Il-2 variants and uses thereof

Abstract

The application provides an IL-2 variant and application thereof, and particularly, the IL-2 variant has at least one of the following site mutations: the 3rd, 39th, 88th and 125th amino acids. The IL-2 variant provided by the application has weak binding with IL2R alpha and extremely weak binding with IL2R beta gamma, the variant has higher activity and lower immunotoxicity compared with IL-2 wild type and other types of mutants, is not prone to causing immune storm, and has good clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to IL-2 variants and their applications. Background Technology

[0002] Cancer is a major disease affecting human survival and development. Besides surgical resection, traditional cancer treatments such as chemotherapy and radiotherapy have certain side effects and a high recurrence rate. In recent years, immunotherapy, including tumor-targeting antibodies, immune checkpoint antibodies, and bispecific antibodies, has become a new hot topic and a source of new hope in the fight against cancer.

[0003] Interleukin-2 (IL-2) is an immune-activating cytokine that stimulates the body's immune system to fight cancer by activating immune cells such as NK cells and T cells. Currently, IFNa-2b (Intron-A, E. coli), PEG-IFNa-2b (PEG-INTRON, E. coli), and IFNa-2a (Roferon-A, E. coli) have been approved for clinical use in the treatment of melanoma, sarcoma, and pilocytic lymphoma. However, the high immunotoxicity of IL-2 severely limits its widespread application in the treatment of other cancer indications.

[0004] Therefore, further development of IL-2 proteins with low immunotoxicity and high activity is still needed. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art.

[0006] The clinical application of IL-2 is limited by its immunotoxicity. For example, IL-2 can promote the proliferation and activation of T cells, especially CD4⁺ and CD8⁺ T cells. High doses of IL-2 can lead to excessive activation of T cells, releasing large amounts of cytokines and triggering a cytokine storm. IL-2 has three different receptors: IL2Rα, IL2Rβγ, and IL2Rαβγ. Treg cells highly express IL-2Rα (CD25) and can preferentially consume IL-2, thereby inhibiting the activation of other immune cells. In low-dose IL-2 treatment, the activity of Treg cells may limit the therapeutic effect, while in high-dose IL-2 treatment, the function of Treg cells may be suppressed, leading to immunotoxicity. Some endothelial cells also express IL-2Rα, and IL-2 can directly act on these cells, leading to vascular leakage syndrome. Therefore, through extensive preliminary screening, this invention has discovered a variety of IL-2 variants. These variants have a mutation at amino acid position 39 compared to wild-type IL-2, which results in weak binding to IL2Rα, very weak binding to IL2Rβγ, and the highest affinity for IL2Rαβγ. Compared to wild-type IL-2 and other types of IL-2 mutants, they exhibit higher activity and lower immunotoxicity.

[0007] Therefore, in a first aspect, the present invention provides an IL-2 variant. According to an embodiment of the invention, compared to the wild-type IL-2 protein, the IL-2 variant has at least one of the following site mutations: amino acids at positions 3, 39, 88, and 125. In previous studies of the present invention, various IL-2 variants were designed and investigated, and it was unexpectedly discovered that the IL-2 variant carrying the above-mentioned mutation sites weakly binds to IL2Rα and very weakly binds to IL2Rβγ. This variant exhibits higher activity and lower toxicity compared to wild-type IL-2 and other types of mutants.

[0008] According to embodiments of the present invention, the above-described IL-2 variant may further include at least one of the following additional technical features: According to embodiments of the present invention, compared to wild-type IL-2 protein, the IL-2 variant has at least one of the following mutations: the 3rd amino acid is mutated from T to a neutral amino acid A, N, C, E, G, I, L, M, F, P, S, W, Y, or V; the 39th amino acid is mutated from M to a neutral amino acid A, N, C, E, G, I, L, M, F, P, S, W, Y, or V; the 88th amino acid is mutated from N to a neutral amino acid A, N, C, E, G, I, L, M, F, P, S, W, Y, or V; and the 125th amino acid is mutated from C to a neutral amino acid A, N, C, E, G, I, L, M, F, P, S, W, Y, or V. Thus, compared to wild-type IL-2, the IL-2 variant exhibits lower immunotoxicity and higher activity.

[0009] According to an embodiment of the present invention, the IL-2 variant has at least one of the following mutations compared to the wild-type IL-2 protein: T3A, M39E, N88E, and C125A.

[0010] According to an embodiment of the invention, the IL-2 variant has the amino acid sequence shown in SEQ ID NO:1 or 15, or an amino acid sequence having at least 90% identity with it. An unexpected discovery of the invention is that the IL-2 variant carrying the M39E mutation site binds weakly to IL2Rα and very weakly to IL2Rβγ.

[0011] According to an embodiment of the present invention, the IL-2 variant can also be connected to the Fc region.

[0012] According to an embodiment of the present invention, the C-terminus of the IL-2 variant is connected to the N-terminus of the Fc region.

[0013] According to an embodiment of the present invention, at least a portion of the Fc region is derived from at least one of a mouse antibody, a primate antibody, or a mutant thereof.

[0014] According to an embodiment of the present invention, at least a portion of the Fc region is derived from mouse IgG1 or human IgG1 or a mutant thereof.

[0015] According to an embodiment of the present invention, the Fc region, relative to the amino acid sequence of the Fc fragment of human wild-type IgG1, has at least one of the following mutation sites: Y349C, T366S, L368A, and Y407V.

[0016] According to an embodiment of the present invention, the Fc region, relative to the amino acid sequence of the Fc fragment of human wild-type IgG1, further has at least one of the following mutation sites: L234A and L235A.

[0017] According to an embodiment of the present invention, the Fc region has a P329G mutation site relative to the amino acid sequence of the Fc fragment of human wild-type IgG1.

[0018] It should be noted that the Fc regions represented by "hoss" and "kbss" do not contain the last two amino acid residues G and K of the Fc fragment of the human wild-type IgG1, as shown in Table 1.

[0019] According to an embodiment of the present invention, the IL-2 variant has the amino acid sequence shown in SEQ ID NO:4, 7 or 17 or an amino acid sequence having at least 90% identity with it.

[0020] In a second aspect, the present invention provides a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes the IL-2 variant described in the first aspect. The nucleic acid molecule according to embodiments of the invention is capable of efficiently encoding the aforementioned IL-2 variant.

[0021] It should be noted that in this embodiment of the invention, the Fc region linked to the IL-2 variant acts as a tag. The Fc region is a novel and convenient affinity tag that can facilitate the detection, purification, and localization of proteins.

[0022] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0023] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is also disclosed. Furthermore, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0024] In a third aspect, the present invention provides an expression vector. According to an embodiment of the invention, the vector carries the nucleic acid molecule described in the second aspect. When the nucleic acid molecule is ligated to the vector, the nucleic acid molecule can be directly or indirectly linked to control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. These control elements can be directly derived from the vector itself or are exogenous, i.e., not derived from the vector itself. Of course, the nucleic acid molecule and the control elements need to be operably linked. In this context, "operably linked" means ligating a foreign gene to the vector so that the control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform their intended function of regulating the transcription and translation of the foreign gene. Commonly used vectors include plasmids, bacteriophages, etc. After the expression vector according to some specific embodiments of the present invention is introduced into suitable recipient cells, the aforementioned IL-2 variant can be effectively obtained in large quantities in vitro under the mediation of a regulatory system.

[0025] According to an embodiment of the present invention, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0026] According to an embodiment of the present invention, the expression vector is a plasmid expression vector.

[0027] In a fourth aspect, the present invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell carries the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or expresses the IL-2 variant described in the first aspect. Using this recombinant cell, under suitable conditions, the aforementioned IL-2 variant can be efficiently expressed intracellularly.

[0028] It should be noted that the "suitable conditions" mentioned in this application specification refer to conditions suitable for the expression of the IL-2 variant described in this invention. Those skilled in the art will readily understand that suitable conditions for the expression of the IL-2 variant include, but are not limited to, suitable transformation or transfection methods, healthy host cell status, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the IL-2 variant based on the specific environment of their laboratory.

[0029] According to an embodiment of the present invention, the recombinant cells are obtained by introducing the expression vector described in the third aspect into a host cell.

[0030] According to an embodiment of the present invention, the recombinant cells are eukaryotic cells.

[0031] According to an embodiment of the present invention, the recombinant cell is a mammalian cell.

[0032] In a fifth aspect, the present invention provides a composition comprising at least one of the following, according to an embodiment of the invention: The IL-2 variants described in the first aspect; The nucleic acid molecules described in the second aspect; The expression carrier described in the third aspect; or The recombinant cells described in the fourth aspect.

[0033] According to an embodiment of the invention, the composition further comprises a pharmaceutically acceptable carrier.

[0034] The compositions of this invention can be administered by any acceptable method of administration. The compositions of this invention can be formulated into solid, semi-solid, liquid, or gaseous formulations, such as injections or lyophilized powders, and current methods for preparing these dosage forms are known or readily apparent to those skilled in the art. Typical routes of administration of such compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, oral, rectal, vaginal, and intranasal routes. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. The compositions of this invention are formulated to allow the bioactive components contained therein to be bioavailable after administration to a subject.

[0035] As used herein, a "pharmaceuticalally acceptable" ingredient is a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents.

[0036] The compositions of this invention contain a safe and effective amount of the active ingredient of this invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched with the route of administration, wherein the route of administration can be oral, nasal, intradermal, subcutaneous, intramuscular, intravenous, or intraperitoneal. The dosage forms of the pharmaceuticals of this invention are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. For example, they are prepared using physiological saline or aqueous solutions containing glucose and other excipients by conventional methods. The pharmaceutical compositions are preferably manufactured under sterile conditions. The pharmaceutical compositions can be administered by intravenous infusion or injection, or by intramuscular or subcutaneous injection.

[0037] In a sixth aspect, the invention provides a coupling. According to an embodiment of the invention, the coupling comprises the IL-2 variant described in the first aspect; and a coupling portion connected to the IL-2 variant.

[0038] According to an embodiment of the invention, the coupling portion includes a purification tag or label.

[0039] According to an embodiment of the present invention, the purification label or marker includes: a luminescent substance, a colored substance, an affinity label, or an enzyme.

[0040] According to an embodiment of the present invention, the marker is selected from radioactive markers, fluorescent markers, chromophore markers, electron-dense markers, or spin markers.

[0041] According to embodiments of the present invention, the enzyme is selected from one or more of the following: radioisotopes, fluorophores, rhodamine, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, glucose-6-phosphate dehydrogenase, and biotin / antibiotin protein.

[0042] According to an embodiment of the present invention, the purification label or marker is selected from one or more of colloidal gold, magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0043] According to a specific embodiment of the present invention, the affinity label includes an Fc region.

[0044] In a seventh aspect, the present invention provides the use of the IL-2 variant described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, or the composition described in the fifth aspect in the preparation of a medicament for the prevention and / or treatment of cancer. As previously described, the IL-2 variant according to embodiments of the present invention has a mutation at amino acid position 39 compared to wild-type IL-2, resulting in weak binding to IL2Rα, very weak binding to IL2Rβγ, and the highest affinity for IL2Rαβγ. Compared to wild-type IL-2 and other types of IL-2 mutants, it exhibits higher activity and lower immunotoxicity, and can be effectively used for the treatment and / or prevention of IL-2-mediated cancers.

[0045] According to embodiments of the present invention, the cancer includes at least one selected from colorectal cancer, bowel cancer, melanoma, sarcoma, pilocellular lymphoma, kidney cancer, lung cancer, gastric cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, tongue cancer, nasopharyngeal carcinoma, multiple myeloma, and bladder cancer.

[0046] In an eighth aspect, the present invention provides a method for treating and / or preventing cancer. According to embodiments of the invention, the method comprises administering to a subject a pharmaceutically effective amount of the aforementioned IL-2 variant or composition. The method of the present invention can effectively treat or prevent IL-2-mediated cancer.

[0047] According to embodiments of the present invention, the cancer includes at least one selected from colorectal cancer, bowel cancer, melanoma, sarcoma, pilocellular lymphoma, kidney cancer, lung cancer, gastric cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, tongue cancer, nasopharyngeal carcinoma, multiple myeloma, and bladder cancer.

[0048] The effective amount of the IL-2 variant or composition described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the subject's weight, the subject's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0049] The IL-2 variants or compositions of the present invention can be incorporated into medicaments suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These medicaments can be prepared in various forms, such as liquids, semi-solids, and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions) or lyophilized powders. Typical medicaments are in the form of injection solutions or infusion solutions. The aforementioned IL-2 variants or compositions can be administered by intravenous infusion or injection, or by intramuscular or subcutaneous injection.

[0050] According to an embodiment of the present invention, the method is administered via subcutaneous injection or intravenous injection.

[0051] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0052] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0053] Figure 1 This is a sequence comparison diagram of an IL-2 variant and wild-type IL-2 in one embodiment of the present invention; Figure 2 This is an ELISA detection result diagram of hIgG1, IL-2wt-Fc, IL-2v04-Fc, and IL-2v88 binding with IL-2Rα or IL-2Rβγ in one embodiment of the present invention. Figure 3 This is an ELISA detection result diagram of the binding of hIgG1, IL-2wt-Fc, IL-2v8804-Fc, IL-2v2149-Fc and IL-2Rα in one embodiment of the present invention; Figure 4 This is an ELISA detection result diagram of the binding of hIgG1, IL-2wt-Fc, IL-2v8804-Fc, IL-2v2149-Fc and IL-2Rβγ in one embodiment of the present invention; Figure 5 This is a flow cytometry result of hIgG1, IL-2v8804-Fc, and IL-2v2149-Fc promoting STAT5 phosphorylation in T cells in one embodiment of the present invention. Figure 6This is an ELISA detection result of the binding of hIgG1, IL-2v8804×mPD-1, IL-2v2149×mPD-1, and IL-2wt×mPD-1 fusion protein to IL-2Rα in one embodiment of the present invention. Figure 7 The image shows the ELISA detection results of the binding of hIgG1, IL-2v8804×mPD-1, IL-2v2149×mPD-1, and IL-2wt×mPD-1 fusion protein to IL-2Rβγ in one embodiment of the present invention. Figure 8 The figure shows the survival rate of IL-2wt×mPD-1, IL-2v8804×mPD-1, IL-2v88×mPD-1, and IL-2v2149×mPD-1 fusion proteins in C57BL / 6 mice after administration in one embodiment of the present invention. Figure 9 The figure shows the results of detecting the effects of PBS, IL-2v8804×mPD-1, IL-2v2214×mPD-1 fusion protein and known PD1 antibody on tumor volume and body weight in a C57BL / 6 mouse MC38 tumor model in one embodiment of the present invention. Detailed Implementation

[0054] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0055] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0056] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0057] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0058] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0059] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0060] In this paper, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). Numerous algorithms exist for aligning sequences and determining sequence identity, including: the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul...). See, Meth.Enzym., 266:460-480 (1996); or GAP, BESTFIT, BLAST Altschul, etc., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0061] In this paper, the term "at least 90% identity" means at least 90% identity with each reference sequence, which may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0062] In this document, the amino acid numbering of the IgG1 Fc portion is based on the EU numbering system. For example, position 366 refers to position 366 according to the EU numbering system; "T366W" means that threonine at position 366 according to the EU numbering system is replaced by tryptophan; and "L368A" means that leucine at position 368 according to the EU numbering system is replaced by alanine.

[0063] In this document, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule to host cells and / or between host cells. The expression vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The expression vector also includes vectors having multiple of the aforementioned functions. The expression vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the expression vector, the expression vector can produce the desired expression product.

[0064] In this document, the term "recombinant cell" generally refers to a cell in which the genetic material of a host cell is modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of this invention and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this invention include immortalized hybridoma cells, NS / O myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0065] In this document, the term "composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely chopped solid carrier, or both.

[0066] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients, except those that are incompatible with the compounds of the present invention, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0067] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The fusion protein or pharmaceutical composition of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, the compositions of the present invention are administered via intravenous or subcutaneous injection.

[0068] In this document, the term "treatment" refers to the administration of a drug or compound to an individual to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in an individual who is susceptible but has not yet been diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0069] Without substantially affecting IL-2 activity (retaining at least 95% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequence of the present invention to obtain variants of the sequence of said IL-2 or its functional fragments. These are all considered to be included within the scope of protection of the present invention. For example, amino acids with similar properties can be substituted in the variable region. The variant sequences of the present invention can have at least 90%, 85%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence. The sequence identity described in the present invention can be measured using sequence analysis software, such as the computer program BLAST using default parameters, especially BLASTP or TBLASTN. The amino acid sequences mentioned in the present invention are shown in N-terminus to C-terminus arrangement.

[0070] The nucleic acid or amino acid sequences used in this application are shown in Table 1: Table 1:

[0071] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0072] Unless otherwise specified, the practice of this disclosure will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994) and "Current Protocols in Immunology" (JEColigan et al., ed., 2011), each of which is explicitly incorporated herein by reference.

[0073] In this embodiment of the invention, the nucleotide sequence used to prepare the expression vector can be obtained from its amino acid sequence using conventional methods or conventional software (such as the online program Vectorbuilder (website: https: / / www.vectorbuilder.cn / tool / codon-optimization.html), GeneOptimizer online program, etc.).

[0074] Example 1: Protein Preparation This invention modifies wild-type IL-2 to obtain variants with higher activity and lower immunotoxicity, less likely to induce an immune storm. After multiple screenings, variants named IL-2v8804 and IL-2v04 were obtained, and their specific sequences are shown in Table 1. For testing, this invention links the C-terminus of the above variants to the Fc region of an affinity tag. A series of fusion proteins were also prepared, such as wild-type IL-2, the known IL-2v88 variant, and the known IL-2v2149 and IL-2v2214 variants (all from patent CN118119635A) fused with the Fc region, to demonstrate the effectiveness of the IL-2 variants obtained in this application.

[0075] The specific experimental procedures for protein preparation are as follows: (1) ExpiCHO cells (purchased from Thermo Fisher) were cultured in ExpiCHO Expression Medium (purchased from Thermo Fisher) and the cell concentration was adjusted to 6×10⁻⁶. 6 / mL, to obtain ExpiCHO cell solution. (2) When the protein has 1 strand, add the pcDNA3.4 vector containing the coding sequence (commissioned to Nanjing GenScript) to 2mL OptiSFM medium (purchased from Thermo Fisher) to obtain solution a; or, when the protein has 2 strands, add two pcDNA3.4 vectors containing three coding sequences (commissioned to Nanjing GenScript) in a 1:1 ratio to 2mL OptiSFM medium (purchased from Thermo Fisher) to obtain solution a; or, when the protein has 3 strands, add three pcDNA3.4 vectors containing three coding sequences (commissioned to Nanjing GenScript) in a 1:1:1 ratio to 2mL OptiSFM medium (purchased from Thermo Fisher) to obtain solution a; (3) Add 160μL ExpiFectamineCHO transfection reagent (purchased from Thermo Fisher) to 2mL OptiSFM medium (purchased from Thermo Fisher) to obtain solution b. (4) Then mix solutions a and b to obtain a transfection mixture, and add the entire transfection mixture to 50 mL of ExpiCHO cell solution within 5 minutes. (5) After culturing at 37°C and 5% CO2 for 1 day, add 8 mL of Feed and 300 μL of Lenhancer (purchased from Thermo Fisher), and then transfer to 32°C and 5% CO2 for 9 days. Harvest the culture supernatant, adding 8 mL of Feed on day 5. (6) Use a Protein A purification column (purchased from Nanomicro) to affinity purify the target cytokine variant and fusion protein from the culture supernatant.

[0076] In this embodiment, a total of 12 proteins were prepared to examine the various properties of the fusion protein of the present invention. The amino acid sequence of each protein is shown in Table 2.

[0077] Table 2: Cytokine variants and fusion proteins and their corresponding amino acid sequences

[0078] Example 2: IL-2v-Fc protein ELISA binding assay This embodiment uses ELISA to detect the binding characteristics of IL-2v04-Fc, IL-2v88-Fc, IL-2v8804-Fc, IL-2wt-Fc, and IL-2v2149-Fc. IL-2 receptors IL-2Rα and IL-2Rβγ proteins were coated into 96-well plates, and the signal strength after the addition of these fusion proteins was measured to determine the binding characteristics of the proteins to various IL-2 receptors. hIgG1 was used as a control. The specific experimental procedures are as follows: Dilute IL-2Rα and IL-2Rβγ proteins (purchased from Acro) to 2 μg / ml with PBS buffer, and add 100 μl / well to each well of a 96-well plate. Incubate overnight at 4°C. Remove the PBS buffer from the 96-well plate, wash 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20), and add 200 μl / well of PBS / 10% BSA. Incubate at 37°C for 2 h for blocking. Remove the blocking buffer, wash 6 times with PBST, and add 100 μl / well of the target IL-2wt-Fc, IL-2v04-Fc, IL-2v88-Fc, IL-2v8804-Fc, and IL-2v2149-Fc, serially diluted with PBST / 0.05% BSA. Incubate at 37°C for 1 h. hIgG1 was used as a control group. Remove the reaction mixture, wash the plate 6 times with PBST, and then dilute HRP (horseradish peroxidase)-labeled anti-human IgG antibody secondary antibody (purchased from Jackson Lab) with PBST / 0.05% BSA at 100 μl / well. Incubate at 37°C for 1 h. After washing the plate 6 times with PBST, add 80 μl / well of TMB (tetramethylbenzidine), incubate at room temperature for 3 min, and then stop the reaction by adding 80 μl / well of 4M sulfuric acid. Read the absorbance at 450 nm using a microplate reader.

[0079] Figure 2 The results show that the binding of IL-2v04-Fc to IL-2Rα and IL-2Rβγ is significantly weaker than that of IL-2wt-Fc; while the binding of IL-2v88-Fc to IL-2Rβγ is significantly weaker than that of IL-2wt-Fc, and the binding of IL-2v88-Fc to IL-2Rα is similar to that of IL-2wt-Fc.

[0080] Figure 3 The results show that the binding affinity of IL-2v8804-Fc to IL-2Rα is weaker than that of IL-2wt-Fc and IL-2v2149-Fc.

[0081] Figure 4 The results showed that the binding affinity of IL-2v8804-Fc of the present invention to IL-2Rβγ was weaker than that of IL-2wt-Fc and IL-2v2149-Fc.

[0082] Based on the mutation sites of each IL-2 variant and the above results, it can be concluded that the mutation at amino acid M at position 39 is the key site where the binding affinity of the IL-2 variant changes.

[0083] Example 3: Detection of the activity of the IL-2v-Fc protein in promoting T cell STAT5 phosphorylation according to the present invention. Flow cytometry was used to detect the properties of IL-2v8804-Fc and IL-2v2149-Fc in promoting STAT5 phosphorylation in T cells. In this embodiment, the fusion protein was added to human peripheral blood T cells, and the strength of the pSTAT5 signal after the addition of the fusion protein was used to determine the effect of the fusion protein on T cells. The aforementioned hIgG1 was used as a control.

[0084] Human peripheral blood T cells were diluted to 2 × 10⁶ cells using complete RPMI 1640 medium. 6 / ml (purchased from Selene Biotech), 100μl / tube was added to a 96-well plate, and serially diluted IL-2v8804-Fc and IL-2v2149-Fc (obtained in Example 1 of this invention) and control hIgG1 (purchased from BioLegend Biotech) were added. The plate was incubated at 37°C for 1 h. The cells were then labeled with pSTAT5 fluorescent antibody (purchased from Biolegend), and then resuspended in 200μl / tube of PBS for flow cytometry analysis.

[0085] The results are as follows Figure 5 As shown, the IL-2v8804-Fc of the present invention promotes T cell STAT5 phosphorylation much weaker than IL-2v2149-Fc.

[0086] Example 4: ELISA binding assay of IL-2v×mPD-1 fusion protein In this embodiment, the binding characteristics of the fusion proteins IL-2v8804×mPD-1, IL-2wt×mPD-1, and IL-2v2149×mPD-1 were detected using an ELISA assay. The IL-2 receptors IL-2Rα and IL-2Rβγ were coated into 96-well plates. The signal strength after the addition of the fusion proteins was used to determine the binding characteristics of the fusion proteins to various IL-2 receptors. hIgG1 was used as a control.

[0087] Dilute IL-2Rα and IL-2Rβγ proteins (purchased from Acro) to 2 μg / ml with PBS buffer, and add 100 μl / well to each well of a 96-well plate. Incubate overnight at 4°C. Remove the PBS buffer from the 96-well plate, wash 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20), and add 200 μl / well of PBS / 10% BSA. Incubate at 37°C for 2 h for blocking. Remove the blocking buffer, wash 6 times with PBST, and add 100 μl / well of serially diluted IL-2wt×mPD-1, IL-2v8804×mPD-1, and IL-2v2149×mPD-1 (PBST / 0.05% BSA). Incubate at 37°C for 1 h. Remove the reaction mixture, wash the plate 6 times with PBST, and then dilute HRP (horseradish peroxidase)-labeled anti-human IgG antibody secondary antibody (purchased from Jackson Lab) with PBST / 0.05% BSA at 100 μl / well. Incubate at 37°C for 1 h. After washing the plate 6 times with PBST, add 80 μl / well of TMB (tetramethylbenzidine), incubate at room temperature for 3 min, and then stop the reaction by adding 80 μl / well of 4M sulfuric acid. Read the absorbance at 450 nm using a microplate reader.

[0088] Figure 6 The results show that the binding affinity of IL-2v8804×mPD-1 to IL-2Rα is weaker than that of IL-2v2149×mPD-1 and IL-2wt×mPD-1.

[0089] Figure 7 The results show that the binding affinity of IL-2v8804×mPD-1 to IL-2Rβγ is weaker than that of IL-2wt×mPD-1.

[0090] Example 5: Toxicity of IL-2v×mPD-1 fusion protein in C57BL / 6 mice In this embodiment, based on the survival of mice after drug administration, the toxicity of the IL-2v8804×mPD-1, IL2wt×mPD-1, IL2v88×mPD-1 and IL-2v2149×mPD-1 fusion proteins of the present invention and their different doses in mice was tested.

[0091] (1) Based on body weight, 7-week-old female C57BL / 6 mice were randomly divided into 6 groups of 5 or 6 mice each; (2) After grouping, mice were injected twice a week via tail vein with the above-mentioned fusion protein and the solvent control PBS, 250 μl per mouse. The specific injection dosage is as follows: Figure 7 As shown; (3) Observe the survival status of the mice every day, and weigh and record the weight of the mice twice a week.

[0092] The results are as follows Figure 8 As shown, administration of 5 mg / kg IL-2wt×mPD-1 (all 6 mice died), 5 mg / kg IL-2v88×mPD-1 (2 out of 5 mice died), 20 mg / kg IL-2v2149×mPD-1 (all 6 mice died), and 40 mg / kg IL-2v2149×mPD-1 (all 6 mice died) all caused death in mice. However, all mice survived administration of 20 mg / kg IL-2v8804×mPD-1 and 40 mg / kg IL-2v8804×mPD-1. These results indicate that the toxicity of IL-2v8804 is significantly lower than that of IL-2wt, IL-2v88, and IL-2v2149.

[0093] Example 6: Anti-cancer experiment of IL-2v×mPD-1 fusion protein in C57BL / 6 mouse model In this embodiment, an in vivo pharmacodynamic experiment was used to detect the function of the IL-2wt×mPD-1, IL-2v8804×mPD-1, and IL-2v2214×mPD-1 fusion proteins of the present invention in promoting anti-cancer activity in mice.

[0094] (1) Subcutaneous tumors were implanted on the right ventral side of 7-week-old female C57BL / 6 mice. Each mouse was injected with 3×10 6 One MC38 colorectal cancer cell; (2) The tumor volume grew to approximately 300 mm. 3 On day 0, the tumor volume of the mice was measured, they were weighed, and the mice were randomly grouped according to their tumor volume and body weight. (3) Twice a week, mice were injected via the tail vein with the fusion protein and the solvent control PBS (i.e., the Vehicle group), 250 μl per mouse; (4) After the above fusion protein was injected, the tumor volume was measured twice a week and the mice were weighed.

[0095] The results are as follows Figure 9 As shown, the IL-2v8804×mPD-1 and IL-2v2214×mPD-1 fusion proteins have similar anti-cancer functions; the IL-2v2214×mPD-1 fusion protein caused weight loss and death in mice (4 out of 6 mice died); while PD1×IL-2v8804 did not cause weight loss or death in mice; these results indicate that IL-2v8804 has effective anti-cancer function while having better safety.

[0096] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

[0097] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0098] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An IL-2 variant, characterized in that, The amino acid sequence of the IL-2 variant is shown in SEQ ID NO:1 or 15.

2. The IL-2 variant according to claim 1, characterized in that, The amino acid sequence of the IL-2 variant is shown in SEQ ID NO:4, 7 or 17.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the IL-2 variant as described in any one of claims 1 to 2.

4. An expression carrier, characterized in that, It contains the nucleic acid molecule as described in claim 3.

5. The expression vector according to claim 4, characterized in that, The expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

6. The expression vector according to claim 4, characterized in that, The expression vector is a plasmid expression vector.

7. A recombinant cell, characterized in that, The recombinant cells contain the nucleic acid molecule of claim 3, the expression vector of any one of claims 4 to 6, or express the IL-2 variant of any one of claims 1 to 2.

8. The recombinant cell according to claim 7, characterized in that, The recombinant cells are obtained by introducing the expression vector according to any one of claims 4 to 6 into the host cell.

9. The recombinant cell according to claim 7, characterized in that, The recombinant cells are eukaryotic cells.

10. The recombinant cell according to claim 7, characterized in that, The recombinant cells are mammalian cells.

11. A composition, characterized in that, Includes at least one of the following: The IL-2 variant according to any one of claims 1 to 2; The nucleic acid molecule according to claim 3; The expression vector according to any one of claims 4 to 6; or The recombinant cells according to any one of claims 7 to 10.

12. The composition according to claim 11, characterized in that, The composition further includes a pharmaceutically acceptable carrier.

13. A coupling, characterized in that, It consists of the following: The IL-2 variant according to any one of claims 1 to 2; and The coupling portion is linked to the IL-2 variant, and the coupling portion is a purification tag or label.

14. The coupling according to claim 13, characterized in that, The purification tags or labels include: luminescent substances, colored substances, affinity tags, and enzymes.

15. The coupling according to claim 13, characterized in that, The markers are selected from radioactive markers, fluorescent markers, chromophore markers, electron-dense markers, or spin markers.

16. The coupling according to claim 14, characterized in that, The enzyme is selected from one or more of luciferase, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactosyloxidase, and glucose-6-phosphate dehydrogenase.

17. The coupling according to claim 15, characterized in that, The marker is selected from radioactive isotopes or fluorophores.

18. The coupling according to claim 17, characterized in that, The fluorophore includes one or more of rhodamine and fluorescein.

19. The coupling according to claim 13, characterized in that, The purification label or marker is selected from one or more of colloidal gold, magnetic microspheres, plastic microspheres, and plastic microparticles.

20. The coupling according to claim 14, characterized in that, The affinity tag is an Fc region or a biotin / antibiotin protein.

21. Use of the IL-2 variant of any one of claims 1-2, the nucleic acid molecule of claim 3, the expression vector of any one of claims 4-6, the recombinant cell of any one of claims 7-10, or the composition of any one of claims 11-12 in the preparation of a medicament for treating cancer, wherein the cancer is at least one of intestinal cancer, sarcoma, pilocellular lymphoma, renal cancer, lung cancer, gastric cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, glioma, esophageal cancer, head and neck cancer, multiple myeloma, and bladder cancer.

22. The use according to claim 21, characterized in that, The cancer is at least one of colorectal cancer, oral squamous cell carcinoma, tongue cancer, melanoma, and nasopharyngeal carcinoma.

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