IL2-IL2Ralpha-Fc fusion protein and application thereof

Abstract

The invention discloses an IL2-IL2R alpha-Fc fusion protein as well as a preparation method and application thereof. The fusion protein comprises the following components: (1) IL-2 or a mutant thereof; (2) IL-2R alpha, and (3) an Fc fragment; wherein the IL-2 or the mutant thereof is connected with the IL-2R alpha, and the IL-2R alpha is connected with the Fc fragment through a linker.

Description

Technical Field

[0001] The present disclosure generally relates to the field of biomedicine, and more specifically, the present disclosure relates to fusion proteins and their uses. Background Art

[0002] Interleukin-2 (IL-2) is an important cytokine secreted by activated T cells and belongs to the type I cytokine family. In the immune system, IL-2 has multiple biological functions, mainly by binding with high affinity to the interleukin-2 receptor (IL-2R), regulating the proliferation and differentiation of T cells, and promoting the activation and functional enhancement of natural killer cells (NK cells) and B cells.

[0003] IL-2R is a key cell surface receptor mediating the IL-2 signaling pathway and plays an important role in the regulation of the immune system. IL-2R is composed of three subunits: IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132, common gamma chain).

[0004] Different combinations of these subunits can form low-, medium-, and high-affinity receptor complexes, among which the high-affinity IL-2R (composed of α, β, and γ chains together) is the main functional form. IL-2R regulates the proliferation and survival of effector T cells by activating the JAK-STAT signaling pathway and plays a key role in the maintenance of regulatory T cells (Tregs), thus playing a dual role in immune response and immune tolerance.

[0005] IL-2 not only plays a core role in humoral and cellular immunity, but also participates in the establishment of immune tolerance by maintaining the number and function of regulatory T cells (Tregs) and preventing the immune system from overreacting.

[0006] In addition, IL-2 has shown significant potential in anti-tumor immunity and has been widely used in the immunotherapy research of various cancers (such as melanoma and renal cell carcinoma). The ability of IL-2 to expand T cells and maintain cell functional activity has been translated into the first reproducible and effective immunotherapy for human cancer.

[0007] In recent years, the strategies of modifying IL-2 and applying it at low doses have also been used to treat autoimmune diseases and chronic viral infections, highlighting its important position in the field of immunomodulation. Summary of the Invention

[0009] On the one hand, the present disclosure relates to a fusion protein, which comprises:

[0010] (1) IL-2 or its mutant;

[0011] (2) IL-2Rα; and

[0012] (3) Fc fragment;

[0013] wherein the IL-2 or its mutant is linked to the IL-2Rα, and the IL-2Rα is linked to the Fc fragment through a linker.

[0014] On the other hand, the present disclosure relates to a pharmaceutical composition comprising the fusion protein described in the present disclosure and a pharmaceutically acceptable excipient.

[0015] On yet another aspect, the present disclosure relates to a method for modulating the IL-2 receptor, which comprises contacting the IL-2 receptor with a modulating effective amount of the fusion protein described in the present disclosure or a modulating effective amount of the pharmaceutical composition described in the present disclosure.

[0016] On yet another aspect, the present disclosure relates to a method for treating tumors, which comprises administering to an individual in need of said method a therapeutically effective amount of the fusion protein described in the present disclosure or a therapeutically effective amount of the pharmaceutical composition described in the present disclosure.

[0017] On the other hand, the present disclosure relates to a method for modulating immune cells, which comprises administering to an individual in need of said method a modulating effective amount of the fusion protein described in the present disclosure or a modulating effective amount of the pharmaceutical composition described in the present disclosure.

[0018] On yet another aspect, the present disclosure relates to a polynucleotide encoding the fusion protein described in the present disclosure.

[0019] On yet another aspect, the present disclosure relates to a vector comprising the polynucleotide described in the present disclosure.

[0020] On the other hand, the present disclosure relates to a host cell comprising the vector described in the present disclosure.

[0021] On yet another aspect, the present disclosure relates to a method for expressing a fusion protein, which comprises culturing the host cell described in the present disclosure under conditions in which the polynucleotide expresses the fusion protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Shows the experimental results of SDS-PAGE of the fusion protein of the present disclosure;

[0024] Figure 2 Shows the experimental results of Western Blot of the fusion protein of the present disclosure;

[0025] Figure 3 Shows the amplification effect of different fusion proteins on NK92;

[0026] Figure 4Shows the amplification effect of different fusion proteins on human peripheral blood mononuclear cells;

[0027] Figure 5 Shows the experimental results of the selective amplification of the fusion proteins of the present disclosure;

[0028] Figure 6 Shows the inhibitory effect of the fusion proteins of the present disclosure on the tumor volume of melanoma in mice; and

[0029] Figure 7 Shows the inhibitory effect of the fusion proteins of the present disclosure on the tumor weight of melanoma in mice.

[0030] Detailed description

[0031] In the following description, certain specific details are included to provide a comprehensive understanding of each disclosed embodiment. However, those skilled in the relevant art will recognize that the embodiments can be implemented without one or more of these specific details, and other methods, components, materials, etc. can be used instead.

[0032] Unless otherwise required in this application, throughout the specification and the appended claims, the words "comprising", "including", "containing" and "having" shall be construed in an open, inclusive sense, i.e., "including but not limited to".

[0033] When used in the present disclosure and the appended claims, unless the context clearly dictates otherwise, singular referents without a quantity indicator include plural referents.

[0034] References throughout the specification to "one embodiment", "an embodiment", "in another embodiment" or "in certain embodiments" mean that at least one embodiment includes the specific reference elements, structures or features related to that embodiment described. Thus, the phrases "in one embodiment" or "in an embodiment" or "in another embodiment" or "in certain embodiments" that appear in different places throughout the specification do not necessarily all refer to the same embodiment. In addition, the specific elements, structures or features can be combined in one or more embodiments in any appropriate manner.

[0035] It should be understood that the singular forms of the articles "a" (corresponding to "a", "an" and "the" in English) used in the specification and the appended claims of the present disclosure include plural objects, unless otherwise clearly specified in the text. Thus, for example, a pharmaceutical composition containing "adjuvants" includes a pharmaceutical composition with one adjuvant, or a pharmaceutical composition with two or more adjuvants.

[0036] Definition

[0037] Accordingly, unless otherwise indicated to the contrary, the following terms used in the specification and appended claims shall have the following meanings:

[0038] In the present disclosure, the term "IL-2", ie "interleukin-2 (IL-2)", refers to an important cytokine secreted by activated T cells and belongs to the type I cytokine family.

[0039] In this disclosure, the term "IL-2R" refers to a key cell surface receptor that mediates the IL-2 signaling pathway. IL-2R is composed of three subunits: IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132, the universal γ chain).

[0040] In the present disclosure, the term "CD", ie "cluster of differentiation", refers to cell surface markers that appear or disappear during the normal differentiation, maturation and activation of leukocytes of different lineages.

[0041] In the present disclosure, the term "light chain" refers to the smaller molecular weight peptide chain in immunoglobulin, which is composed of approximately 214 amino acids and is divided into two types: "κ light chain (kappa light chain)" and "λ light chain (lambda light chain)" based on the differences in their structure and constant region antigenicity.

[0042] As used herein, the term "heavy chain" refers to the larger peptide chain in an immunoglobulin, which has a molecular weight of approximately 50 to 75 kDa and is composed of 450 to 550 amino acid residues. Heavy chains can be divided into five categories based on their immunogenicity: μ, γ, α, δ, and ε. Antibodies are classified into five categories: IgM, IgG, IgA, IgD, and IgE, depending on the heavy chain composition.

[0043] In this disclosure, the term "complementarity determining region" or "CDR" in relation to antibodies refers to the hypervariable loops in the heavy or light chain variable region of an antibody.

[0044] In this disclosure, the term "heavy chain variable region" or "V H ” refers to a fragment of a heavy chain that comprises three complementarity determining regions (CDRs) interposed between flanking segments called framework regions, which are more conserved than the complementarity determining regions (CDRs) and form a scaffold to support the complementarity determining regions (CDRs).

[0045] In this disclosure, the term "light chain variable region" or "V L ” refers to a light chain fragment comprising three complementarity determining regions (CDRs) interposed between framework regions.

[0046] In the present disclosure, the term "CDR set" refers to three hypervariable regions of the heavy chain variable region or the light chain variable region. Starting from the N-terminus of the heavy chain or the light chain, these regions are designated as "CDR1", "CDR2", and "CDR3", respectively. Thus, the antigen-binding site contains six CDRs, including the CDR sets from the heavy chain variable region and the light chain variable region, respectively.

[0047] In the present disclosure, the term "Fc fragment" refers to the fragment crystallizable (Fc), which corresponds to the CH2 and CH3 domains of Ig and is the site where Ig interacts with effector molecules or cells.

[0048] In the present disclosure, the term "pharmaceutical composition" refers to a preparation formed by the fusion protein described in the present disclosure and a medium that is commonly accepted in the art for delivering a bioactive substance to mammals such as humans. Such a medium includes all pharmaceutically acceptable excipients.

[0049] In the present disclosure, the term "polynucleotide" refers to a single-stranded or double-stranded nucleic acid polymer. In certain embodiments, the nucleotides contained in the polynucleotide can be ribonucleotides or deoxyribonucleotides or any type of modified form of nucleotides. The modifications include base modifications such as bromouridine, ribose modifications such as arabinose and 2',3'-dideoxyribose, and modifications of the internucleotide linkages such as phosphorothioate, dithiophosphonate, selenophosphonate, diselenophosphonate, phosphoroanilothioate, phoshoraniladate, and aminophosphate. The term "polynucleotide" expressly includes DNA in both single-stranded and double-stranded forms.

[0050] In the present disclosure, the term "isolated polynucleotide" refers to a genomic polynucleotide, cDNA, polynucleotide, or polynucleotide of synthetic origin or some combination thereof. Due to its origin, the "isolated polynucleotide" (1) is not bound to all or part of the polynucleotide related to the polynucleotide isolated in nature, (2) is linked to a polynucleotide that is not linked to it in nature, or (3) does not exist as part of a larger sequence in nature.

[0051] In the present disclosure, the term "operably linked" means that the components to which the term applies are in a relationship that allows them to perform their inherent functions under appropriate conditions. For example, a transcriptional control sequence "operably linked" to a protein-coding sequence is linked to it such that the expression of the protein-coding sequence can be accomplished under conditions compatible with the transcriptional activity of the control sequence.

[0052] In the present disclosure, the term "vector" refers to a vehicle into which a polynucleotide encoding a protein can be covalently inserted to effect protein expression and / or polynucleotide cloning.

[0053] In the present disclosure, the term "mammal" refers to animals including, for example, dogs, cats, cows, sheep, horses, and humans. In certain embodiments, the mammal includes humans.

[0054] In the present disclosure, the term "individual" refers to animals (e.g., humans), companion animals (e.g., dogs, cats, or horses), and livestock (e.g., cows, pigs, and sheep). In certain embodiments, the individual is a mammal including both males and females. In certain embodiments, the individual is a human.

[0055] As used herein, "treating" or "treatment" encompasses treating a relevant disease or disease state in a mammal such as a human suffering from the relevant disease or disorder, and includes:

[0056] (i) preventing the occurrence of a disease or disease state in a mammal, particularly when the mammal is susceptible to the disease state but has not been diagnosed with such disease state;

[0057] (ii) inhibiting a disease or disease state, i.e., preventing its occurrence; or

[0058] (iii) alleviating a disease or disease state, i.e., causing the disease or disease state to regress or not progress. Detailed Description

[0059] In one aspect, the present disclosure relates to a fusion protein comprising:

[0060] (1) IL-2 or a mutant thereof;

[0061] (2) IL-2Rα; and

[0062] (3) an Fc fragment;

[0063] wherein the IL-2 or mutant thereof is linked to the IL-2Rα, and the IL-2Rα is linked to the Fc fragment via a linker.

[0064] In certain embodiments, exemplary instances of Fc fragments that can be used in the present disclosure include, but are not limited to, IgG1, IgG2, IgG3, and IgG4.

[0065] In certain embodiments, the linker is (Gly4Ser)3.

[0066] In certain embodiments, the molecular weight of the IL-2 mutant is about 17.6 kDa.

[0067] In certain embodiments, the molecular weight of the IL-2Rα-Fc formed by linking the IL-2Rα and the Fc fragment through a linker is about 50 kDa.

[0068] In certain embodiments, the IL-2 mutant comprises the sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0069] In certain embodiments, exemplary examples of the IL-2 mutant that can be used in the present disclosure include, but are not limited to, the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

[0070] In certain embodiments, the IL-2Rα-Fc comprises the sequence SEQ ID NO:5.

[0071] In certain embodiments, exemplary examples of the IL-2Rα-Fc that can be used in the present disclosure include, but are not limited to, the sequence SEQ ID NO:5.

[0072] In certain embodiments, the IL-2 mutant is called T119.120, and its sequence is:

[0073] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLECELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0074] In certain embodiments, the IL-2 mutant is called T121.122, and its sequence SEQ ID NO:1 is:

[0075] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLECELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRCCTFCQSIISTLT

[0076] In certain embodiments, the IL-2 mutant is called T123.127, and its sequence SEQ ID NO:2 is:

[0077] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLECELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWICFCQCIISTLT

[0078] In certain embodiments, the IL-2 mutant is designated T124.126 and its sequence, SEQ ID NO:3, is:

[0079] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLECELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITCCCSIISTLT

[0080] In certain embodiments, the IL-2 mutant is designated T125.128 and its sequence, SEQ ID NO:4, is:

[0081] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLECELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSCISTLT

[0082] In certain embodiments, the sequence SEQ ID NO:5 is:

[0083] ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKCGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0084] In certain embodiments, the complementarity determining regions (CDRs) can interact with the antigen structure and largely determine the binding to the antigen (although some framework regions are known to be involved in binding). Each of the heavy chain variable region and the light chain variable region contains three complementarity determining regions (CDRs). The complementarity determining regions (CDRs) can be defined or identified by conventional methods, such as by the sequences of Kabat et al (Wu, T.T. and Kabat, E.A., J Exp Med. 132(2):211-50, (1970); Borden, P. and Kabat E.A., PNAS, 84:2440-2443 (1987); Kabat, E.A. et al, Sequences of proteins of immunological interest, Published by DIANE Publishing, 1992), or by the structures of Chothia et al (Choithia, C. and Lesk, A.M., J Mol. Biol., 196(4):901-917 (1987), Choithia, C. et al, Nature, 342:877-883 (1989)).

[0085] In certain embodiments, the fusion proteins of the present disclosure are capable of enhancing the binding efficiency of IL-2 to its receptor.

[0086] In certain embodiments, the fusion proteins of the present disclosure have excellent biological activities.

[0087] In certain embodiments, the fusion proteins of the present disclosure are capable of selectively activating CD8 + T cells and natural killer cells.

[0088] On the other hand, the present disclosure relates to pharmaceutical compositions comprising the fusion proteins described herein, and pharmaceutically acceptable excipients.

[0089] In certain embodiments, the pharmaceutical compositions of the present disclosure may be administered in a therapeutically effective amount, which depends on a variety of factors, including the activity of the particular compound being applied; the metabolic stability and duration of action of the compound; the age, weight, general health, sex and diet of the patient, the mode and time of administration; the rate of excretion; drug combinations; the severity of the particular disease or disorder; and the circumstances of the individual being treated. In certain embodiments, the therapeutically effective daily dose (for a 70 kg mammal) is from about 0.001 mg / kg (i.e., 0.07 mg) to about 100 mg / kg (i.e., 7.0 g). In certain embodiments, the therapeutically effective daily dose (for a 70 kg mammal) is from about 0.01 mg / kg (i.e., 0.7 mg) to about 50 mg / kg (i.e., 3.5 g). In certain embodiments, the therapeutically effective daily dose (for a 70 kg mammal) is from about 1 mg / kg (i.e., 70 mg) to about 25 mg / kg (i.e., 1.75 g).

[0090] In yet another aspect, the present disclosure relates to a method of modulating an IL-2 receptor, which comprises contacting the IL-2 receptor with a modulating effective amount of the fusion proteins described herein or a modulating effective amount of the pharmaceutical compositions described herein.

[0091] In certain embodiments, the contacting is carried out in vitro.

[0092] In certain embodiments, the contacting is carried out ex vivo in a mammal.

[0093] In certain embodiments, the contacting is carried out ex vivo in a human.

[0094] In certain embodiments, the contacting is carried out in vivo.

[0095] In certain embodiments, the contacting is carried out in vivo in a mammal.

[0096] In certain embodiments, the contacting is carried out in vivo in a human.

[0097] In yet another aspect, the present disclosure relates to a method of treating a tumor, which comprises administering to an individual in need of said method a therapeutically effective amount of the fusion proteins described herein or a therapeutically effective amount of the pharmaceutical compositions described herein.

[0098] In certain embodiments, exemplary instances of individuals that can be used in the present disclosure include, but are not limited to, mammals.

[0099] In certain embodiments, exemplary instances of mammals that can be used in the present disclosure include, but are not limited to, humans.

[0100] In certain embodiments, exemplary instances of tumors that can be used in the present disclosure include, but are not limited to, melanoma, metastatic melanoma, and renal cell carcinoma.

[0101] In certain embodiments, the method for treating a tumor of the present disclosure further comprises administering another therapeutic agent to the individual.

[0102] In certain embodiments, exemplary instances of another therapeutic agent of the present disclosure include, but are not limited to, Nemvaleukin alfa (ALKS 4230), mechlorethamine, aziridine, methylmelamine, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purine analogs, vinca alkaloids, epipodophyllotoxins, antibiotics, topoisomerase inhibitors, anticancer vaccines, acivicin, aclarubicin, acodazole hydrochloride, acronine, adozelesin, aldesleukin, ambomycin, ametantrone acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate, bizelesin, bleomycin sulfate, busulfan, actinomycin C, calusterone, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, chlorambucil, cirolemycin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, actinomycin D, daunorubicin hydrochloride, decitabine, docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, epirubicin hydrochloride, esorubicin hydrochloride, estramustine, etanidazole, etoposide, floxuridine, fluorouracil, fludarabine, gemcitabine, idarubicin hydrochloride, ifosfamide, interleukin II, interferon α-2a, interferon α-2b, irinotecan hydrochloride, letrozole, mercaptopurine, methotrexate, metopterin, mitomycin, mitoxantrone, paclitaxel, procarbazine, thiotepa, vinblastine, vincristine, angiogenesis inhibitors, camptothecin, dexamethasone, aspirin, acetaminophen, indomethacin, ibuprofen, ketoprofen, meloxicam, and corticosteroids.

[0103] On the other hand, the present disclosure relates to a method of modulating immune cells, which comprises administering to an individual in need of the method a modulating effective amount of the fusion protein described in the present disclosure or a modulating effective amount of the pharmaceutical composition described in the present disclosure.

[0104] In certain embodiments, exemplary instances of immune cells that can be used in the present disclosure include, but are not limited to, T-lymphocytes, natural killer cells, macrophages, and dendritic cells (DC).

[0105] In certain embodiments, exemplary instances of T-lymphocytes that can be used in the present disclosure include, but are not limited to, helper T cells, cytotoxic T cells, and regulatory T cells.

[0106] In certain embodiments, exemplary instances of T-lymphocytes that can be used in the present disclosure include, but are not limited to, CD8 + T cells, memory precursor CD8 + T cells (MP CD8 + cells), and CD4 + CD25 + regulatory T cells (CD4 + CD25 + Tregs).

[0107] In certain embodiments, exemplary instances of natural killer cells that can be used in the present disclosure include, but are not limited to, NK92 cells and CD69 + NK cells.

[0108] In certain embodiments, exemplary instances of individuals that can be used in the present disclosure include, but are not limited to, mammals.

[0109] In certain embodiments, exemplary instances of mammals that can be used in the present disclosure include, but are not limited to, humans.

[0110] In another aspect, the present disclosure relates to a polynucleotide encoding the fusion protein described in the present disclosure.

[0111] In certain embodiments, the polynucleotide is an isolated polynucleotide.

[0112] In certain embodiments, the polynucleotide variant may have substantial sequence identity with a polynucleotide sequence encoding a fusion protein or a domain thereof described herein. For example, the polynucleotide may be a polynucleotide that, using the methods described herein (e.g., BLAST analysis with standard parameters as described below, as described herein), may have at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity compared to a reference polynucleotide sequence (e.g., a sequence encoding a fusion protein or a domain thereof described herein). Those skilled in the art will recognize that these values can be appropriately adjusted by considering codon degeneracy, amino acid similarity, reading frame positioning, etc. to determine the corresponding identity of the proteins encoded by two nucleotide sequences. Generally, the polynucleotide variant will contain one or more substitutions, additions, deletions, and / or insertions, preferably such that the binding affinity of the binding domain, or the binding affinity of the fusion protein, or the function of the fusion protein encoded by the variant polynucleotide is substantially not reduced compared to the unmodified reference protein encoded by the polynucleotide sequence specifically shown herein.

[0113] In certain embodiments, the polynucleotide fragment may comprise or consist of contiguous sequence segments of varying lengths that are identical or complementary to the sequences encoding the fusion proteins or domains thereof described herein. For example, the provided polynucleotides comprise at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500, or 1000 or more contiguous nucleotides of the sequence encoding the fusion proteins or domains thereof described herein (such as its binding domain), and all intermediate lengths of contiguous nucleotides therebetween, or consist of such contiguous nucleotides. It is readily understood that in the context of the present disclosure "intermediate length" refers to any length between the recited values, e.g., 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers from 200 to 500, 500 to 1,000, etc. The polynucleotide sequences described herein may be extended at one or both ends by adding nucleotides not present in the native sequence. The added sequence may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides at either or both ends of the polynucleotide encoding the fusion proteins or domains thereof described herein or the polynucleotide encoding the fusion proteins or domains thereof described herein.

[0114] In certain embodiments, provided are polynucleotides capable of hybridizing, under medium to high stringency conditions, to the polynucleotide sequences encoding the fusion proteins or domains thereof (such as its binding domain) provided herein, or fragments thereof, or complementary sequences thereof. Hybridization techniques are well known in the art of molecular biology. For purposes of illustration, suitable medium stringency conditions for testing hybridization of the polynucleotides provided herein to other polynucleotides include: prewashing in a solution of 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization overnight at 50°C to 60°C in 5X SSC; followed by washing twice for 20 minutes each at 65°C with 2X SSC, containing 0.1% SDS, 0.5X SSC, and 0.2X SSC. Those skilled in the art will appreciate that the stringency of hybridization can be readily manipulated, e.g., by varying the salt concentration of the hybridization solution and / or the temperature at which hybridization is performed. For example, in certain embodiments, suitable high stringency hybridization conditions include those described above, except that the temperature of hybridization is increased to, e.g., 60°C to 65°C or 65°C to 70°C.

[0115] In certain embodiments, determination of the three-dimensional structure of a representative polypeptide (e.g., a fusion protein provided by the present disclosure) can be carried out by conventional methods such that substitution, addition, deletion, or insertion of one or more amino acids with selected natural or unnatural amino acids can be virtually modeled for the purpose of determining whether the resulting structural variant retains the space-filling properties of the species of the present disclosure. Many computer programs are known to those skilled in the art for determining suitable amino acid substitutions (or suitable polynucleotides encoding amino acid sequences) within, for example, an antibody or its antigen-binding fragment so as to, for example, maintain affinity or achieve better affinity. The polynucleotides or fragments thereof described in the present disclosure, regardless of the length of the coding sequence itself, can be combined with other DNA sequences (such as promoters, polyadenylation signals, additional restriction enzyme sites, polylinker sites, other coding fragments, etc.) such that their total length can vary widely. Thus, it is expected that nucleic acid fragments of almost any length can be applied, the total length of which is preferably limited by convenience of preparation and use in the intended recombinant DNA protocol. For example, exemplary polynucleotide fragments of lengths of about 10,000, about 5,000, about 3,000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs, etc. (including all intermediate lengths) can be considered.

[0116] When comparing polynucleotide sequences, two sequences can be considered "identical" if the nucleotide sequences in the two sequences are the same when maximally aligned as described below. Usually, comparison between two sequences is carried out by comparing the sequences in a comparison window so as to identify and compare local regions of sequence similarity. The "comparison window" used in the present disclosure refers to a segment of at least about 20 consecutive positions, usually 30 to about 75, 40 to about 50 consecutive positions, where the sequence can be compared with a reference sequence after the sequence is optimally aligned with a reference sequence having the same number of consecutive positions.

[0117] The Megalign program in the Lasergene bioinformatics software suite (DNASTAR, Inc., Madison, WI) can be used with default parameters to perform the optimal alignment of sequences for comparison. This program includes several alignment schemes described in the following references: Dayhoff, M.O. (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345 - 358; Hein J., Unified Approach to Alignment and Phylogenes, pp. 626 - 645 (1990); Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M., CABIOS 5:151 - 153 (1989); Myers, E.W. and Muller W., CABIOS 4:11 - 17 (1988); Robinson, E.D., Comb. Theor 77:105 (1971); Santou, N. Nes, M., Mol. Biol. Evol. 4:406 - 425 (1987); Sneath, P.H.A. and Sokal, R.R., Numerical Taxonomy - the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA (1973); Wilbur, W.J. and Lipman, D.J., Proc. Natl. Acad., Sci. USA 80:726 - 730 (1983).

[0118] In certain embodiments, the optimal alignment of sequences for comparison can be conducted by: the local homology algorithm of Smith and Waterman, Add. APL. Math 2:482 (1981); the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970); the similarity method search of Pearson and Lipman, Proc. Nat’l Acad. Sci. USA 85:2444 (1988); the computer execution of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or inspection.

[0119] Exemplary instances of algorithms suitable for determining the percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nucl. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. For example, BLAST and BLAST 2.0 can be used with the parameters described herein to determine the percent sequence identity between two or more polynucleotides. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. In one exemplary instance, for nucleotide sequences, the cumulative score can be calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). The extension of character hits in each direction is stopped when: the cumulative alignment score drops by amount X from its maximum achieved value; the cumulative score reaches 0 or below 0 due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses the following as defaults: word size (W) of 11, expectation (E) of 10, BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignment, (B) of 50, expectation (E) of 10, M = 5, N = -4, and comparison of both strands.

[0120] In certain embodiments, “percent sequence identity” is determined by comparing two optimally aligned sequences in a comparison window of at least 20 positions, wherein a portion of the polynucleotide sequence in the comparison window may include 20% or less, typically 5%-15%, or 10%-12% additions or deletions (i.e., gaps) as compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases exist in both sequences to yield the number of paired positions, dividing the number of paired positions by the total number of positions in the reference sequence (i.e., the size of the window), and multiplying the result by 100 to yield the percentage of sequence identity.

[0121] Those skilled in the art will understand that due to the degeneracy of the genetic code, there are multiple nucleotide sequences encoding the fusion proteins described in the present disclosure. Some of these polynucleotides have minimal sequence identity with the nucleotide sequence of the native or original polynucleotide sequence encoding the fusion protein. However, polynucleotides that differ due to codon usage differences are expressly covered by the present disclosure. In certain embodiments, sequences that have been codon-optimized for mammalian expression are specifically contemplated.

[0122] In another aspect, the present disclosure relates to vectors that contain the polynucleotides described in the present disclosure.

[0123] In certain embodiments, the isolated polynucleotide is inserted into a vector. Any suitable method known in the art can be utilized to insert the isolated polynucleotide into a vector, for example but not limited to, the vector can be digested with a suitable restriction enzyme and then ligated with the isolated polynucleotide having matching restriction ends.

[0124] Exemplary instances of vectors that can be used in the present disclosure include but are not limited to plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. Exemplary instances of animal virus types that can be used as vectors in the present disclosure include but are not limited to: retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and polyomaviruses (such as SV40).

[0125] For the expression of a polypeptide, a vector can be introduced into a host cell to allow the polypeptide to be expressed in the host cell. In certain embodiments, the expression vector can contain various elements for controlling expression, including but not limited to a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selectable marker, and a signal sequence. Those skilled in the art can select these elements as appropriate. For example, a promoter sequence can be selected to promote the transcription of the polynucleotide in the vector. Suitable promoter sequences include but are not limited to the T7 promoter, the T3 promoter, the SP6 promoter, the β-actin promoter, the EF1a promoter, the CMV promoter, and the SV40 promoter. An enhancer sequence can be selected to enhance the transcription of the polynucleotide. A selectable marker can be selected to allow host cells into which the vector has been inserted to be selected from host cells that have not inserted the vector. For example, the selectable marker can be a gene that confers antibiotic resistance. A signal sequence can be selected to allow the expressed polypeptide to be transported outside the host cell.

[0126] In certain embodiments, the vector can also contain substances that help it enter the cell, including but not limited to virus particles, liposomes, or protein capsids.

[0127] For the cloning of a polynucleotide, a vector can be introduced into a host cell (an isolated host cell) to allow the vector to replicate itself and thereby amplify the copies of the polynucleotide contained therein. A cloning vector can contain sequence components, generally including but not limited to: an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selectable marker. Those skilled in the art can select these elements as appropriate. For example, an origin of replication can be selected to promote the autonomous replication of the vector in the host cell.

[0128] On the other hand, the present disclosure relates to a host cell that contains the vector described in the present disclosure.

[0129] In certain embodiments, exemplary instances of host cells that can be used in the present disclosure include but are not limited to prokaryotic cells, fungal cells, yeast cells, and higher eukaryotic cells. In certain embodiments, exemplary instances of higher eukaryotic cells include but are not limited to mammalian cells.

[0130] In certain embodiments, exemplary instances of suitable prokaryotic cells for this purpose include, but are not limited to, eubacteria, such as Gram-negative organisms or Gram-positive organisms, e.g., Enterobacteriaceae, such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescens, Shigella, Bacilli, such as Bacillus subtilis and Bacillus licheniformis, Pseudomonas, such as Pseudomonas aeruginosa, and Streptomyces.

[0131] The expression of antibodies and antigen-binding fragments in prokaryotic cells such as E. coli has been established in the art. For a review, see, e.g., Pluckthun, A. Bio / Technology 9:545-551 (1991). The expression in cultured eukaryotic cells has also been used by those skilled in the art as an option for generating antibodies or their antigen-binding fragments. See recent reviews, e.g., Ref, M. E. (1993) Curr. Opinion Biotech. 4:573-576; Trill J. J. et al. (1995) Curr. Opinion Biotech 6:553-560.

[0132] In certain embodiments, exemplary instances of suitable fungal cells for this purpose include, but are not limited to, filamentous fungi and yeasts. Exemplary instances of fungal cells include Saccharomyces cerevisiae, common baker's yeast, Schizosaccharomyces pombe, Kluyveromyces hosts such as, for example, Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces wickeramii (ATCC 24,178), Kluyveromyces waltii (ATCC 56,500), Kluyveromyces drosophilarum (ATCC 36,906), Kluyveromyces thermotolerans, and Kluyveromyces marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesei (EP 244,234); Neurospora crassa; Schwanniomyces hosts such as Schwanniomyces occidentalis; and filamentous fungi such as, for example, Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as Aspergillus nidulans and Aspergillus niger.

[0133] Higher eukaryotic cells, particularly those derived from multicellular organisms, can be used for the expression of the glycosylated polypeptides provided by the present disclosure. In certain embodiments, exemplary instances of suitable higher eukaryotic cells include, but are not limited to, invertebrate cells and insect cells, as well as vertebrate cells. In certain embodiments, exemplary instances of invertebrate cells include plant cells and insect cells. A large number of baculovirus strains and variants and their corresponding host insect host cells have been identified from the following hosts: for example, Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. A variety of virus strains for transfection are publicly available, for example, the K-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and these viruses can be used as the viruses herein, particularly for the transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts. Examples of vertebrate cells include mammalian host cell lines such as the monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL1651); the human embryonic kidney cell line (293 or subclones of 293 cells grown in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRK-1587); human cervical cancer cells (HELA, ATCC CCL2); dog kidney cells (MDCK, ATCC CCL34); Buffalo rat hepatocytes (BRL3A, ATCC CRL1442); human lung cells (W138, ATCC CCL75); human hepatocytes (Hep G2, HB8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells; and the human hepatoma cell line (Hep G2).

[0134] Any suitable method known in the art can be used to introduce the vector into the host cell, including but not limited to: DEAE-dextran-mediated delivery, calcium phosphate precipitation, cationic lipid-mediated delivery, liposome-mediated transfection, electroporation, particle bombardment, receptor-mediated gene delivery, and delivery mediated by polylysine, histone, chitosan, and peptides.

[0135] Standard methods for cell transfection and transformation for the expression of the vector of interest are known in the art.

[0136] In certain embodiments, the host cell contains a first vector encoding a first polypeptide and a second vector encoding a second polypeptide. In certain embodiments, the first vector and the second vector may be the same or different. In certain embodiments, the first polypeptide and the second polypeptide may be the same or different.

[0137] In certain embodiments, the first vector and the second vector may be introduced simultaneously or non-simultaneously. In certain embodiments, the first vector and the second vector can be introduced into the host cell together. In certain embodiments, the first vector can be introduced into the host cell first, and then the second vector is introduced. In certain embodiments, the first vector can be introduced into the host cell, and then a stable cell line expressing the first polypeptide is established, and then the second vector is introduced into the stable cell line.

[0138] In certain embodiments, the host cell contains a vector encoding a first polypeptide and a second polypeptide. In certain embodiments, the first polypeptide and the second polypeptide may be the same or different.

[0139] In another aspect, the present disclosure relates to a method for expressing a fusion protein, which includes culturing the host cell of the present disclosure under conditions for expressing the fusion protein in a polynucleotide.

[0140] Hereinafter, the present disclosure will be explained in detail by the following examples to better understand various aspects and advantages of the present disclosure. However, it should be understood that the following examples are non-limiting and are only used to illustrate certain embodiments of the present disclosure.

[0141] Example

[0142] The reagents and equipment used in the examples of the present disclosure are all conventional and commercially available.

[0143] Reagents and Consumables

[0144]

[0145]

[0146] Instrument

[0147] Name Model Manufacturer Gene amplifier TC-96 / G / H(b)C Hangzhou Bioer Technology Electrophoresis apparatus PES300 Tianneng Protein purification system SCG100 Sepax Instruments pH meter pHS-3C Shanghai Yueping

[0148] Example 1

[0149] Preparation of the fusion protein

[0150] (1) Obtaining the target gene

[0151] Using the synthetic plasmid containing the target fragment synthesized by Zhongmei Taihe Biotechnology (Beijing) Co., Ltd. as a template, PCR amplification was carried out according to the primers described in Table 1, the reaction system described in Table 2, and the reaction conditions described in Table 3 to obtain the corresponding target gene fragment.

[0152] Table 1

[0153]

[0154] Table 2

[0155] Reaction system Volume 2×RapidTaq Master Mix (Vazyme; P222-01) 10 μL Forward primer (10 μM) 1 μL Reverse primer (10 μM) 1 μL Template (0.1 μg / μL) 1 μL <![CDATA[H2O]]> Make up to 20 μL

[0156] Table 3

[0157]

[0158] * The annealing temperature needs to be adjusted according to the Tm value of the primer, and it is generally set to be 3 to 5 °C lower than the primer T m value.

[0159] (2) Construction of the expression vector: The pcDNA3.4 plasmid for expressing the fusion protein was constructed using the pcDNA3.4-TOPOTA cloning kit (purchased from Invitrogen (Shanghai) Trading Co., Ltd.). Among them, the pcDNA3.4 plasmid constructed in this example contains the fusion gene of cytokine IL-2, cytokine IL-2 receptor α, and the Fc fragment of the antibody.

[0160] (3) Transfecting cells for expression; Transfecting the vector for expressing the fusion protein constructed above into ExpiCHO-S cells, where the ExpiCHO-S cells were purchased from Invitrogen (Shanghai) Trading Co., Ltd., and transfection was carried out according to the method of the ExpiCHO expression system kit (purchased from Invitrogen (Shanghai) Trading Co., Ltd.); The cell supernatant was collected on the 10th day after transfection according to the instructions of the kit.

[0161] (4) Expression of the fusion protein: The fusion protein was expressed using the ExpiCHO expression system kit (purchased from Invitrogen (Shanghai) Trading Co., Ltd.), denoted as the IL2-IL2R-Fc fusion protein.

[0162] (5) Purification of fusion protein: The supernatant was filtered with 0.45 μm and 0.22 μm filter membranes to remove cell debris; the protein A affinity column HiTrap MabSelect SuRe (purchased from GE General) was treated with 5 column volumes of equilibration buffer (5.6 mM NaH2PO4, 14.4 mM Na2HPO4, 0.15 M NaCl, pH 7.2), and the supernatant was loaded. After loading, the loosely bound impurities were washed to the baseline with buffer (5.6 mM NaH2PO4·H2O, 14.4 mM Na2HPO4, 0.5 M NaCl, pH 7.2); the protein was eluted with 50 mM citric acid / sodium citrate buffer (containing 0.02% Tween-80 + 5% mannitol, pH 3.2); and the pH was adjusted to 7.0 with 1 M Tris-Cl (pH 8.0). The purified samples were sterilized by filtration through a 0.22 μm filter membrane and stored at 4°C.

[0163] Example 2

[0164] Structural verification of fusion proteins

[0165] SDS-PAGE: 5 μg of target protein was analyzed by SDS-PAGE on a 10% SDS-PAGE gel at 120 V for 60 minutes. After electrophoresis, the gel was stained with Coomassie Brilliant Blue to visualize the protein bands. Based on the molecular weights of the target proteins and marker protein controls, the molecular weights of the target proteins were confirmed to be 17.6 kDa for IL-2 and 50 kDa for IL-2RαFc.

[0166] Western Blot: Verify the correctness of the fusion protein using Western Blot. The electrophoresis process is the same as SDS-PAGE, and the membrane is transferred using the wet transfer method. The following antibodies are used for detection:

[0167] Primary antibody: Anti-IL-2Antibody, Rabbit Polyclonal (SinoBiological);

[0168] Secondary antibody: Goat Anti-Rabbit IgG H&L (Dylight800) (Abbkine);

[0169] Color development: Gel Doc TM XR+System(BIO-RAD).

[0170] The molecular weight and structure of the IL-2-IL-2Rα fusion protein were successfully verified by SDS-PAGE and Western Blot experiments.

[0171] Example 3

[0172] In vitro immune cell stimulation experiment

[0173] To evaluate the activation effect on lymphocytes, the activation effect of recombinant proteins T119.120, T121.122, T123.127, T124.126, T125.128 and hIL-2 (6 different concentration gradients) on CD25 on the surface of NK92 cells was verified using NK-92 cells (ATCC). The absorbance at 450 nm was measured using a CD25 ELISA kit. hIL-2 activates NK92 cells at high concentrations. T119.120, T121.122, T123.127, T124.126, and T125.128 are fusion proteins of IL-2 - IL-2α, which stimulate NK-92 cells to express CD122 and CD132, and the expression level of CD25 is lower compared to hIL-2.

[0174] Example 4

[0175] Amplification experiment of human peripheral blood mononuclear cells

[0176] To compare the differences in the activation of effector immune cells and regulatory immune cells incubated with IL2-IL2R fusion protein and rhIL-2, a cell proliferation assay was performed.

[0177] Healthy human peripheral blood mononuclear cells (Peripheral blood mononuclear cells, PBMCs) were isolated and co-cultured with Anti-CD3 antibody to activate T cells. hIL-2 and three IL2-IL2R fusion proteins (T119.120, T121.122, T123.127) were used together with CD69 + NK cells; memory precursor CD8 + T cells (MP CD8 + cells), CD4 + CD25 + Regulatory T cells (CD4 + CD25 + Tregs).

[0178] CD69 + NK cells: IL-2 has a certain effect on the activation of CD69 + NK cells, but the activation degree is not significant. The IL2-IL2R fusion proteins (T119.120, T121.122) have a lower activation effect on CD69 + NK cells compared to IL-2. T123.127 has a higher activation effect on CD69 + NK cells than hIL-2.

[0179] MP CD8 + Cells: IL-2 can significantly promote the activation of these cells. IL-2 can significantly promote the activation or expansion of subsets of these cells. The activation of these cells by three IL2-IL2R fusion proteins (T119.120, T121.122, T123.127) is lower than that of hIL-2.

[0180] CD4 + CD25 + Tregs: IL-2 significantly promotes the expansion of regulatory T cells (Tregs). The expansion of regulatory T cells (Tregs) by T119.120 is close to that of IL-2 (about 30% to 35%). The expansion of regulatory T cells (Tregs) by T121.122 and T123.127 is lower than that of IL-2.

[0181] The activation of CD69 by T123.127 + is higher than that of IL-2 for NK cells, and is close to that of IL-2 for the activation of MP CD8 + cells, and is lower than that of IL-2 for the activation of CD4 + CD25 + Tregs. T123.127 effectively and selectively activates CD8 + T cells and natural killer (NK) cells.

[0182] Example 5

[0183] Pharmacokinetics and pharmacodynamics experiments in mice

[0184] C57BL / 6 mice were injected subcutaneously to determine the pharmacokinetics of IL2-IL2R fusion proteins and hIL-2.

[0185] Female C57BL / 6 mice were injected once with a fixed dose daily for 5 consecutive days, and the results were analyzed on the 6th day.

[0186] Compared with the control group, the total number of spleen cells significantly increased for IL-2 and IL2-IL2R fusion proteins (T121.122, T123.127), but the effect of T123.127 on the total number of spleen cells was more obvious, approaching 2 times that of IL-2 and T121.122.

[0187] The effects of IL-2 and IL2-IL2R fusion proteins (T121.122, T123.127) on the cell proliferation of NK cells, CD8 + T cells, and regulatory T cells (Tregs) were also analyzed.

[0188] Compared with IL-2 and T121.122, T123.127 has a significant proliferative effect on NK cells and CD8 + T cells, but has no obvious proliferative effect on Tregs.

[0189] Example 6

[0190] Experiment on inhibiting the growth of melanoma in C57BL / 6 mice

[0191] Female C57BL / 6 mice aged 6 to 8 weeks (purchased from the Institute of Laboratory Animal Resources, National Institutes for Food and Drug Control) were used.

[0192] 5×10 5 mouse melanoma B16F10 cells / 100 μL were injected subcutaneously into the right axilla of the mice, and then the mice were randomly divided into 3 groups. Administration started on the 6th day after tumor inoculation (two groups of mice were respectively given the fusion protein IL2-IL2Rα-Fc, the control protein IL-2, and the third group of mice were not given drugs as a control), by subcutaneous injection. The drugs in each group were adjusted to an equimolar amount, and the drugs were administered twice a week for a total of 5 times. When the tumor grew to be visible to the naked eye, the length and width of the tumor were measured, and the volume of the tumor was calculated. The volume calculation method was length × width × width / 2. The specific detection results are shown in Figure 6.

[0193] It can be seen from Figure 6 that the tumor volumes of the two drug-administered groups were significantly smaller than those of the control group, and the tumor volume of the IL2-IL2Rα-Fc treatment group was the smallest at 20 days after treatment, significantly smaller than that of the IL-2 drug-administered group.

[0194] After 20 days of treatment, the mice were sacrificed by cervical dislocation, the tumors were dissected and weighed. The results are shown in Figure 7.

[0195] It can be seen from Figure 7 that the tumor weights of the two drug-administered groups were significantly smaller than those of the control group, and the tumor weight of the fusion protein IL2-IL2Rα-Fc was significantly smaller than that of the IL-2 drug-administered group.

[0196] In the present disclosure, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations.

[0197] It can be understood from the foregoing that, although specific embodiments of the present disclosure have been described for purposes of illustrative explanation, those skilled in the art can make various modifications or improvements without departing from the spirit and scope of the present disclosure. These modifications or improvements should all fall within the scope of the appended claims of the present disclosure.

Claims

1. A fusion protein, comprising: (1) IL-2 or a mutant thereof; (2) IL-2Rα; and (3) an Fc fragment; wherein the IL-2 or its mutant is linked to the IL-2Rα, and the IL-2Rα is linked to the Fc fragment through a linker.

2. The fusion protein according to claim 1, wherein the linker is (Gly4Ser)3.

3. The fusion protein according to claim 1 or 2, wherein the molecular weight of the IL-2 mutant is about 17.6 kDa.

4. The fusion protein according to any one of claims 1 to 3, wherein the molecular weight of the IL-2Rα-Fc is about 50 kDa.

5. The fusion protein according to any one of claims 1 to 4, wherein the IL-2 mutant comprises the sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:

4.

6. The fusion protein according to any one of claims 1 to 5, wherein the IL-2Rα-Fc comprises the sequence SEQ ID NO:

5.

7. A pharmaceutical composition, comprising the fusion protein according to any one of claims 101 to 106, and a pharmaceutically acceptable excipient.

8. Use of the fusion protein according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating tumors.

9. The use according to claim 8, wherein the tumor is selected from melanoma, metastatic melanoma and renal cell carcinoma.

10. Use of the fusion protein according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 7 in the preparation of a medicament for regulating immune cells.

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