-rhIL2-K64, and 30KD PEG-rhIL2-P65 obtained in Example 3 were each adjusted to pH 3.0±0.2 and conductivity ≦5 mS / cm with equilibration buffer. The sample was then loaded onto a Capto MMC and subjected to linear elution (0-100% elution, 20 CV) with elution buffer to collect the target protein components. This resulted in a target protein sample with a purity of approximately 95%. Taking 30KD PEG-rhIL2-Y45 as an example, typical RP-HPLC spectra of the purified conjugate 30KD PEG-rhIL2-Y45 and rhIL2-Y45 before the coupling reaction are shown in Figure 5. The purification results of other protein samples were similar.

[0146] Example 5: Evaluation of in vitro activity of site-specifically modified 30KD PEG-rhIL2 (STAT5 phosphorylation experiment) This method uses two cell lines: mouse CTLL-2 cells, a cell line containing IL-2Rαβγ, and human YT cells, a cell line containing IL-2Rβγ. rhIL-2 binds to the IL-2R on the cell surface and activates the JAK-STAT signaling pathway. Each sample has a different modification site, resulting in different relative activities on the two cell types. The lower the percentage change in the EC50 ratio of YT cells to CTLL-2 cells, the greater the immune-stimulating effect of the sample; conversely, the greater the immune-suppressing effect.

[0147] The specific process was as follows: mouse CTLL-2 cells (purchased from American Type Culture Collection) and human YT cells were cultured in their respective media (CTLL-2 cell media: RPMI 1640 + 10% FBS + 400 IU / mL rhIL-2, 2 mM L-glutamine, 1 mM sodium pyruvate; YT cell media: RPMI 1640 + 10% FBS + 1 mM Non-Essential Amino Acids Solution (purchased from Gibco, catalog number 11140050)) at 37°C under 5% CO2 conditions until sufficient volume was reached. Then, cells were starved for 4 hours before measurement, and the cell density was subsequently reduced to 1 × 10 6The concentration was adjusted to 1000 cells / mL and then placed in standby. 30KD PEG-rhIL2-K35, 30KD PEG-rhIL2-T41, 30KD PEG-rhIL2-Y45, 30KD PEG-rhIL2-E61, 30KD PEG-rhIL2-P65, rhIL2-K35, rhIL2-T41, rhIL2-Y45, rhIL2-E61, and rhIL2-P65 samples before the coupling reaction, and reference rhIL-2 (purchased from Beijing Suo Laibao Science and Technology Co., Ltd., product number P00020) were each subjected to gradient dilution, resulting in a total of six concentrations for each sample. (In the CTLL-2 cell experiment, the reference was diluted 4-fold over the concentration range of 0.004 to 4 ng / mL, while in the YT cell experiment, the reference was diluted 3-fold over the concentration range of 2.1 to 510 ng / mL.) The concentration ranges of other samples were obtained by screening in preliminary experiments and correspond to the corresponding EC50 values ​​in Table 2. After 10 minutes of stimulation at 37°C, the cells were lysed and subjected to Western blot analysis using a pSTAT5 antibody (purchased from CST, catalog number 9359L) and β-actin (purchased from CST, catalog number 8457S) to measure the amount of pSTAT5 and β-actin protein in the cell lysate. The EC50 values ​​were calculated based on the grayscale results of pSTAT5 / β-actin and the sample concentrations. The results are shown in Table 2. The results clearly show that the rhIL2 with the unnatural amino acid NBOK inserted at K35, T41, Y45, E61, and P65, coupled with 30KD PEG, all met the initial design requirements (the EC50 ratios were lower than those of the reference).

[0148] [Table 3]

[0149] Example 6: Pharmacokinetic studies in mice Female C57 mice (SPF grade, purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used in this experiment. Quanqi rhIL-2 (purchased from Shandong Quangang Pharmaceutical Co., Ltd.) was used as a positive control to examine the metabolic status of the test substance, 30KD PEG-rhIL2-Y45, in mice. 30KD PEG-rhIL2-Y45 and Quanqi rhIL-2 were each administered intravenously at 1 mg / kg. Blood samples were taken at the following times: pre-dose, 0.0833 hours, 0.5 hours, 1 hour, 4 hours, 8 hours, 16 hours, and 24 hours post-dose. 0.5 mL of blood (n=5) was collected at each time point. 30KD PEG-rhIL2-Y45 was sampled at five additional times: 48 hours, 72 hours, 96 hours, 120 hours, and 144 hours. The blood samples were left at room temperature for 15 minutes and then centrifuged at 6800 g / min for 6 minutes to obtain serum, which was then analyzed for drug blood concentration using the following method.

[0150] (1) Coating: 50 μL of 1 μg / mL anti-IL-2 antibody (purchased from Abcam, catalog number ab9618) working solution was added to a high-adsorption 96-well plate and incubated overnight at 2–8°C. (2) Washing: The wells were discarded and washed three times with 300 μL / well of 1x PBST (0.05% Tween-20). (3) Sealing: 200 μL / well of casein sealing solution (purchased from Thermo, catalog number 37528) was added and the plate was left to stand at room temperature for 90 minutes. (4) Washing: The wells were discarded and washed three times with 300 μL / well of 1x PBST. (5) Sample addition: Quanqi rhIL-2, 30KD PEG-rhIL2-Y45, and serum samples to be measured were diluted with mouse serum and transferred to a microwell plate at 50 μL / well and left to stand at room temperature for 120 minutes. (6) Washing: The liquid in the wells was discarded, and the wells were washed three times with 300 μL / well of 1x PBST. (7) Primary antibody: 50 μL of a working solution of 0.25 μg / mL IL-2 Monoclonal Antibody (BG5) and Biotin (purchased from Invitrogen, catalog number M600B) was added per well and allowed to stand at room temperature for 60 minutes. (8) Washing: The liquid in the wells was discarded, and the wells were washed three times with 300 μL / well of 1x PBST. (9) Secondary antibody: Pierce TM High Sensitivity Streptavidin-HRP (purchased from Thermo, catalog number 21130) was diluted 4000-fold with casein blocking solution, and 50 μL was added per well and allowed to stand at room temperature for 60 minutes. (10) Washing: The liquid in the wells was discarded, and the wells were washed four times with 300 μL of 1×PBST per well. (11) Substrate: 1-Step TM50 μL of Turbo TMB-ELISA Substrate Solution (purchased from Thermo, catalog number 34022) was added per well. (12) Stop Value Reading: After 25 minutes, 2M sulfuric acid stop solution was added, and the absorbance values ​​at 450 nm and 650 nm were read using an enzyme labeling instrument (purchased from Perkin Elmer, model number EnSight). (13) Analysis: Four-parameter fitting was performed using Dazdaq Ltd. WorkOut 1.5 analysis software. The concentrations and units corresponding to Senki rhIL-2 and 30KD PEG-rhIL2-Y45 were entered, and one curve was fitted for each of them to calculate the drug blood concentration in the serum to be measured. The mean half-lives (t) of Senki rhIL-2 and 30KD PEG-rhIL2-Y45 were calculated using the non-ventricular model (statistical moment parameters) of the DAS software. 1 / 2 The pharmacokinetic parameters of Quanqi rhIL-2 and 30KD PEG-rhIL2-Y45 are shown in Table 3.

[0151] [Table 4]

[0152] Example 7: Pharmacodynamic studies in mice - antitumor activity In the experiment, female Balb / c mice (SPF grade, purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used, and CT26.WT (purchased from ATCC, catalog number CRL-2638) cell suspension was inoculated at 2 × 10 5 4 × 10 cell suspension in H22 (purchased from CTCCC, catalog number GDC0091) / 0.1 mL / mouse 5 0.1 mL / mouse was inoculated subcutaneously on the right back of the mouse, and the tumor volume was 50 mm 3When tumor size reached a certain level, mice were randomly grouped into groups of 7 and administered vehicle (1xPBS), 0.7mg / kg 30KD PEG-rhIL2-T41, 5.0mg / kg 30KD PEG-rhIL2-T41, 0.7mg / kg 30KD PEG-rhIL2-Y45, or 5.0mg / kg 30KD PEG-rhIL2-Y45 (administration volume: 10mL / kg). Animal weights and tumor volumes were measured three times a week during the experiment, and the administration methods and experimental results are shown in Table 4. Relative inhibition rate TGI TW The formula for (%) is (T WC -T WT ) / T WC × 100%, where T WC is the mean tumor weight in the vehicle control group, T WT is the mean tumor weight of the treatment group.

[0153] As is clear from the results, compared to the vehicle group, both the high and low dose groups of the two test compounds exhibited significant inhibitory effects on the allograft tumors of mouse colon cancer CT26.WT and mouse hepatic cancer H22.

[0154] [Table 5]

[0155] Example 8: Pharmacodynamic study in mice - expression of immune cell populations In the experiment, female Balb / c mice (SPF grade, purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used, and CT26.WT cell suspension was inoculated at 2 × 10 5 0.1 mL / mouse was inoculated subcutaneously on the right back of the mouse, and the tumor volume was 100 mm 3 When the tumor reached the target level, the mice were randomly grouped into groups of 3 mice each, and administered each test substance (Quanqi® is a commercially available recombinant human IL-2 injection) at a dose of 10 mL / kg according to Table 5. On the fifth day, tumor tissue samples were taken from each group to determine the CD8 + T cells and CD4 + The changes in the proportion of Treg cell population were obtained by flow detection, and the results are shown in Table 5.

[0156] [Table 6]

[0157] Compared with the control, 30KD PEG-rhIL2-T41 and 30KD PEG-rhIL2-Y45 significantly increased CD8 + The proportion of T cells increased significantly, and CD4 + The proportion of Treg cells was significantly reduced, and CD8 + T / CD4 + The proportion of Tregs was significantly increased, and excellent immune-enhancing efficacy was discovered.

[0158] Example 9: Lys-azido active group reduction phenomenon Using the method described in Example 1 as a reference, a strain expressing rhIL-2 (abbreviated as rhGH-V91) in which the codon for the 91st amino acid (valine) was mutated to an amber codon was constructed. The primers used in the construction process are shown below.

[0159] V91-F:5'-GATTTCCAATATCAACTAGATTGTTCTGGAACTGA-3'(SEQ ID NO:48), V91-R:5'-TCAGTTCCAGAACAATCTAGTTGATATTGGAAATC-3' (SEQ ID NO:49).

[0160] Using the rhGH-V91-expressing strain described above, rhGH-2 mutated at position 91 to Lys-azido was expressed by adding Lys-azido during the fermentation process, and the rhGH-2 was purified by the same method as in Example 2. The molecular weight of this rhGH-2 mutated at position 91 to Lys-azido was analyzed thoroughly using liquid chromatography and mass spectrometry (high-resolution mass spectrometer: XevoG2-XS Q-Tof, Waters; ultra-high performance liquid chromatography: UPLC (Acquity UPLC I-Class), Waters) as shown in Figure 6. The results clearly indicated that the sample contained a component approximately 26 Da smaller than the theoretical molecular weight (15,672.75 Da). This component was presumably the product of reduction of the azide structure (-N3) at the end of Lys-azido to (-NH2).

[0161] The reduction of lys-azido group resulted in the inability of rhIL-2 mutated to lys-azido to couple with BCN-PEG, resulting in a decreased coupling rate. However, when rhIL-2 containing unnatural amino acids of the present invention was coupled with PEG, the coupling efficiency was significantly improved, resulting in a significant improvement in reaction efficiency.

[0162] Example 10: Expression and purification of rhIL-2 with different unnatural amino acids inserted by site-directed mutagenesis 1. Expression of mutant rhIL-2 incorporating unnatural amino acids The expression strain rhIL2-T41-BL21 obtained in Example 1 was inoculated into LB medium (containing 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, and 100 mg / L spectinomycin) and cultured at 37°C for 4 to 6 hours. 600 Secondary propagation was carried out (LB medium) until the β-glucan content reached 2.0±0.2 to obtain secondary seed solution.

[0163] The secondary seed solution was inoculated into a fermentation medium and cultured in a 3 L quadruple fermentation tank. The culture volume was 1 L, the medium was 2xYT medium (16 g / L yeast extract, 10 g / L tryptone, 5 g / L NaCl), the inoculum amount was 5% (v / v), the culture temperature was 37°C, the pH was controlled at 6.90±0.05, ammonia water or H3PO4 was automatically added as needed, and the DO was controlled at 30% by setting the DO-related rotation speed. 600 When the pH reached 20.0 ± 2.0, IPTG and the unnatural amino acids obtained in Production Example 2 (NPAK), Production Example 3 (NBPK), Production Example 4 (NPOK), Production Example 5 (NBGK), Production Example 6 (NPOK-2), and Production Example 7 (NBGK-2) were added to a final concentration of 1 mM. Simultaneously, 50% glycerol was added at a rate of 0.5 ± 0.1 mL / min. After 5–6 hours of induced expression, the cells were harvested. SDS-PAGE electrophoresis of recombinant IL-2 containing different unnatural amino acids is shown in Figure 7.

[0164] 2. Separation and extraction of mutant rhIL-2 The collected bacterial cells were subjected to separation and extraction using the method described in Example 2 to obtain mutant rhIL-2 crude proteins incorporating different unnatural amino acids (see Figure 8). These proteins were named rhIL2-T41NPAK, rhIL2-T41NBPK, rhIL2-T41NPOK, rhIL2-T41NBGK, rhIL2-T41NPOK-2, and rhIL2-T41NBGK-2, respectively, and could be used directly for the subsequent PEG coupling.

[0165] Example 11: Site-specific coupling of PEG to rhIL-2 into which the unnatural amino acid NPAK has been inserted by site-directed mutagenesis [ka] The synthetic route for the site-specific coupling of PEG with rhIL-2 into which NPAK had been inserted by site-directed mutagenesis (rhIL2-T41NPAK) was as shown in Scheme 3 (where the direction from P1 to P2 is from the N-terminus to the C-terminus of the amino acid sequence).

[0166] Taking rhIL-2 coupled with 30 kD aminooxy PEG (i.e., hydroxyamine PEG) via oximation as an example, the coupling procedure was the same as in Example 3. After the coupling reaction, the coupling status was analyzed using RP-HPLC under the same RP-HPLC analysis conditions as in Example 3. The analysis results are shown in Figures 9A and 9B. As can be seen from the results in Figure 9B, the rhIL-2 peak significantly decreased compared to before the reaction, and the product peak (at 21.175 minutes) significantly increased accordingly, indicating that PEG was coupled to the target protein. Furthermore, the coupling rate (100% - concentration of rhIL-2 remaining after completion of coupling / concentration of rhIL-2 at time zero of the coupling reaction × 100%) was close to 80%.

[0167] The PEG-coupled mutant rhIL-2 protein was designated 30KD PEG-rhIL2-T41NPAK.

[0168] Example 12: Purification of site-specifically modified 30KD PEG-rhIL2-T41NPAK The 30KD PEG-rhIL2-T41NPAK obtained in Example 11 was purified by the purification method described in Example 4 to obtain a target protein sample with a purity of approximately 95%. A typical RP-HPLC spectrum of the purified conjugate is shown in Figure 10.

[0169] Example 13: Evaluation of in vitro activity of site-specifically modified 30KD PEG-rhIL2-T41NPAK (STAT5 phosphorylation experiment) The in vitro activity of the 30KD PEG-rhIL2-T41NPAK obtained in Example 12 was evaluated using the evaluation method described in Example 5. The only difference from the evaluation method of Example 5 was that the reference concentration range in the YT cell experiment was 6.3 to 1530 ng / mL.

[0170] The results are shown in Figures 11A to 11D and Table 6. As can be seen from the results, 30KD PEG-rhIL2-T41NPAK met the initial design requirements (when the reference was used as a control, the rate of change in EC50 ratio was lower than that of the reference).

[0171] [Table 7]

[0172] Unless otherwise specified, all terms used herein have the meanings that are commonly understood by those skilled in the art.

[0173] The embodiments described in the present invention are merely illustrative and do not limit the protection scope of the present invention; those skilled in the art may make various other substitutions, changes and improvements within the scope of the present invention; therefore, the present invention is not limited to the above embodiments, but is limited only by the claims.

Claims

1. A conjugate of human interleukin-2 and polyethylene glycol, comprising recombinant human interleukin-2 containing one unnatural amino acid and PEG coupled to said one unnatural amino acid, the unnatural amino acid is a carbonyl terminal group-containing compound having a structure represented by formula (I-3), formula (I-4), formula (I-5), or formula (I-6), or an enantiomer thereof, and PEG is coupled to the one unnatural amino acid by forming an oxime bond between the carbonyl terminal group and a hydroxylamine terminal group-containing PEG; 【Chemistry 1】 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 In the formula, X' represents a C0-C6 linear alkylene group, one of -CH 2 - of which can be replaced by -O- or -NH-; R １ , R ２ each independently represents hydrogen, The recombinant human interleukin-2 is a protein shown in SEQ ID NO: 3 or a functionally active fragment thereof; the position of the unnatural amino acid in the recombinant human interleukin-2 containing one unnatural amino acid is one selected from positions K35, T41, Y45, E61, and P65 of SEQ ID NO: 2; Conjugate of human interleukin-2 and polyethylene glycol.

2. The X' represents a C0 to C4 linear alkylene group, or X' represents a C0 to C4 linear alkylene group, one of which is -CH ２ - can be replaced by -O- or -NH-; A conjugate of human interleukin-2 and polyethylene glycol according to claim 1.

3. The unnatural amino acid is a compound having any one of the following structures: 【Transformation 5】 A conjugate of human interleukin-2 and polyethylene glycol according to claim 1.

4. The molecular weight of the hydroxylamine-terminated PEG is 20-50 KD. A conjugate of human interleukin-2 and polyethylene glycol according to claim 1.

5. The conjugate of human interleukin-2 and polyethylene glycol according to claim 1 is used as a drug for immunostimulation, a drug for the prevention and / or treatment of solid tumors and hematological tumors, and / or a drug for the treatment of CD8 ＋ Use in the manufacture of a drug for T cell proliferation.

6. the solid tumor is bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, renal cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer; The hematological tumors include 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, Hodgkin's lymphoma, high-grade B-cell non-Hodgkin's lymphoma, 6. The use according to claim 5, wherein the tumor is primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-cell lymphoblastic lymphoma, B-cell juvenile lymphocytic 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 lymphomatous granuloma.

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