IL-2 variant, PD-1 antibody and VEGF receptor fusion protein and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to an IL-2 variant, PD-1 antibody and VEGF receptor fusion protein and application thereof. The invention provides a fusion protein which comprises a first functional binding region, the first functional binding region is provided with an IL-2 mutant, the IL-2 variant is weakly bound with IL2R alpha and extremely weakly bound with IL2R beta gamma, and compared with IL-2 wild type and other types of mutants, the variant has higher activity and lower immunotoxicity and is not prone to causing immune storm. Compared with fusion proteins prepared from IL-2 wild type and other types of mutants, the fusion protein prepared from the IL-2 variant has a stronger anti-tumor immune function, is lower in immunotoxicity and has a good clinical application value. Compared with the PD-1 antibody, the fusion protein of the PD-1 antibody and the IL-2 variant, and the fusion protein of the PD-1 antibody and the VEGF antibody or the VEGF receptor, the IL-2 variant, the PD-1 antibody and the VEGF receptor can simultaneously block the combination of the PD-1 and the PDL1 and the combination of the VEGF and the VEGF receptor, and have stronger anti-cancer activity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to IL-2 variants, PD-1 antibodies, and VEGF receptor fusion proteins 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 and their fusion proteins with low immunotoxicity and high activity is still needed. Summary of the Invention

[0005] This invention aims to at least partially address one of the technical problems in related technologies. Therefore, one object of this invention is to provide a fusion protein comprising a first functional binding region having an IL-2 mutant. The IL-2 variant of this invention weakly binds to IL-2Rα and very weakly binds to IL-2Rβγ. Compared to wild-type IL-2 and other types of mutants, this variant exhibits higher activity and lower immunotoxicity, and is less likely to induce an immune storm. The fusion protein prepared using this IL-2 variant, compared to fusion proteins prepared using wild-type IL-2 and other types of mutants, possesses stronger anti-tumor immune function and lower immunotoxicity, demonstrating good clinical application value.

[0006] Therefore, a first aspect of the present invention provides a fusion protein, characterized in that it includes a first functional binding region, wherein the first functional binding region has an IL-2 mutant. The amino acid sequence of the IL-2 mutant was obtained by mutating at least one of the following positions in the amino acid sequence of the wild-type IL-2 protein: The 3rd, 39th, 88th and 125th positions.

[0007] 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 T cell overactivation, releasing large amounts of cytokines and triggering a cytokine storm. Furthermore, Treg cells highly express IL-2Rα (CD25) and preferentially consume IL-2, thereby inhibiting the activation of other immune cells. In low-dose IL-2 treatment, Treg cell activity may limit the therapeutic effect, while in high-dose IL-2 treatment, Treg cell function 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 discovered an IL-2 variant that weakly binds to IL-2Rα and very weakly binds to IL-2Rβγ. Compared to wild-type IL-2 and other types of IL-2 mutants, this variant exhibits higher activity and lower immunotoxicity. Fusion proteins prepared using this IL-2 variant have stronger anti-tumor immune function and lower immunotoxicity compared to fusion proteins prepared from wild-type IL-2 and other types of mutants, demonstrating good clinical application value.

[0008] According to an embodiment of the present invention, compared with the amino acid sequence of the wild-type IL-2 protein, the amino acid sequence of the IL-2 mutant has at least one of the following mutations (1) to (4): (1) The third amino acid is mutated from T to A, N, C, E, G, I, L, M, F, P, S, W, Y or V; (2) The 39th amino acid is mutated from M to A, N, C, E, G, I, L, M, F, P, S, W, Y or V; (3) The 88th amino acid is mutated from N to A, N, C, E, G, I, L, M, F, P, S, W, Y or V; (4) The amino acid at position 125 is mutated from C to A, N, C, E, G, I, L, M, F, P, S, W, Y or V.

[0009] Therefore, compared with wild-type IL-2, the IL-2 mutant has lower immunotoxicity and higher activity.

[0010] According to an embodiment of the present invention, the amino acid sequence of the IL-2 mutant has at least one of the following mutations: p.Thr3Ala, p.Met39Glu, p.Asn88Glu, and p.Cys125Ala.

[0011] According to an embodiment of the present invention, the amino acid sequence of the IL-2 mutant has the following mutations: p.Thr3Ala, p.Met39Glu, p.Asn88Glu, and p.Cys125Ala.

[0012] According to an embodiment of the present invention, the amino acid sequence of the IL-2 mutant is the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 90% identity with it.

[0013] According to an embodiment of the present invention, the fusion protein further includes a second functional binding region having PD-1 binding activity.

[0014] According to an embodiment of the present invention, the fusion protein further includes a third functional binding region, which has the activity of blocking the binding of VEGF to VEGFR.

[0015] Based on the low-toxicity and high-efficiency IL-2 variant of the present invention, the present invention provides an IL-2 variant fusion protein with PD-1 antibody and VEGF receptor. Compared with PD-1 antibody, PD-1 antibody fusion protein with IL-2 variant, PD-1 antibody fusion protein with VEGF antibody, or PD-1 antibody fusion protein with VEGF receptor, the IL-2 variant fusion protein with PD-1 antibody and VEGF receptor of the present invention can simultaneously block the binding of PD-1 to PDL1 and the binding of VEGF to VEGF receptor, and has stronger anti-cancer activity, thus having good clinical application value.

[0016] According to embodiments of the present invention, the fusion protein has at least one of the following characteristics: 1) The first functional binding region is connected to the N-terminus or C-terminus of the Fc segment; 2) The second functional binding region is connected to the N-terminus or C-terminus of the Fc segment; 3) The second functional binding region is selected from the Fab fragment; 4) The second functional combination area includes HCDRs and LCDRs, wherein the HCDRs and / or LCDRs are defined by Kabat, Chothia, AbM, Contact or IMGT; 5) The second functional integration area includes: HCDR1, HCDR2, and HCDR3 are represented by the amino acid sequences of SEQ ID NO: 31-33, respectively. LCDR1, LCDR2, and LCDR3 are shown in the amino acid sequences of SEQ ID NO: 34-36, respectively; 6) The third functional binding region is connected to the N-terminus or C-terminus of the Fc segment; 7) The third functional binding region includes at least one fusion protein unit formed by linker peptides connecting the extracellular domains of VEGFR1 and VEGFR2.

[0017] According to embodiments of the present invention, the fusion protein has at least one of the following characteristics: a) The first functional binding region is connected to the N end of the Fc segment; b) The second functional binding region is connected to the N-terminus of the Fc segment; c) The third functional binding region is connected to the C-terminus of the Fc segment; d) The second functional binding region has a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO: 37 and a light chain variable region as shown in the amino acid sequence of SEQ ID NO: 38; e) The amino acid sequence of the extracellular domain of VEGFR1 in the third functional binding region is shown in SEQ ID NO: 39, and the amino acid sequence of the extracellular domain of VEGFR2 is shown in SEQ ID NO: 40. f) The third functional binding region comprises two of the fusion protein units.

[0018] According to an embodiment of the present invention, the C-terminus of the first functional binding region is connected to the N-terminus of the Fc segment.

[0019] According to an embodiment of the present invention, the first functional binding region further includes a first Fc region, and the C-terminus of the IL-2 mutant is connected to the N-terminus of the first Fc region.

[0020] According to an embodiment of the present invention, the second functional binding region further includes a heavy chain constant region and / or a light chain constant region.

[0021] According to embodiments of the present invention, at least a portion of the heavy chain constant region and / or light chain constant region is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, red junglefowl antibodies, turkey antibodies, fighting rooster antibodies, and mutants thereof.

[0022] According to embodiments of the present invention, at least a portion of the heavy chain constant region and / or light chain constant region is derived from at least one of mouse antibodies, human antibodies, and primate antibodies.

[0023] According to embodiments of the present invention, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or the light chain constant region includes a light chain constant region selected from κ or λ types.

[0024] According to embodiments of the present invention, the heavy chain constant region and / or light chain constant region are derived from mouse antibodies, human antibodies, primate antibodies, or mutants thereof.

[0025] According to an embodiment of the present invention, the N end of the heavy chain constant region is connected to the C end of the heavy chain variable region; and / or the N end of the light chain constant region is connected to the C end of the light chain variable region.

[0026] According to an embodiment of the present invention, the Fab fragment includes a heavy chain variable region, a light chain variable region, a CH1 region, and a CL region.

[0027] According to an embodiment of the present invention, the C-terminus of the heavy chain variable region is connected to the N-terminus of the CH1 region, and the C-terminus of the CH1 region is connected to the N-terminus of the second Fc region.

[0028] According to an embodiment of the present invention, the C-end of the light chain variable region is connected to the N-end of the CL region.

[0029] According to an embodiment of the present invention, the Fab fragments are internally connected by covalent bonds.

[0030] According to an embodiment of the present invention, the Fab fragments are internally connected by disulfide bonds.

[0031] According to an embodiment of the present invention, at least a portion of the first Fc region and the second Fc region are derived from at least one of a mouse antibody, a primate antibody, or a mutant thereof.

[0032] According to an embodiment of the present invention, at least a portion of the first Fc region and the second Fc region are derived from mouse IgG1 or human IgG1 or mutants thereof.

[0033] According to an embodiment of the present invention, the first Fc region has at least one of the following mutation sites relative to the amino acid sequence of the Fc fragment of human wild-type IgG1: Y349C, T366S, L368A and Y407V.

[0034] According to an embodiment of the present invention, the second Fc region has at least one of the following mutation sites relative to the amino acid sequence of the Fc fragment of human wild-type IgG1: S354C and T366W.

[0035] According to an embodiment of the present invention, the first 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.

[0036] According to an embodiment of the present invention, the second Fc region, relative to the amino acid sequence of the Fc fragment of human wild-type IgG1, also has at least one of the following mutation sites: L234A and L235A.

[0037] According to embodiments of the present invention, the fusion protein has at least one of the following characteristics: i) At least a portion of the framework regions of the light chain variable region and the heavy chain variable region in the Fab fragment are each independently derived from at least one of the following: mouse antibody, primate antibody, bovine antibody, equine antibody, dairy bovine antibody, porcine antibody, sheep antibody, goat antibody, canine antibody, feline antibody, rabbit antibody, camel antibody, donkey antibody, deer antibody, mink antibody, chicken antibody, duck antibody, goose antibody, turkey antibody, fighting rooster antibody, or mutants thereof; ii) The CH1 and CL fragments in the Fab fragment are each independently derived from at least one of the following: mouse antibody, primate antibody, bovine antibody, equine antibody, dairy bovine antibody, porcine antibody, sheep antibody, goat antibody, canine antibody, feline antibody, rabbit antibody, camel antibody, donkey antibody, deer antibody, mink antibody, chicken antibody, duck antibody, goose antibody, turkey antibody, fighting rooster antibody, or mutants thereof; iii) The CH1 fragment in the Fab fragment is selected from the CH1 fragment of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD; and / or The CL fragments in the Fab fragments are selected from κ-type or λ-type CL fragments; iv) The Fc fragment is derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof, preferably a human Fc fragment; v) The two chains of the Fc fragment are connected by a knock-into-hole structure; vi) The fusion protein further includes a linker peptide, through which at least one of the following segments are linked: a. The first functional integration region, the second functional integration region, and the third functional integration region are respectively connected to the Fc segment; b. The connection between the VEGFR1 extracellular domain and the VEGFR2 extracellular domain in the third functional binding region.

[0038] According to an embodiment of the present invention, the linker peptide has an amino acid sequence as shown in (GGGGS)n, where n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0039] According to embodiments of the present invention, the Fc fragment is derived from the Fc fragment of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

[0040] According to an embodiment of the present invention, the Fc fragment is a human IgG1 Fc fragment or a mutant thereof.

[0041] According to an embodiment of the present invention, one chain of the Fc fragment has an amino acid sequence as shown in SEQ ID NO: 42, and the other chain has an amino acid sequence as shown in SEQ ID NO: 43.

[0042] According to an embodiment of the present invention, the fusion protein comprises: Having a first polypeptide chain as shown in the amino acid sequence of SEQ ID NO: 20, Having a second polypeptide chain as shown in the amino acid sequence of SEQ ID NO: 27, and It has a third polypeptide chain as shown in the amino acid sequence of SEQ ID NO: 28.

[0043] A second aspect of the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes the fusion protein described in the first aspect. The nucleic acid molecule according to embodiments of the present invention can effectively encode the aforementioned fusion protein.

[0044] According to embodiments of the present invention, the nucleic acid molecule is DNA or RNA.

[0045] 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.

[0046] A third aspect of the present invention provides an expression vector. According to embodiments of the present invention, the expression vector comprises the nucleic acid molecule described in the second aspect.

[0047] According to an embodiment of the present invention, the expression vector is a eukaryotic vector or a prokaryotic vector.

[0048] When ligating the aforementioned nucleic acid molecules to a vector, the nucleic acid molecules 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 molecules. These control elements can originate directly from the vector itself or be exogenous, i.e., not derived from the vector itself. Of course, the nucleic acid molecules and control elements only need to be operatively linked. In this context, "operatively linked" means linking a foreign gene to a vector so that the control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform their intended functions 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 fusion protein can be effectively obtained in large quantities in vitro under the mediation of a regulatory system.

[0049] According to an embodiment of the present invention, the expression vector includes at least one selected from plasmid vectors, adenovirus vectors, lentivirus vectors, and adeno-associated virus vectors.

[0050] A fourth aspect of the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell carries the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect; or expresses the fusion protein described in the first aspect.

[0051] According to an embodiment of the present invention, the recombinant cell is obtained by introducing the expression vector described in the second aspect into a host cell. Using this recombinant cell, under suitable conditions, the aforementioned fusion protein can be effectively expressed intracellularly.

[0052] It should be noted that the "suitable conditions" mentioned in this application specification refer to conditions suitable for the expression of the fusion protein described in this invention. Those skilled in the art will readily understand that suitable conditions for the expression of the fusion protein include, but are not limited to, suitable transformation or transfection methods, healthy host cell state, 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 fusion protein according to the specific environment of their laboratory.

[0053] According to an embodiment of the present invention, the recombinant cells are prokaryotic cells or eukaryotic cells.

[0054] According to an embodiment of the present invention, the recombinant cell is a mammalian cell.

[0055] A fifth aspect of the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises at least one of the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect.

[0056] According to embodiments of the present invention, pharmaceutically acceptable excipients are further included.

[0057] 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.

[0058] 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.

[0059] 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 fusion protein can be administered by intravenous infusion or injection, or by intramuscular or subcutaneous injection.

[0060] The effective amount of the fusion protein 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.

[0061] The fusion proteins or compositions of the present invention can be incorporated into pharmaceuticals suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These pharmaceuticals 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 pharmaceuticals are in the form of injection solutions or infusion solutions. The aforementioned fusion proteins or compositions can be administered by intravenous infusion or injection, or by intramuscular or subcutaneous injection.

[0062] According to embodiments of the present invention, the administration route of the method is subcutaneous injection or intravenous injection.

[0063] A sixth aspect of the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate includes the fusion protein described in the first aspect and a conjugation portion, the conjugation portion being linked to the fusion protein.

[0064] According to an embodiment of the present invention, the coupling portion includes a purification tag or label.

[0065] According to embodiments of the present invention, the marker is selected from radioactive markers, fluorescent markers, chromophore markers, electron-dense markers, or spin markers.

[0066] According to embodiments 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.

[0067] According to embodiments of the present invention, the coupling portion includes, but is not limited to, one or more of the following: a radioisotope, a fluorophore, 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.

[0068] A seventh aspect of this invention provides the use of the fusion protein 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, and the pharmaceutical composition described in the fifth aspect in the preparation of a medicament. According to embodiments of the invention, the medicament is used for the prevention and / or treatment of cancer.

[0069] According to embodiments of the present invention, the cancer includes at least one selected from colorectal cancer, lung cancer, intestinal cancer, melanoma, kidney 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, multiple myeloma, and bladder cancer.

[0070] 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

[0071] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is a schematic diagram of the IL-2v×PD1, PD1×VEGFR and IL-2v×PD1×VEGFR fusion proteins in one embodiment of the present invention; Figure 2 This is a comparative diagram of the amino acid sequences of IL-2wt, IL-2v8804, IL-2v04 and IL-2v88 in one embodiment of the present invention; Figure 3 The image shows the ELISA detection results of IL-2wt-Fc, IL-2v04-Fc and IL-2v88-Fc binding with IL-2Rα and IL-2Rβγ, respectively, in one embodiment of the present invention. Figure 4 This is an ELISA detection result diagram of the binding of IL-2wt-Fc, IL-2v8804-Fc and IL-2v2149-Fc with IL-2Rα in one embodiment of the present invention; Figure 5 This is an ELISA detection result diagram of the binding of IL-2wt-Fc, IL-2v8804-Fc and IL-2v2149-Fc with IL-2Rβγ in one embodiment of the present invention; Figure 6 Flow cytometry results showing how IL-2v8804-Fc and IL-2v2149-Fc promote STAT5 phosphorylation in T cells in one embodiment of the present invention. Figure 7The image shows the ELISA detection results of the binding of IL-2Rα to the fusion proteins IL-2v8804×mPD-1, IL-2v2149×mPD-1 and IL-2wt×mPD-1 in one embodiment of the present invention. Figure 8 The image shows the ELISA detection results of the binding of IL-2Rβγ to the fusion proteins IL-2v8804×mPD-1, IL-2v2149×mPD-1 and IL-2wt×mPD-1 in one embodiment of the present invention. Figure 9 The figure shows the survival rate of IL-2v8804×mPD-1, IL-2v88×mPD-1, IL-2wt×mPD-1, and IL-2v2149×mPD-1 fusion proteins in C57BL / 6 mice after administration in one embodiment of the present invention. Figure 10 This is a graph showing the efficacy and body weight of IL-2v8804×mPD-1, IL-2v2149×mPD-1 fusion protein and a known PD1 antibody in a C57BL / 6 mouse MC38 tumor model, according to one embodiment of the present invention. Figure 11 This is a diagram showing the efficacy results of the IL-2v8804×mPD-1×VEGFR, IL-2v8804×mPD-1, and mPD-1×VEGFR fusion proteins in a C57BL / 6 mouse B16F10 tumor model according to one embodiment of the present invention. Figure 12 This is a graph showing the SPR results of the binding of the IL-2v8804×PD-1×VEGFR fusion protein to IL-2Rα in one embodiment of the present invention. Figure 13 This is a graph showing the SPR results of the binding of the IL-2v8804×PD-1×VEGFR fusion protein to IL-2Rβγ in one embodiment of the present invention. Figure 14 This is a graph showing the SPR results of the IL-2v8804×PD-1×VEGFR fusion protein binding to PD-1 in one embodiment of the present invention. Figure 15 This is a graph showing the SPR results of the IL-2v8804×PD-1×VEGFR fusion protein binding to VEGF in one embodiment of the present invention. Figure 16 This is an ELISA result diagram of the IL-2v8804×PD-1×VEGFR fusion protein, PD-1×VEGF bispecific antibody AK112, and IL-2v2149×PD-1 fusion protein IBI363 blocking the binding of PD-1 to PDL1 in one embodiment of the present invention. Figure 17This is an ELISA result diagram of the IL-2v8804×PD-1×VEGFR fusion protein and the PD-1×VEGF bispecific antibody AK112 blocking the binding of VEGF and VEGFR1 in one embodiment of the present invention. Figure 18 This is an ELISA result diagram of the IL-2v8804×PD-1×VEGFR fusion protein and the PD-1×VEGF bispecific antibody AK112 blocking the binding of VEGF and VEGFR2 in one embodiment of the present invention; Figure 19 This is a diagram showing the efficacy results of IL-2v8804×PD-1×VEGFR fusion protein and PD-1 antibody Pembrolizumab in an hPD-1 transgenic mouse Colon26 tumor model, according to one embodiment of the present invention. Figure 20 This is a diagram showing the efficacy results of the IL-2v8804×PD-1×VEGFR, IL-2v8804×PD-1, and PD-1×VEGFR fusion proteins in an hPD-1 transgenic mouse Colon26 tumor model, according to one embodiment of the present invention. Figure 21 This is a diagram showing the efficacy results of the IL-2v8804×PD-1×VEGFR, IL-2v8804×PD-1, and PD-1×VEGFR fusion proteins in an hPD-1 transgenic mouse MC38 tumor model according to one embodiment of the present invention. Figure 22 This is a diagram showing the efficacy results of the IL-2v8804×PD-1×VEGFR and PD-1×VEGFR fusion proteins in the hPD-1 transgenic mouse B16F10 tumor model in one embodiment of the present invention. Figure 23 The figure shows the efficacy results of the IL-2v8804×PD-1×VEGFR and IL-2v8804×PD-1 fusion protein in the hPD-1 transgenic mouse B16F10 tumor model in one embodiment of the present invention. Detailed Implementation

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] In this document, the term "mutant" or "variant" may refer to a molecule obtained by mutating one or more nucleotides or amino acids into any naturally occurring or engineered molecule.

[0079] 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.

[0080] 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%.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] In this article, the term "antibody" is used in the broadest sense, encompassing full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, with no specific structural restrictions as long as they exhibit the desired biological activity. Antibody molecules typically consist of a lighter light chain and a heavier heavy chain, linked by disulfide bonds. The amino-terminal (N-terminus) amino acid sequence of the peptide chain varies considerably and is called the variable region (V-terminus); the carboxyl-terminus (C-terminus) is relatively stable and changes very little, and is called the constant region (C-terminus). The V-termini of the L-chain and H-chain are referred to as VL and VH, respectively.

[0089] As used herein, the terms “complementarity-determining region,” “CDR,” “CDR region,” or “CDRs” refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of antibodies or functional fragments of them to the antigens or epitopes they recognize. For example, these typically include amino acid residues near 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and near 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health). Health, Bethesda, MD. (1991); and / or amino acid residues from “hypervariant rings” (e.g., amino acid residues near 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and amino acid residues near 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). In specific embodiments of this disclosure, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0090] In this document, the term "antigen-binding fragment" refers to a fragment containing part or all of an antibody that lacks at least some of the amino acids present in the full-length chain but still possesses the performance activity of specifically binding to an antigen. For example, the fragment may contain part or all of the antibody's CDR. Such fragments are biologically active because they bind to the antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from Fab, Fv, scFv, or single-domain antibodies. Such fragments can be generated using recombinant nucleic acid technology or through enzymatic or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0091] Without substantially affecting antibody activity (retaining at least 95% 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 antibody or its functional fragment sequence. 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.

[0092] The amino acid sequences used in this application are shown in Table 1: Table 1:

[0093] In Table 1 above, "hoss" refers to the chain containing "hole" in the "knob into hole" structure, and "kbss" refers to the chain containing "knob" in the "knobinto hole" structure.

[0094] 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.

[0095] 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.

[0096] In this embodiment of the invention, the nucleotide sequence used to prepare the expression vector can be obtained using conventional methods or conventional software based on its amino acid sequence.

[0097] Example 1: Protein Preparation In this embodiment, wild-type IL-2 was modified 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 detection, the present invention links the C-terminus of the above variants to the Fc region of the affinity tag. A series of fusion proteins of the above proteins and PD-1 antibodies were also prepared; the specific structures are described in [reference needed]. Figure 1Furthermore, a series of known proteins, such as wild-type IL-2, known IL-2v88 variants, and known IL-2v2149 and IL-2v2214 variants (the specific sequences of which are referenced in patent CN118119635A), were prepared as Fc fusion proteins to demonstrate the effectiveness of the IL-2 variant IL-2v8804 obtained in this application. The amino acid sequences of IL-2wt, IL-2v8804, IL-2v04, and IL-2v88 are, for example... Figure 2 As shown.

[0098] 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 (2) When the protein has one strand, add the pcDNA3.4 vector containing the coding sequence (synthesized by Nanjing GenScript) to 2 mL of OptiSFM medium (purchased from Thermo Fisher) to obtain solution a; or, when the protein has two strands, add two pcDNA3.4 vectors containing three coding sequences (synthesized by Nanjing GenScript) in a 1:1 ratio to 2 mL of OptiSFM medium (purchased from Thermo Fisher) to obtain solution a; or, when the protein has three strands, add three pcDNA3.4 vectors containing three coding sequences (synthesized by Nanjing GenScript) in a 1:1:1 ratio to 2 mL of OptiSFM medium (purchased from Thermo Fisher) to obtain solution a; (3) Add 160 μL of LExpiFectamineCHO transfection reagent (purchased from Thermo Fisher) to 2 mL of 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 enhancer (purchased from Thermo Fisher), and 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.

[0099] In this embodiment, a total of 19 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.

[0100] Table 2: Cytokine variants and fusion proteins and their corresponding amino acid sequences

[0101] Example 2: IL-2v-Fc protein ELISA binding assay This embodiment uses ELISA to detect the binding characteristics of IL-2v04-Fc (SEQ ID NO: 17), IL-2v88-Fc (SEQ ID NO: 18), IL-2v8804-Fc (SEQ ID NO: 4), IL-2wt-Fc (SEQ ID NO: 6), and IL-2v2149-Fc (SEQ ID NO: 5). 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. 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.

[0102] Figure 3The results show that the binding of IL-2v04-Fc to IL-2Rα and IL-2Rβγ is weaker than that of IL-2wt-Fc; while the binding of IL-2v88-Fc to IL-2Rβγ is 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. Figure 4 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.

[0103] Figure 5 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.

[0104] Example 3: Detection of the activity of 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.

[0105] 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, hIgG1 (purchased from Bio-Legend Biotech), obtained in Example 1 of this invention, 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.

[0106] The results are as follows Figure 6 As shown, the IL-2v8804-Fc of the present invention promotes T cell STAT5 phosphorylation much less strongly than IL-2v2149-Fc.

[0107] 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, and 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.

[0108] 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.

[0109] Figure 7 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.

[0110] Figure 8 The results show that the binding affinity of IL-2v8804×mPD-1 to IL-2Rβγ is weaker than that of IL-2wt×mPD-1.

[0111] 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-2v88×mPD-1, IL-2v8804×mPD-1, IL2wt×mPD-1 and IL-2v2149×mPD-1 fusion proteins of the present invention and their different doses in mice was tested.

[0112] (1) Thirty 7-week-old female C57BL / 6 mice were randomly divided into groups according to their body weight; (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 9 As shown; (3) Observe the survival status of the mice every day, and weigh and record the weight of the mice twice a week.

[0113] The results are as follows Figure 9 As shown, administration of 5 mg / kg IL-2wt×mPD-1, 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 mouse mortality, while all mice survived administration of 20 mg / kg IL-2v8804×mPD-1 and 40 mg / kg IL-2v8804×mPD-1. This indicates that the IL-2v8804 of this invention has lower toxicity than IL-2wt, IL-2v88, and IL-2v2149. 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 for changes in the binding affinity of the IL-2 variant.

[0114] 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-2v8804×mPD-1, IL-2v2214×mPD-1 fusion protein and the anti-mouse PD-1 antibody mPD1 in promoting anti-cancer activity in mice.

[0115] (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.

[0116] The results are as follows Figure 10 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 IL-2v8804×mPD-1 did not cause weight loss or death in mice, and has better safety.

[0117] Example 7: Anti-cancer experiment of IL-2v×mPD-1×VEGFR fusion protein in C57BL / 6 mouse model In this embodiment, an in vivo pharmacodynamic experiment was used to detect the function of the IL-2v8804×mPD-1×VEGFR, IL-2v2214×mPD-1, and mPD-1×VEGFR fusion proteins of the present invention in promoting anti-cancer activity in mice.

[0118] (1) Subcutaneous tumors were implanted on the right ventral side of 7-week-old female C57BL / 6 mice. Each mouse was injected with 1×10 6 One B16F10 melanoma cell; (2) The tumor volume grew to approximately 140 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.

[0119] The results are as follows Figure 11 As shown, the IL-2v8804×mPD-1×VEGFR, IL-2v8804×mPD-1, and mPD-1×VEGFR fusion proteins all have anti-cancer functions, and the anti-cancer function of IL-2v8804×mPD-1×VEGFR is superior to that of IL-2v2214×mPD-1 and mPD-1×VEGFR.

[0120] Example 8: Protein Affinity Detection Biacore is a method for analyzing biomolecular interactions based on the principle of optical surface plasmon resonance (SPR). It can not only detect the specific binding between antigens and antibodies, but also obtain data that are very important in drug development, such as the binding rate constant (Ka), dissociation rate constant (Kd), and equilibrium dissociation constant (KD) between molecules, thereby calculating the affinity of the antibody.

[0121] In the Biacore 1K (Cytiva) system, the antibody was diluted to 10 μg / mL with run buffer (HBS-EP), and the fusion protein was coupled to protein A (Cytiva, 29127556) chip at a flow rate of 10 μL / min. The kinetics and affinity data of the protein binding to the fusion protein were detected at a flow rate of 30 μL / min, with a binding time of 120 s and a dissociation time of 800 s.

[0122] The kinetics and affinity data of the binding of the fusion protein IL-2v8804×PD-1×VEGFR to IL-2Rα, IL-2Rβγ, PD-1, and VEGF proteins were obtained.

[0123] The results are as follows Figure 12 As shown, the KD value of IL-2v8804×PD-1×VEGFR and IL-2Rα is 24.3 nM.

[0124] The results are as follows Figure 13 As shown, IL-2v8804×PD-1×VEGFR does not show significant binding to IL-2Rβγ.

[0125] The results are as follows Figure 14 As shown, the KD value of IL-2v8804×PD-1×VEGFR and PD-1 is 14.2 nM.

[0126] The results are as follows Figure 15 As shown, the KD value of IL-2v8804×PD-1×VEGFR and VEGF is 0.86 nM.

[0127] Note: Ka represents the binding rate constant (the larger the value, the stronger the affinity); Kd represents the dissociation rate constant (the smaller the value, the stronger the affinity), reflecting the affinity of the compound for the target; KD represents Kd / Ka, which is the equilibrium dissociation constant (affinity constant). The smaller the KD, the less dissociation, indicating a stronger affinity.

[0128] Example 9: Experiment on the blocking of PD-1 and PDL1 binding by IL-2v8804×PD-1×VEGFR fusion protein In this embodiment, the blocking properties of the IL-2v8804×PD-1×VEGFR fusion protein, the PD-1×VEGF bispecific antibody AK112, and the IL-2v2149×PD-1 fusion protein IBI363 were detected using an ELISA assay. PD-1 protein was coated into 96-well plates, and the fusion protein and biotinylated PDL1 protein were added. The signal strength was used to determine the ability of the fusion protein to block the binding of PD-1 and PDL1.

[0129] Dilute PD-1 protein (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), add 200 μl / well of PBS / 10% BSA, and 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-2v8804×PD-1×VEGFR fusion protein, PD-1×VEGF bispecific antibody AK112, IL-2v2149×PD-1 fusion protein IBI363, and 10 μg / ml Biotin-PDL1, and incubate at 37°C for 1 h. Remove the reaction mixture, wash the plate 6 times with PBST, and dilute HRP (horseradish peroxidase)-labeled Streptavidin 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 terminate the reaction by adding 80 μl / well of 4M sulfuric acid. Read the absorbance at 450 nm using a microplate reader.

[0130] Figure 16 The results show that the IL-2v8804×PD-1×VEGFR of the present invention has the ability to block the binding of PD-1 and PDL1.

[0131] Example 10: Experiment on the blocking of VEGF-VEGFR binding by IL-2v8804×PD-1×VEGFR fusion protein In this embodiment, the blocking properties of the IL-2v8804×PD-1×VEGFR fusion protein and the PD-1×VEGF bispecific antibody AK112 were detected using an ELISA assay. VEGFR1 and VEGFR2 proteins were coated into 96-well plates, and the fusion protein and biotinylated VEGF protein were added. The signal strength was used to determine the ability of the fusion protein to block the binding of VEGF to VEGFR.

[0132] VEGFR1 or VEGFR2 protein (purchased from Acro) was diluted to 1 μg / ml with PBS buffer and added to 96-well plates at a volume of 100 μl / well. The plates were incubated overnight at 4°C. The PBS buffer was then removed from the 96-well plates. The plates were washed 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer, and then 200 μl / well of PBS / 10% BSA was added for blocking. The plates were incubated at 37°C for 2 h. The blocking buffer was removed, and the plates were washed 6 times with PBST. Then, 100 μl / well of the target IL-2v8804×PD-1×VEGFR fusion protein, PD-1×VEGF bispecific antibody AK112, and 0.4 μg / ml Biotin-VEGF were added in a serially diluted PBST / 0.05% BSA solution. The plates were incubated at 37°C for 1 h. Remove the reaction mixture, wash the plate 6 times with PBST, and dilute HRP (horseradish peroxidase)-labeled Streptavidin 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 terminate the reaction by adding 80 μl / well of 4M sulfuric acid. Read the absorbance at 450 nm using a microplate reader.

[0133] Figure 17 The results show that the IL-2v8804×PD-1×VEGFR of the present invention has the ability to block the binding of VEGF and VEGFR1, and the blocking ability is stronger than that of AK112.

[0134] Figure 18 The results show that the IL-2v8804×PD-1×VEGFR of the present invention has the ability to block the binding of VEGF and VEGFR2, and the blocking ability is stronger than that of AK112.

[0135] Example 11: Anti-cancer experiment of IL-2v8804×PD-1×VEGFR fusion protein in hPD-1 transgenic mouse Colon26 colorectal cancer model In this embodiment, an in vivo pharmacodynamic experiment was conducted to detect the anti-cancer effects of the IL-2v8804×PD-1×VEGFR, IL-2v8804×PD-1, PD-1×VEGFR fusion protein, and PD-1 antibody Pembrolizumab in mice.

[0136] (1) Subcutaneous tumors were implanted on the right ventral side of 7-week-old female hPD-1 transgenic mice. Each mouse was injected with 2×10 6 One Colon26 colorectal cancer cell; (2) The tumor volume grew to approximately 200 mm. 3On 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.

[0137] The results are as follows Figure 19 As shown, 20 mg / kg of PD-1 antibody Pembrolizumab had virtually no anti-cancer effect in this model, while IL-2v8804×PD-1×VEGFR showed significant anti-cancer activity after administration of 20 mg / kg.

[0138] The results are as follows Figure 20 As shown, 20 mg / kg of IL-2v8804×PD-1×VEGFR, IL-2v8804×PD-1, and PD-1×VEGFR fusion proteins all exhibited significant anticancer activity, and the anticancer activity of IL-2v8804×PD-1×VEGFR was stronger than that of IL-2v8804×PD-1 and PD-1×VEGFR.

[0139] Example 12: Anti-cancer experiment of IL-2v8804×PD-1×VEGFR fusion protein in hPD-1 transgenic mouse MC38 colorectal cancer model In this embodiment, an in vivo pharmacodynamic experiment was used to detect the function of the IL-2v8804×PD-1×VEGFR, IL-2v8804×PD-1, and PD-1×VEGFR fusion proteins of the present invention in promoting anti-cancer activity in mice.

[0140] (1) Subcutaneous tumors were implanted on the right ventral side of 7-week-old female hPD-1 transgenic mice. Each mouse was injected with 3×10 6 One MC38 colorectal cancer cell; (2) The tumor volume grew to approximately 350 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.

[0141] The results are as follows Figure 21As shown, 20 mg / kg of IL-2v8804×PD-1×VEGFR, IL-2v8804×PD-1, and PD-1×VEGFR fusion protein all showed significant anticancer activity. Furthermore, the anticancer activity of IL-2v8804×PD-1×VEGFR was similar to that of IL-2v8804×PD-1 and stronger than that of PD-1×VEGFR.

[0142] Example 13: Anti-cancer experiment of IL-2v8804×PD-1×VEGFR fusion protein in hPD-1 transgenic mouse B16F10 melanoma model In this embodiment, an in vivo pharmacodynamic experiment was used to detect the function of the IL-2v8804×PD-1×VEGFR, IL-2v8804×PD-1, and PD-1×VEGFR fusion proteins of the present invention in promoting anti-cancer activity in mice.

[0143] (1) Subcutaneous tumors were implanted on the right ventral side of 7-week-old female hPD-1 transgenic mice. Each mouse was injected with 1×10 6 One B16F10 melanoma cell; (2) The tumor volume grew to approximately 150 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.

[0144] The results are as follows Figure 22 As shown, both 20 mg / kg IL-2v8804×PD-1×VEGFR and PD-1×VEGFR fusion protein showed significant anticancer activity after administration, and the anticancer activity of IL-2v8804×PD-1×VEGFR was stronger than that of PD-1×VEGFR.

[0145] The results are as follows Figure 23 As shown, both IL-2v8804×PD-1×VEGFR and IL-2v8804×PD-1 fusion protein showed significant anticancer activity after administration of 20 mg / kg, and the anticancer activity of IL-2v8804×PD-1×VEGFR was stronger than that of IL-2v8804×PD-1.

[0146] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is 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.

[0147] 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. A fusion protein, characterized in that, Includes a first functional binding region, wherein the first functional binding region has an IL-2 mutant. The amino acid sequence of the IL-2 mutant was obtained by mutating at least one of the following positions in the amino acid sequence of the wild-type IL-2 protein: The 3rd, 39th, 88th and 125th positions.

2. The fusion protein according to claim 1, characterized in that, Compared with the amino acid sequence of wild-type IL-2 protein, the amino acid sequence of the IL-2 mutant has at least one of the following mutations (1) to (4): (1) The third amino acid is mutated from T to A, N, C, E, G, I, L, M, F, P, S, W, Y or V; (2) The 39th amino acid is mutated from M to A, N, C, E, G, I, L, M, F, P, S, W, Y or V; (3) The 88th amino acid is mutated from N to A, N, C, E, G, I, L, M, F, P, S, W, Y or V; (4) The amino acid at position 125 is mutated from C to A, N, C, E, G, I, L, M, F, P, S, W, Y or V.

3. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the IL-2 mutant has at least one of the following mutations: p.Thr3Ala, p.Met39Glu, p.Asn88Glu, and p.Cys125Ala.

4. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the IL-2 mutant has the following mutations: p.Thr3Ala, p.Met39Glu, p.Asn88Glu, and p.Cys125Ala.

5. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the IL-2 mutant is the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence that has at least 90% identity with it.

6. The fusion protein according to claim 1, characterized in that, The fusion protein further includes a second functional binding region having PD-1 binding activity.

7. The fusion protein according to claim 6, characterized in that, The fusion protein further includes a third functional binding region, which has the activity of blocking the binding of VEGF to VEGFR.

8. The fusion protein according to claim 6 or 7, characterized in that, The fusion protein has at least one of the following characteristics: 1) The first functional binding region is connected to the N-terminus or C-terminus of the Fc segment; 2) The second functional binding region is connected to the N-terminus or C-terminus of the Fc segment; 3) The second functional binding region is selected from the Fab fragment; 4) The second functional combination area includes HCDRs and LCDRs, wherein the HCDRs and / or LCDRs are defined by Kabat, Chothia, AbM, Contact or IMGT; 5) The second functional integration area includes: HCDR1, HCDR2, and HCDR3 are represented by the amino acid sequences of SEQ ID NO: 31-33, respectively. LCDR1, LCDR2, and LCDR3 are shown in the amino acid sequences of SEQ ID NO: 34-36, respectively; 6) The third functional binding region is connected to the N-terminus or C-terminus of the Fc segment; 7) The third functional binding region includes at least one fusion protein unit formed by linker peptides connecting the extracellular domains of VEGFR1 and VEGFR2.

9. The fusion protein according to claim 8, characterized in that, The fusion protein has at least one of the following characteristics: a) The first functional binding region is connected to the N-terminus of the Fc segment; b) The second functional binding region is connected to the N-terminus of the Fc segment; c) The third functional binding region is connected to the C-terminus of the Fc segment; d) The second functional binding region has a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO: 37 and a light chain variable region as shown in the amino acid sequence of SEQ ID NO: 38; e) The amino acid sequence of the extracellular domain of VEGFR1 in the third functional binding region is shown in SEQ ID NO: 39, and the amino acid sequence of the extracellular domain of VEGFR2 is shown in SEQ ID NO:

40. f) The third functional binding region comprises two of the fusion protein units.

10. The fusion protein according to claim 8, characterized in that, The fusion protein has at least one of the following characteristics: i) At least a portion of the framework regions of the light chain variable region and the heavy chain variable region in the Fab fragment are each independently derived from at least one of the following: mouse antibody, primate antibody, bovine antibody, equine antibody, dairy bovine antibody, porcine antibody, sheep antibody, goat antibody, canine antibody, feline antibody, rabbit antibody, camel antibody, donkey antibody, deer antibody, mink antibody, chicken antibody, duck antibody, goose antibody, turkey antibody, fighting rooster antibody, or mutants thereof; ii) The CH1 and CL fragments in the Fab fragment are each independently derived from at least one of the following: mouse antibody, primate antibody, bovine antibody, equine antibody, dairy bovine antibody, porcine antibody, sheep antibody, goat antibody, canine antibody, feline antibody, rabbit antibody, camel antibody, donkey antibody, deer antibody, mink antibody, chicken antibody, duck antibody, goose antibody, turkey antibody, fighting rooster antibody, or mutants thereof; iii) The CH1 fragment in the Fab fragment is selected from the CH1 fragment of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD; and / or The CL fragments in the Fab fragments are selected from κ-type or λ-type CL fragments; iv) The Fc fragment is derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof, preferably a human Fc fragment; v) The two chains of the Fc fragment are connected by a knock-into-hole structure; vi) The fusion protein further includes a linker peptide, through which at least one of the following segments are linked: a. The first functional integration region, the second functional integration region, and the third functional integration region are respectively connected to the Fc segment; b. The connection between the VEGFR1 extracellular domain and the VEGFR2 extracellular domain in the third functional binding region; Optionally, the linker peptide has an amino acid sequence as shown in (GGGGS)n, where n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Optionally, the Fc fragment is derived from the Fc fragment of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; Optionally, the Fc fragment is a human IgG1 Fc fragment or a mutant thereof; Optionally, one chain of the Fc fragment has an amino acid sequence as shown in SEQ ID NO: 42, and the other chain has an amino acid sequence as shown in SEQ ID NO:

43.

11. The fusion protein according to claim 1, characterized in that, The fusion protein includes: Having a first polypeptide chain as shown in the amino acid sequence of SEQ ID NO: 20, Having a second polypeptide chain as shown in the amino acid sequence of SEQ ID NO: 27, and It has a third polypeptide chain as shown in the amino acid sequence of SEQ ID NO:

28.

12. A nucleic acid molecule, characterized in that, Encodes the fusion protein according to any one of claims 1 to 11.

13. An expression vector, characterized in that it comprises the nucleic acid molecule of claim 12; Optionally, the expression vector is a eukaryotic vector or a prokaryotic vector; Optionally, the expression vector includes at least one selected from plasmid vectors, adenovirus vectors, lentivirus vectors, and adeno-associated virus vectors.

14. A recombinant cell, characterized in that, Carrying the nucleic acid molecule of claim 12 or the expression vector of claim 13; or expressing the fusion protein of any one of claims 1 to 11; Optionally, the recombinant cells are obtained by introducing the expression vector of claim 13 into host cells; Optionally, the recombinant cells are prokaryotic cells or eukaryotic cells.

15. A pharmaceutical composition, characterized in that, It includes at least one of the fusion protein according to any one of claims 1 to 11, the nucleic acid molecule according to claim 12, the expression vector according to claim 13, or the recombinant cell according to claim 14; Optionally, pharmaceutically acceptable excipients may be further included.

16. A coupling, characterized in that, It includes the fusion protein according to any one of claims 1 to 11 and the coupling portion, wherein the coupling portion is linked to the fusion protein; Optionally, the coupling portion includes a purification tag or label; Optionally, the marker is selected from radioactive markers, fluorescent markers, chromophore markers, electron-dense markers, or spin markers.

17. The use of the fusion protein of any one of claims 1 to 11, the nucleic acid molecule of claim 12, the expression vector of claim 13, the recombinant cell of claim 14, and the pharmaceutical composition of claim 15 in the preparation of a medicament, characterized in that, The drug is used for the prevention and / or treatment of cancer.

18. The use according to claim 17, characterized in that, The cancers include at least one selected from colorectal cancer, lung cancer, bowel cancer, melanoma, kidney cancer, stomach cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, multiple myeloma, and bladder cancer.

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