Novel fusion proteins and uses thereof

Abstract

The present invention provides a fusion protein formed by variants of human interleukin-2 (IL-2) and interferon alpha (IFNα) and Fc, as well as the design, preparation and use of the fusion protein. By changing the binding ability of IFNα to its receptor through genetic mutation, a superior fusion protein is obtained that can overcome the shortcomings of existing drugs, such as poor specificity and severe side effects.

Description

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to Chinese Patent Application No. 202210521837.5, filed on May 13, 2022, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing, the entire contents of which are incorporated herein by reference. [Technical field]

[0003] The present invention belongs to the field of biomedical technology, and in particular relates to the design, preparation and use of a novel IL-2 fusion protein with IFNα and Fc. [Background technology]

[0004] IL-2, also known as T cell growth factor, is a gene located on chromosome 4 with a total sequence of 7 kb. The IL-12 protein consists of 133 amino acids and has a molecular weight of approximately 15 kD. IL-2 acts through the IL-2R, which contains three subunits: IL-2Rα (i.e., CD25), IL-2Rβ (i.e., CD122), and IL-2Rγ (i.e., CD132). These three subunits can form three receptor forms: a high-binding affinity receptor consisting of all three subunits IL-2Rα / β / γ, a medium-binding affinity receptor consisting of two subunits IL-2Rβ / γ, and a low-binding affinity receptor, which is IL-2Rα. Of these, IL-2Rβ and IL-2Rγ are required for IL-2 to activate downstream signaling pathways. When IL-2 binds to both IL-2Rβ and IL-2Rγ, the two receptor subunits form a heterodimer and phosphorylate STAT5 in the cell, which then enters the cell nucleus and triggers the transcription and expression of the corresponding genes. IL-2Rα is not required for signal transduction, but it can promote the binding of IL-2 to IL-2Rβ and IL-2Rγ. IL-2Rγ is expressed in all immune cells; IL-2Rβ is expressed in CD8+ T cells, NK cells, and regulatory T cells, and its expression level increases upon T cell activation; IL-2Rα is persistently highly expressed in regulatory T cells and transiently expressed in activated CD8+ T cells, after which its expression level is downregulated [1,2]. IL-2 was the second immunotherapy approved for the treatment of metastatic melanoma (1988) and renal cell carcinoma (1992), while IFNα was the first immunotherapy approved for the treatment of hairy cell leukemia (1986) and was subsequently approved for the treatment of metastatic melanoma, renal cell carcinoma, non-Hodgkin's lymphoma, and Kaposi's sarcoma.

[0005] IL-2 preparations approved as tumor treatment drugs require high doses to be effective because of their relatively short half-life. However, high doses of IL-2 result in obvious toxic side effects, preventing their widespread use. The selectivity for NK cells and CD8+ T cells expressing IL-2Rβ and IL-2Rγ is relatively low, so the ability of NK cells and CD8+ T cells to kill tumors cannot be fully exerted [3,4].

[0006] Meanwhile, IFNα is a cytokine produced by immune cells in the body, a group of small glycoproteins with similar structure and function that are produced by immune cells through antiviral responses when infected with a virus. Interferons play a very important role in the body's immune system [5,6]. Recombinant human interferon α has been approved as a treatment for viral infections and tumors. Interferon α receptors are widely distributed and expressed in many normal cells, making them prone to causing serious toxic side effects. Thus, there is a need in the art to generate new cytokine constructs that provide more effective therapeutic effects while minimizing side effects. Summary of the Invention

[0007] To address the above problems, the present invention discloses the design, preparation and use of a novel IL-2 fusion protein with IFNα and Fc, which alters the binding ability of IL-2 to a receptor(s) and the binding ability of IFNα to a receptor(s) by genetic mutation, to overcome the shortcomings of existing drugs, such as weak specificity, short half-life and severe side effects.

[0008] In some aspects, the disclosure provides a fusion protein comprising an IL-2 portion and an Fc portion, where the IL-2 portion comprises an amino acid sequence having one or more mutations compared to a wild-type IL-2 protein, and the amino acid sequence has at least 90% identity to SEQ ID NO:2.

[0009] In some embodiments, the mutation comprises one or more substitutions selected from the following, with reference to the amino acid positions of SEQ ID NO:1: R38A, L80F, R81D, L85V, I86V and I91F.

[0010] In some embodiments, the Fc portion consists of a human IgG Fc, such as human IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, or a variant thereof. For example, the Fc variant comprises one or more mutations selected from the following: L234A and L235A mutations, and M252Y, S254T, and T256E mutations.

[0011] In some embodiments, the fusion protein further comprises an IFNα portion. The IFNα portion may comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 6 or 5. In some embodiments, the IFNα portion comprises one or more substitutions selected from the following, with reference to amino acid positions in SEQ ID NO: 5: R144A and R149A.

[0012] In some embodiments, the fusion protein comprises: (a) an IL-2 portion operably linked to an Fc portion; (b) an IL-2 moiety operably linked to an Fc moiety, and an Fc moiety operably linked to an IFNα moiety; (c) an IL-2 portion operably linked to an IFNα portion, and an IFNα portion operably linked to an Fc portion; or (d) an IFNα portion operably linked to an IL-2 portion, and an IL-2 portion operably linked to an Fc portion. In some further embodiments, the fusion protein consists of: (a) an IL-2 portion operably linked to an Fc portion; (b) an IL-2 moiety operably linked to an Fc moiety, and an Fc moiety operably linked to an IFNα moiety; (c) an Fc portion operably linked to an IL-2 portion; or (d) an IFNα portion operably linked to an Fc portion, and an Fc portion operably linked to an IL-2 portion.

[0013] In some embodiments, the operable linkage is either a direct linkage or a linkage via a peptide linker. Optionally, the peptide linker is a GS series linker, such as (GS)n, where n=1-5. In some embodiments, the linker is as set forth in SEQ ID NO:11.

[0014] In some embodiments, the fusion protein consists of the amino acid sequence set forth in SEQ ID NO:3, 4, 9 or 10.

[0015] In some aspects, the disclosure provides a fusion protein comprising an IFNα portion and an Fc portion, wherein the IFNα portion comprises an amino acid sequence having one or more mutations compared to a wild-type IFNα protein, and the amino acid sequence has at least 90% identity to SEQ ID NO:6.

[0016] In some embodiments, the IFNα portion comprises one or more substitutions selected from the following with reference to amino acid positions in SEQ ID NO:5: R144A and R149A.

[0017] In some embodiments, the Fc portion consists of a human IgG Fc, such as human IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, or a variant thereof. For example, the Fc variant comprises one or more mutations selected from the following: L234A and L235A mutations, and M252Y, S254T, and T256E mutations. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: (a) an IFNα portion operably linked to an Fc portion; or (b) an Fc portion operably linked to an IFNα portion.

[0018] In some embodiments, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:7 or 8.

[0019] In some aspects, the disclosure provides a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein disclosed herein.

[0020] In some aspects, the present disclosure provides a vector comprising the nucleic acid molecule disclosed herein.

[0021] In some embodiments, the disclosure provides a host cell comprising a nucleic acid molecule or vector disclosed herein.

[0022] In some aspects, the present disclosure provides a pharmaceutical composition comprising a fusion protein disclosed herein or a nucleic acid molecule encoding same, and a pharma- ceutically acceptable carrier.

[0023] In some embodiments, the present disclosure provides a method for producing a fusion protein disclosed herein, comprising the steps of: - expressing the fusion protein in a host cell containing a vector encoding the fusion protein; and - isolating the fusion protein from the host cell culture.

[0024] In some aspects, the present disclosure provides a method for modulating an immune response in a subject, comprising administering to the subject a fusion protein or pharmaceutical composition disclosed herein.

[0025] In some embodiments, the present disclosure provides a method for treating or preventing cancer or an infectious disease in a subject, comprising administering to the subject an effective amount of a fusion protein or pharmaceutical composition disclosed herein.

[0026] In some embodiments, the cancer is selected from the group consisting of breast cancer, gastric cancer, melanoma, lymphoma, lung cancer, colon cancer, ovarian cancer, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, pancreatic cancer and leukemia.

[0027] In some embodiments, the present disclosure provides for the use of a fusion protein disclosed herein in the manufacture of a medicament for the prevention, treatment and / or management of cancer or an infectious disease.

[0028] In some embodiments, the present disclosure provides a fusion protein as disclosed herein for use in the treatment or prevention of cancer or an infectious disease.

[0029] In some aspects, the disclosure provides a kit comprising a container that includes a fusion protein or pharmaceutical composition disclosed herein.

[0030] The present invention also relates to the following embodiments: 1. A novel fusion protein of IL-2 with IFNα and Fc, comprising: The fusion protein includes a fusion protein of dual factors of IL-2 and IFNα with Fc, a fusion protein of IL-2 with Fc, and a fusion protein of IFNα with Fc; IL-2 and IFNα dual factor-Fc fusion proteins: mutant IL-2 and mutant IFNα are fused to the N-terminus or C-terminus of Fc, respectively, resulting in the polypeptide chains IL-2-Fc-IFNα or IFNα-Fc-IL-2; IL-2 and Fc fusion protein: a mutant IL-2 is fused to the N-terminus or C-terminus of Fc, resulting in the polypeptide chain Fc-IL-2 or IL-2-Fc; Fusion proteins of IFNα and Fc: The mutant IFNα is fused to the N-terminus or C-terminus of Fc, resulting in the polypeptide chains Fc-IFNα or IFNα-Fc. 2. A novel fusion protein of IL-2 with IFNα and Fc as described in embodiment 1, characterized in that in the fusion protein of IL-2 and IFNα dual factor with Fc, the Fc fragment comprises the hinge region of the heavy chain constant region, the second domain CH2 of the heavy chain constant region, and the third domain CH3 of the heavy chain constant region. 3. A novel fusion protein of IL-2 with IFNα and Fc as described in embodiment 1, characterized in that in the fusion protein of IL-2 with Fc, the Fc fragment contains the hinge region of the heavy chain constant region, the second domain CH2 of the heavy chain constant region, and the third domain CH3 of the heavy chain constant region. 4. A novel fusion protein of IL-2 with IFNα and Fc as described in embodiment 1, characterized in that in the fusion protein of IFNα and Fc, the Fc fragment contains the hinge region of the heavy chain constant region, the second domain CH2 of the heavy chain constant region, and the third domain CH3 of the heavy chain constant region. 5. A novel fusion protein of IL-2 with IFNα and Fc according to any one of embodiments 1 to 2, characterized in that in the fusion protein of IL-2 and IFNα dual factor with Fc, L234A and L235A mutations are further introduced into the Fc fragment together with M252Y, S254T and T256E mutations. 6. A novel fusion protein of IL-2 with IFNα and Fc according to any one of embodiments 1 or 3, characterized in that in the fusion protein of IL-2 with Fc, the mutations L234A and L235A are further introduced into the Fc fragment, together with the mutations M252Y, S254T and T256E. 7. A novel fusion protein of IL-2 with IFNα and Fc according to any one of embodiments 1 to 4, characterized in that in the fusion protein of IFNα with Fc, the mutations L234A and L235A are further introduced into the Fc fragment, together with the mutations M252Y, S254T and T256E. 8. A novel fusion protein of IL-2 with IFNα and Fc according to embodiment 1, characterized in that in the fusion protein of IL-2 with IFNα dual factor and Fc, the IL-2 mutant is obtained by introducing mutations based on wild-type IL-2 by molecular biological means, and the introduced mutations include, but are not limited to, R38A, L80F, R81D, L85V, I86V and I91F. 9. A novel fusion protein of IL-2 with IFNα and Fc according to embodiment 3, characterized in that in the fusion protein of IL-2 and Fc, the IL-2 mutant is obtained by introducing mutations based on wild-type IL-2 by molecular biological means, and the introduced mutations include, but are not limited to, R38A, L80F, R81D, L85V, I86V and I91F. 10. The novel fusion protein of IL-2, IFNα and Fc described in embodiment 1, characterized in that in the fusion protein of the dual factor IL-2, IFNα and Fc, the IFNα derivative is obtained by introducing mutations based on wild-type IFNα by molecular biological means, and the mutations introduced include, but are not limited to, R144A or R149A. 11. A novel fusion protein of IL-2, IFNα and Fc as described in embodiment 4, characterized in that in the fusion protein of IFNα and Fc, the IFNα derivative is obtained by introducing mutations based on wild-type IFNα by molecular biological means, and the introduced mutations include, but are not limited to, R144A or R149A. 12. A method for preparing a novel fusion protein of IL-2 with IFNα and Fc according to any one of the embodiments 1-2, characterized in that the method for preparing a fusion protein of IL-2 and IFNα dual factor with Fc comprises the following steps: (1) linking an IL-2 mutant to the N-terminus or C-terminus of an Fc fragment via a flexible linker and linking an IFNα derivative to the C-terminus or N-terminus of the Fc fragment via a flexible linker to obtain a polypeptide chain IL-2-Fc-IFNα or IFNα-Fc-IL-2; introducing the mutations L234A, L235A, M252Y, S254T and T256E into the Fc fragment; (2) cloning the DNA fragment obtained in (1) into a pcDNA series vector or other vectors used in mammalian cell expression systems to obtain a recombinant vector; (3) Transfecting the recombinant vector obtained in (2) into mammalian cells to express the fusion protein, and purifying the resulting product to obtain a fusion protein of IL-2 and IFNα dual factor with Fc. 13. A method for preparing a novel fusion protein of IL-2 with IFNα and Fc according to any one of embodiments 1 or 3, characterized in that the method for preparing a fusion protein of IL-2 with Fc comprises the following steps: (1) linking an IL-2 mutant to the N-terminus or C-terminus of an Fc fragment via a flexible linker to obtain a polypeptide chain IL-2-Fc or Fc-IL-2; introducing the mutations L234A, L235A, M252Y, S254T and T256E into the Fc fragment; (2) cloning the DNA fragment obtained in (1) into a pcDNA series vector or other vectors used in mammalian cell expression systems to obtain a recombinant vector; (3) A step of transfecting the recombinant vector obtained in (2) into mammalian cells to express the fusion protein, and purifying the resulting product to obtain a fusion protein of IL-2 and Fc. 14. A method for preparing a novel fusion protein of IL-2 with IFNα and Fc according to any one of embodiments 1 to 4, characterized in that the method for preparing a fusion protein of IFNα and Fc comprises the following steps: (1) linking an IFNα derivative to the C-terminus or N-terminus of an Fc fragment via a flexible linker to obtain a polypeptide chain Fc-IFNα or IFNα-Fc; and introducing the mutations L234A, L235A, M252Y, S254T and T256E into the Fc fragment; (2) cloning the DNA fragment obtained in (1) into a pcDNA series vector or other vectors used in mammalian cell expression systems to obtain a recombinant vector; (3) A step of transfecting the recombinant vector obtained in (2) into mammalian cells to express the fusion protein, and purifying the resulting product to obtain a fusion protein of IFNα and Fc. 15. A method for preparing a novel IL-2 fusion protein having IFNα and Fc according to any one of embodiments 12 to 14, characterized in that in step (3), the mammalian cells include HEK293 cells, CHO cells or cells derived therefrom. 16. A method for preparing a novel IL-2 fusion protein having IFNα and Fc described in any one of embodiments 12 to 14, characterized in that the introduction of mutations into the Fc fragment in step (1) is defined as the introduction of L234A and L235A mutations into the Fc fragment, together with the introduction of M252Y, S254T and T256E mutations into the Fc fragment. 17. Use of a novel fusion protein of IL-2 with IFNα and Fc according to any one of embodiments 1 to 11 in the manufacture of a broad-spectrum anti-tumor medicament, including monotherapy for the treatment of approved tumor therapeutic indications of IL-2 and IFNα, as well as broad-spectrum tumor therapy in combination with other tumor therapeutic methods. 18. Use of a novel fusion protein of IL-2 with IFNα and Fc prepared by the method according to any one of embodiments 12 to 16 in the manufacture of an Fc fusion protein pharmaceutical, including monotherapy for the treatment of approved oncology therapeutic indications of IL-2 and IFN-α, as well as broad-spectrum oncology therapy in combination with other oncology therapeutic methods.

[0031] The foregoing is a summary and thus contains, where necessary, simplifications, generalizations, and omissions of details; as a result, those skilled in the art will appreciate that the summary is illustrative only and is not intended to be in any way limiting. Other aspects, features, and advantages of the methods, compositions, and uses described herein, and / or other subject matter, will become apparent in the teachings set forth herein. [Brief description of the drawings]

[0032] [Figure 1A] 1 illustrates structural diagrams of various Fc fusion proteins encompassed by the present invention. The top-to-bottom orientation of each panel indicates the order from N-terminus to C-terminus, and the trapezoid indicates the hinge region. [Figure 1B] 1 illustrates the SDS PAGE expression profile of an Fc fusion protein of the present invention. [Figure 1C] 1 illustrates an HPLC analysis graph of an Fc fusion protein of the present invention. [Diagram 2] 1 illustrates a graph for detecting the binding ability of the Fc fusion protein of the present invention to the receptor IL-2Rα protein. [Diagram 3] 1 illustrates a graph for detecting the binding ability of the Fc fusion protein of the present invention to the receptor IL-2Rβ protein. [Figure 4] 1 shows graphs for detecting the binding ability of the Fc fusion protein of the present invention to the receptor IFNαR2 protein. [Diagram 5] 1 illustrates a graph of the binding ability detection of Fc fusion proteins of the present invention to receptor IL-2Rα proteins across different species. [Figure 6] 1 illustrates a graph for detecting the binding ability of the Fc fusion protein of the present invention to receptor IL-2Rβ protein across different species. [Figure 7] 1 illustrates a graph for detecting the binding ability of the Fc fusion protein of the present invention to the receptor IFNαR2 protein across different species. [Figure 8A]1 illustrates a graph of the binding ability results of the Fc fusion proteins of the present invention to FcRn protein, detected by ELISA. [Figure 8B] 1 illustrates a graph of the binding ability results of the Fc fusion proteins of the present invention to FcRn protein as detected by Fortebio. [Figure 9] 1 illustrates a distribution chart of specific cell subsets of PBMCs stimulated with Fc fusion proteins of the present invention. [Figure 10] Exemplifying the detection of the ability of the Fc fusion proteins of the invention to stimulate proliferation of CTLL-2 murine T lymphocytes. [Figure 11A] 1 illustrates the detection of the in vitro direct killing ability of the Fc fusion protein of the present invention against tumor cells (A: NCI-N87). [Figure 11B] 1 illustrates detection of the in vitro direct killing ability of the Fc fusion protein of the present invention against tumor cells (B: MDA-MB-231). [Figure 11C] 1 illustrates the detection of the in vitro direct killing ability of the Fc fusion protein of the present invention against tumor cells (C:A375). [Figure 11D] 1 illustrates detection of the in vitro direct killing ability of the Fc fusion protein of the present invention against tumor cells (D: Burkitt's lymphoma cells). [Figure 12A] 12A-12E illustrate the detection of the comprehensive killing ability of the Fc fusion protein of the present invention against tumor cells in vitro (A: NCI-N87). [Figure 12B] FIG. 1 illustrates the detection of the comprehensive killing ability of the Fc fusion protein of the present invention against tumor cells in vitro (B: MDA-MB-231). [Figure 12C] 1 illustrates the detection of the comprehensive killing ability of the Fc fusion protein of the present invention against tumor cells in vitro (C:A375). [Figure 12D] 1 illustrates the detection of the comprehensive killing ability of the Fc fusion protein of the present invention against tumor cells in vitro (D: Burkitt's lymphoma cells). [Figure 12E] FIG. 1 illustrates the detection of the comprehensive killing ability of the Fc fusion protein of the present invention against tumor cells in vitro (E: Burkitt's lymphoma cells). [Figure 13] Illustrates the pharmacodynamic activity of Fc-fusion proteins of the invention in an Ex Vivo organoid system. Description of the Invention

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.All patents, patent applications, and other publications cited herein are incorporated by reference in their entirety.To the extent that the definitions set forth herein conflict with the definitions set forth in the patents, patent applications, and other publications incorporated herein by reference, the definitions set forth herein shall control.

[0034] As used herein, the term "IL-2" refers to interleukin-2 and is intended to include any form of IL-2, such as: 1) the native unprocessed IL-2 molecule, full-length IL-2 protein, or a naturally occurring IL-2 variant; 2) any form of IL-2 produced by intracellular processing; or 3) full-length or modified forms. In this disclosure, the term "IL-2" or "IL-2 domain" includes wild-type IL-2 and IL-2 mutants.

[0035] As used herein, the term "IFNα" refers to interferon alpha and is intended to include any form of IFNα, such as: 1) a native unprocessed IFNα molecule, a full-length IFNα protein, or a naturally occurring IFNα variant; 2) any form of IFNα produced by intracellular processing; or 3) full-length or modified forms. In this disclosure, the term "IFNα" or "IFNα domain" includes wild-type IFNα and IFNα variants.

[0036] The term "variant" with respect to a polypeptide or protein refers to a biologically active polypeptide that contains one or more amino acid mutations relative to the native protein sequence. Optionally, the one or more amino acid mutations include amino acid substitutions and / or insertions at specific positions in the amino acid sequence. Preferably, the variant shares at least about 80% amino acid sequence identity with the corresponding native sequence polypeptide. Such variants include, for example, polypeptides with one or more amino acid (naturally occurring and / or non-naturally occurring) residues added to the N-terminus and / or C-terminus of the polypeptide. Variants for use in the present disclosure can be prepared by a variety of methods well known in the art, such as site-directed mutagenesis or phage display techniques for nucleotides in DNA encoding the native protein, generating DNA encoding the variant, and then expressing the DNA in recombinant cell culture. In certain embodiments disclosed herein, the IL-2 variant contains one or more substitutions compared to the wild-type IL-2 protein. In certain embodiments disclosed herein, the IFNα variant contains one or more substitutions compared to the wild-type IFNα protein.

[0037] The term "Fc" as used herein has the same meaning as it does with respect to an antibody and refers to the portion of an antibody that consists of the second (CH2) and third (CH3) constant regions of a first heavy chain linked to the second and third constant regions of a second heavy chain via disulfide bonds. The Fc region may also include part or all of the hinge region. The Fc region of an antibody is responsible for various effector functions, such as ADCC and CDC, but does not have antigen binding function. In this disclosure, the term "Fc" includes wild-type Fc and Fc variants.

[0038] As used herein, the term "operably linked" refers to a juxtaposition (with or without a spacer or linker or insertion sequence) of two or more biological sequences of interest in a relationship that allows them to function in their intended manner. When used in reference to a polypeptide, it means that the polypeptide sequences are linked in such a way that allows the linked product to have its intended biological function. For example, a fusion protein may be operably linked to a constant region of an immunoglobulin to provide a stable product with ligand binding activity. As another example, a fusion protein may be operably linked to a constant region of an immunoglobulin via an insertion sequence therebetween, which may be a spacer or may include a longer sequence.

[0039] As used herein, the term "fusion" or "fused" when used in reference to an amino acid sequence (e.g., a peptide, polypeptide, or protein) refers to the combination of two or more amino acid sequences into a single non-naturally occurring amino acid sequence, for example, by chemical conjugation or recombinant means. A fused amino acid sequence can be produced by genetic recombination of two coding polynucleotide sequences and expressed by a method in which a construct containing the recombinant polynucleotide is introduced into a host cell.

[0040] The term "fusion protein" as used herein refers to a polypeptide having two (or more) operably linked portions, where each of the portions is a polypeptide having a different property. The property can be a biological property, such as activity in vitro or in vivo. The property can also be a simple chemical or physical property, such as binding to a target antigen, catalysis of a reaction, and the like. The two portions can be directly linked by a single peptide bond or can be linked via a peptide linker comprising one or more amino acid residues. Generally, the two portions and the linker are in reading frame with each other. In certain embodiments, the fusion protein is a fusion protein of an IL-2 portion, an Fc portion, and an IFNα portion. In certain embodiments, the fusion protein is a fusion protein of an IL-2 portion and an Fc portion. In certain embodiments, the fusion protein is a fusion protein of an Fc portion and an IFNα portion.

[0041] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. If the vector allows for the expression of a protein encoded by a polynucleotide inserted therein, the vector is called an expression vector. A vector can have genetic material elements expressed in a host cell by transformation, transduction, or transfection into the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda bacteriophage or M13 bacteriophage, animal viruses, and the like. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, papova viruses (such as SV40), and the like. The vector may contain multiple elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain an origin of replication.

[0042] As used herein, the term "host cell" refers to a cell line that can be engineered to produce a protein, protein fragment, or peptide of interest. Host cells include, but are not limited to, cultured cells, e.g., mammalian cultured cells from rodents (rat, mouse, guinea pig, hamster), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; or human tissue or hybridoma cells, yeast cells, insect cells, as well as cells constructed in transgenic animals or cultured tissue. The term encompasses not only the particular subject cell, but also the progeny of such cells. Because certain modifications may occur in the progeny due to mutations or environmental influences, such progeny may not be identical to the parent cell, but are still within the scope of the term "host cell."

[0043] The term "identity" as used herein refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity" refers to the percent of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest of the molecules being compared. For these calculations, alignment gaps (if any) are preferably addressed by a specific mathematical model or computer program (i.e., "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Sequence Analysis Primer, (Gribskov, M. and Devereux, J. eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAMJ.Applied Math.48:1073.

[0044] The term "subject" includes human or non-human animals, but preferably is a human.

[0045] As used herein, the term "cancer" refers to any tumor or malignant cell proliferation that induces a disease state, which may be a solid tumor or a non-solid tumor mediated by proliferation or metastasis.

[0046] The terms "treatment" and "treatment" as used herein in the context of treating a condition generally relate to treatments and therapies in which some desired therapeutic effect is achieved, for example, inhibition of progression of a condition, whether in a human or animal, including reduction in the rate of progression of a condition, halting the rate of progression, regression of a condition, amelioration of a condition, and curing a condition. Treatment as a preventative measure (i.e., prevention, prophylaxis) is also included. In the case of cancer, "treatment" refers to inhibition or slowing of tumor or malignant cell growth, proliferation or metastasis, or a combination thereof. In the case of tumors, "treatment" includes removal of all or part of the tumor, inhibition or slowing of tumor growth and metastasis, prevention or delay of tumor progression, or a combination thereof.

[0047] As used herein, the term "effective amount" relates to an amount of an active compound, or an amount of a material, composition or dosage form containing an active compound, that is effective, when administered in accordance with a desired treatment regimen, to produce a specific desired therapeutic effect commensurate with a reasonable benefit / risk ratio.

[0048] IL-2 and IFNα mutants In certain aspects, the present disclosure provides an IL-2 variant that includes one or more modifications, such as an insertion, deletion, and / or substitution, compared to a wild-type IL-2 protein, such as a human wild-type IL-2 protein. The present disclosure further provides a fusion protein that includes an IL-2 protein portion fused to an Fc portion. The IL-2 protein portion may include a wild-type IL-2 protein or may include an IL-2 variant that includes one or more modifications (e.g., insertions and / or substitutions) compared to a wild-type IL-2 protein. In some embodiments, compared to a wild-type IL-2 protein, the IL-2 variant includes one or more substitutions selected from, but not limited to, the following: R38A, L80F, R81D, L85V, I86V, and I91F. For example, the amino acid at position 38 is modified in the IL-2 variant compared to the wild-type IL-2 protein. In some embodiments, the IL-2 variant further includes a conservative substitution(s) compared to the wild-type IL-2 protein. In some embodiments, the IL-2 variant consists of or consists of the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the IL-2 variant consists of or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:2, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2.

[0049] In certain aspects, the present disclosure provides IFNα variants that include one or more modifications, such as insertions, deletions, and / or substitutions, compared to a wild-type IFNα protein, such as a human wild-type IFNα protein. The present disclosure further provides a fusion protein that includes an IFNα protein portion fused to an Fc portion. The IFNα protein portion may include a wild-type IFNα protein or may include an IFNα variant that includes one or more modifications (e.g., insertions and / or substitutions) compared to the wild-type IFNα protein. In some embodiments, amino acids R144 and / or R149 in the IFNα variant are modified compared to the wild-type IFNα protein. In some embodiments, the IFNα variant further includes a conservative substitution compared to the wild-type IFNα protein. In some embodiments, the IFNα variant consists of or consists of the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the IFNα variant consists of or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:6, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6.

[0050] Fusion Proteins of the Invention In certain aspects, the present disclosure provides a fusion protein comprising an IL-2 portion and an Fc portion, a fusion protein comprising an IFNα portion and an Fc portion, and a fusion protein comprising an IL-2 portion, an Fc portion and an IFNα portion. The fusion protein may be a single chain or may have multiple chains. In some embodiments, the fusion protein is a double chain homodimer or heterodimer. The respective IL-2 portion, the Fc portion and the IFNα portion of each chain may be the same or different.

[0051] In some embodiments, a fusion protein of the invention consists of an IL-2 moiety operably linked, from N-terminus to C-terminus, to an Fc moiety (also known as an IL-2-Fc fusion protein).

[0052] In some embodiments, a fusion protein of the invention consists of an Fc portion operably linked, from N-terminus to C-terminus, to an IL-2 portion (also known as an Fc-IL-2 fusion protein).

[0053] In some embodiments, a fusion protein of the invention consists of an IFNα portion operably linked, from N-terminus to C-terminus, to an Fc portion (also known as an IFNα-Fc fusion protein).

[0054] In some embodiments, a fusion protein of the invention consists of an Fc portion operably linked, from N-terminus to C-terminus, to an IFNα portion (also known as an Fc-IFNα fusion protein).

[0055] In some embodiments, a fusion protein of the invention comprises, from N-terminus to C-terminus, an IL-2 portion operably linked to an Fc portion, and an Fc portion operably linked to an IFNα portion (also known as an IL-2-Fc-IFNα fusion protein).

[0056] In some embodiments, a fusion protein of the invention consists, from N-terminus to C-terminus, of an IFNα portion operably linked to an Fc portion, and an Fc portion operably linked to an IL-2 portion (also known as an IFNα-Fc-IL-2 fusion protein).

[0057] The IL-2 portion of the fusion protein may comprise a wild-type IL-2 protein or an IL-2 variant. The IFNα portion of the fusion protein may comprise a wild-type IFNα protein or an IFNα variant. The Fc portion of the fusion protein may comprise a wild-type Fc or an Fc variant.

[0058] In some embodiments, the amino acid at position 38 in the IL-2 portion of the fusion protein is modified. The region adjacent to amino acid 38 has been found to play an important role in the increase in vascular permeability induced by IL-2, and thus modification of amino acid 38 can significantly reduce vascular permeability.

[0059] In some embodiments, the modified interferon alpha portion is configured in a fusion protein and has a significantly reduced affinity for the interferon alpha receptor compared to native interferon alpha. Such a reduction can result in a significant reduction in interferon alpha-induced toxicity and side effects while retaining antitumor activity. In some embodiments, amino acids R144 or R149 of the interferon alpha portion configured in the fusion protein are modified. These amino acids play a key role in the binding of interferon alpha to its receptor and can be mutated to cause a significant reduction in affinity without affecting antitumor activity.

[0060] An operable linkage (or represented as "-") is either a direct linkage of the two moieties or a linkage via a linker, such as a peptide linker. In some embodiments, the IL-2 moiety is operably linked to the Fc moiety via a peptide linker. In some embodiments, the IFNα moiety is operably linked to the Fc moiety via a peptide linker. The peptide linker can be any peptide linker commonly used in the art, such as a GS series linker as set forth in SEQ ID NO:11. The fusion proteins provided herein can improve therapeutic efficacy, reduce toxicity and side effects, and expand the therapeutic safety margin.

[0061] Fusion proteins containing Fc regions In some embodiments, the fusion protein provided herein may further comprise an immunoglobulin constant domain sequence, such as a human IgG constant domain sequence, and more specifically, a hinge region and an Fc region, such as an IgG1, IgG2, IgG3, and IgG4 Fc region sequence. As known in the art, Fc refers to a portion of an antibody consisting of a second constant region and a third constant region of a first heavy chain of the antibody bound to a second constant region and a third constant region of a second heavy chain of the antibody via a disulfide bond, and optionally, the Fc region further comprises all or a portion of the hinge region. The Fc region herein includes both wild-type Fc regions and variants thereof, with different variants being used for multiple purposes. The variants may include one or more amino acid residue modifications, such as substitutions, in the Fc region.

[0062] In certain embodiments, the Fc region variants contain one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn). Such variants can increase pharmacokinetic half-life by binding to FcRn at acidic pH, thereby allowing them to escape lysosomal degradation and be translocated and released outside the cell. Methods for engineering antibody molecules to improve binding affinity to FcRn are well known in the art, see, for example, Vaughn, D. et al:63-73, 1998; Kontermann, R. et al., Antibody Engineering, Volume 1, Chapter 27:Engineering of the Fc region for improved PK, Springer, 2010; Yeung, Y. et al., Cancer Research, 70: 3269-3277 (2010); and Hinton, P. et al., J. Immunology, 176: 346-356 (2006).

[0063] In certain embodiments, the fusion proteins provided herein comprise L234A / L235A substitutions to reduce the ability to bind to the receptor FcgRIIIa. In certain embodiments, the fusion proteins provided herein comprise one or more amino acid substitutions at the interface of the Fc region to promote and / or facilitate heterodimerization. These modifications consist of the introduction of a protuberance into the first Fc polypeptide and a cavity into the second Fc polypeptide, where the protuberance can be positioned in the cavity to promote the interaction of the first and second Fc polypeptides to form a heterodimer or complex. Methods for generating protein molecules with such modifications are known in the art and are described, for example, in U.S. Pat. No. 5,731,166. No. 5,731,168.

[0064] In certain embodiments, the Fc domain also contains the triple mutation M252Y / S254T / T256E ("YTE"). This triple mutation has been reported to increase binding to the human neonatal Fc receptor (FcRn) by approximately 10-fold and increase the serum half-life of YTE-containing human IgG in cynomolgus monkeys by approximately 4-fold (Oganesyan V. et al. 2009 May; 46 (8-9):1750-5). In some further embodiments, the Fc domain contains additional mutations to enhance the interaction between Fc and human FcRn.

[0065] In some embodiments, the Fc variant consists of, or consists of, an amino acid sequence that is at least 85%, at least 90%, or at least 99% identical to SEQ ID NO:12.

[0066] Features of the Fusion Proteins of the Disclosure 1. Fusion with Fc extends the half-life of the fusion protein, reduces the amount of cytokine used, and reduces toxicity and side effects. 2. Structural modification of IL-2 improves the affinity of the cytokine for IL-2Rβ and IL-2Rγ on the surface of NK cells and CD8+ T cells, enhancing the killing effect of these cells against tumors. 3. The toxicity and side effects of IL-2-induced vascular hyperpermeability were significantly reduced by modifying the amino acid at position 38 of IL-2. 4. The binding ability to the receptor IFNαR2 is reduced by modification of R144 and R149, reducing toxicity and side effects while retaining antitumor activity. 5. Based on the above characteristics, the shortcomings of current IL-2 and IFNα drugs are overcome by the structurally modified fusion protein of IL-2 with IFNα and Fc, improving the therapeutic effect, reducing toxicity and side effects, and expanding the therapeutic safety margin.

[0067] Polynucleotides, Vectors, and Host Cells In some aspects, the present invention also provides polynucleotides encoding such fusion proteins. The polynucleotides can be used to express the fusion proteins. In some embodiments, the polynucleotides can also be used as therapeutic agents in an active form to achieve in vivo expression of the polypeptide fusion proteins.

[0068] The polynucleotide encoding the fusion protein can be easily isolated and sequenced using conventional procedures known in the art. The polynucleotide can also be obtained by synthetic methods. Preferably, the polynucleotide is codon-optimized for expression in eukaryotic host cells, particularly mammalian cells.

[0069] The polynucleotide encoding the fusion protein can be inserted into a vector for further cloning (amplification of DNA) or expression using known recombinant techniques. The components of a vector typically include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0070] In some embodiments, the present disclosure provides a vector (e.g., an expression vector) comprising a polynucleotide encoding a fusion protein provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the polynucleotide, and at least one selectable marker. Exemplary vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, papoviruses (e.g., SV40), lambda and M13 bacteriophages, liposomes, and plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-H, and the like. yg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT , pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM, pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0071] The vector containing the polynucleotide sequence encoding the fusion protein can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vector herein are prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes suitable for this purpose include gram-negative or gram-positive bacteria, eubacteria such as Escherichia coli. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are also suitable as cloning or expression hosts for the provided vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used of lower eukaryotic host microorganisms. However, numerous other genera, species and strains are commonly available and useful herein.

[0072] The host cell suitable for expressing the fusion protein provided herein can also be derived from a multicellular organism. Examples of invertebrate cells include plant cells and insect cells. In some preferred embodiments, the host cell is a Chinese Hamster Ovary (CHO) cell. In some other preferred embodiments, the host cell is another mammalian cell line, such as a human cell line.

[0073] Host cells are transformed with the above-described expression or cloning vectors to produce the fusion protein and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0074] The host cells used to produce the fusion proteins provided herein may be cultured in a variety of media. Any of these media may be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN), and / or other cellular and biological properties. TMFormulations, trace elements (usually defined as inorganic compounds with final concentrations in the micromolar range), and glucose or an equivalent energy source. Other necessary supplements may also be included at appropriate concentrations known to those of skill in the art. Culture conditions such as temperature, pH, etc. will be those previously used with the host cell selected for expression and will be apparent to those of skill in the art.

[0075] The fusion protein prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, or affinity chromatography, with affinity chromatography being the preferred purification technique.

[0076] Pharmaceutical Compositions In some aspects, the present disclosure provides compositions, such as pharmaceutical compositions, comprising the fusion proteins herein formulated with a pharma- ceutically acceptable carrier. Such compositions comprise at least one fusion protein of the invention or a polynucleotide encoding the fusion protein.

[0077] As used herein, a "pharmaceutical acceptable carrier" includes physiologically compatible, pharma-ceutically acceptable liquid, gel or solid carriers, aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonicity agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestering or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other ingredients known in the art, or various combinations thereof, and the like. The carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, transepidermal, intravitreal injection or implant administration, and the like. Depending on the route of administration, the active compound, i.e., the components of the present invention, can be coated with a material to protect the compound from acids and other natural conditions that may inactivate the compound.

[0078] The pharmaceutical composition of the present invention may further comprise a pharma-ceutically acceptable antioxidant.The examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical composition of the present invention include water, ethanol, polyol (glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oil such as olive oil, and injectable organic ester such as ethyl oleate.Proper fluidity can be maintained, for example, by using coating agents (such as lecithin), maintaining the required particle size in the case of dispersion, and using surfactants.

[0079] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, dispersing agents, etc. The pharmaceutical composition may be in the form of a solid, paste, ointment, gel, liquid, aerosol, spray, polymer, film, emulsion or suspension.

[0080] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. The injectable pharmaceutical composition may be prepared in any conventional form, such as a liquid solution, suspension, emulsion, or a solid form suitable for producing a liquid solution, suspension, or emulsion. The injectable preparation may include a sterile and / or nonpyrogenic solution ready for injection, a sterile dry soluble preparation such as a lyophilized powder ready for mixing with a solvent immediately before use, including hypodermic tablets, a sterile suspension ready for injection, a sterile dry insoluble preparation ready for combining with a vehicle immediately before use, and a sterile and / or nonpyrogenic emulsion. The solution may be aqueous or nonaqueous. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions.

[0081] The actual dosage of the active ingredient and small molecule in the pharmaceutical composition of the present invention can be varied to obtain an amount of active ingredient effective for a particular patient, composition and mode of administration to achieve a desired therapeutic response without toxicity to the patient. The dosage selected will depend on various pharmacokinetic factors, including the activity of the particular combination of the present invention used, or its esters, salts or amides, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular ingredient used, the age, sex, weight, disorder, general health and past medical history of the patient being treated, and similar factors well known in the medical field.

[0082] Applications of the present disclosure The fusion proteins, pharmaceutical compositions and methods of the present disclosure have numerous in vitro and in vivo utilities, including, for example, those involving the enhancement of immune responses. For example, these molecules can be administered to cultured cells, in vitro or ex vivo, or to human subjects, e.g., in vivo, to enhance immunity in a variety of contexts. The immune response can be modulated, e.g., enhanced, stimulated, upregulated, etc.

[0083] For example, the subject includes a human patient who needs to regulate immune response. In certain embodiments, the method is particularly suitable for treating cancer in vivo. To achieve immune enhancement, the fusion protein can be administered alone or in combination with another therapy. When the fusion protein is administered together with another agent, the two can be administered sequentially in any order or simultaneously.

[0084] In some aspects, the present disclosure provides a method of treating a disorder or disease in a mammal, the method comprising administering to a subject (e.g., a human) in need of treatment a therapeutically effective amount of a fusion protein as disclosed herein. The disorder or disease can be a cancer, such as a cancer treatable by IL-2 and IFNα. A variety of cancers, whether malignant or benign, primary or secondary, can be treated or prevented by the methods provided by the present disclosure. The cancer can be a solid cancer or a hematological malignancy.

[0085] The fusion proteins disclosed herein can be used as a monotherapy or in combination with cellular immunotherapy, targeted therapy, chemotherapy, radiation therapy, and the like.

[0086] Summary of sequences related to the present invention SEQ ID NO:1 (Wild type IL-2 amino acid sequence) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0087] SEQ ID NO:2 (IL-2 derivative, amino acid sequence) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT

[0088] SEQ ID NO:3 (IL-2-Fc fusion protein amino acid sequence) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITRE PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPK

[0089] SEQ ID NO: 4 (Fc-IL-2 fusion protein amino acid sequence) EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPAGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT

[0090] SEQ ID NO:5 (Wild type IFNα amino acid sequence) CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMNEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE

[0091] SEQ ID NO:6 (IFNα derivative, amino acid sequence) CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMNEDSILAVRKYFQRITLYLKEKKYSPCAWEVVAAEIMRSFSLSTNLQESLRSKE

[0092] SEQ ID NO:7 (IFNα-Fc fusion protein amino acid sequence) CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMNEDSILAVRKYFQRITLYLKEKKYSPCAWEVVAAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPEAAGGPSV FLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPK

[0093] SEQ ID NO:8 (Fc-IFNα fusion protein amino acid sequence) EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPAGGGGSGGGGSGGGGSCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQ IFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMNEDSILAVRKYFQRITLYLKEKKYSPCAWEVVAAEIMRSFSLSTNLQESLRSKE

[0094] SEQ ID NO:9 (IL-2-Fc-IFNα fusion protein amino acid sequence) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSG GGGSGGGGSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPAGGGGSGGGGSGGGGSCDLPQTHSLGSRRTLMLLAQMRRIS LFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMNEDSILAVRKYFQRITLYLKEKKYSPCAWEVVAAEIMRSFSLSTNLQESLRSKE

[0095] SEQ ID NO: 10 (IFNα-Fc-IL-2 fusion protein amino acid sequence) CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMNEDSILAVRKYFQRITLYLKEKKYS PCAWEVVAAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PAGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT

[0096] SEQ ID NO:11 (flexible linker amino acid sequence) GGGGSGGGGSGGGGS

[0097] SEQ ID NO: 12 (Fc partial amino acid sequence) EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPK

[0098] The present invention will be further elucidated below in conjunction with the accompanying drawings and specific embodiments, but it should be understood that the following specific embodiments are merely used to illustrate the present invention and are not intended to limit the scope of the present invention. EXAMPLES

[0099] Example 1: Molecular construction and production of fusion proteins An IL-2-Fc-IFNα fusion protein was constructed consisting of an IL-2 portion operably linked from N-terminus to C-terminus to an immunoglobulin Fc portion, and an immunoglobulin Fc portion operably linked from N-terminus to C-terminus to an IFNα portion, hereinafter referred to as IL-2-Fc-IFNα, the amino acid sequence of which is set forth in SEQ ID NO: 9. An IL-2-Fc fusion protein was also constructed consisting of an IL-2 portion operably linked from N-terminus to C-terminus to an immunoglobulin Fc portion, hereinafter referred to as IL-2-Fc, the amino acid sequence of which is set forth in SEQ ID NO: 3. Additionally, an Fc-IFNα fusion protein was constructed consisting of an immunoglobulin Fc portion operably linked from N-terminus to C-terminus to an IFNα portion, hereinafter referred to as Fc-IFNα, the amino acid sequence of which is set forth in SEQ ID NO: 8.

[0100] The sequence was amplified by PCR using standard molecular biology techniques, and the gene encoding the amino acid sequence was ligated into the pcDNA3.4 vector by homologous recombination. Plasmids were extracted from the positive clones whose base sequences were determined, and transfected into Expi293F cells. The cells were shake-cultured for 7 days under conditions of 37°C / 5%CO2 / 125 rpm, and at the end of the culture, the supernatant was collected and subjected to Protein A affinity chromatography to obtain purified fusion protein molecules. Protein concentration was measured using a combination of UV280 and theoretical extinction coefficient.

[0101] FIG. 1B is an SDS PAGE gel image of the IL-2-Fc-IFNα fusion protein, and FIG. 1C is an HPLC purity analysis graph thereof.

[0102] Example 2: Binding ability to receptor IL-2Rα protein by ELISA IL-2Rα protein (10165-H08H, Sino Biological) was coated at 100μl / well at a concentration of 2μg / ml and incubated overnight at 4℃; 3% skim milk 300μl / well was used for blocking at 37℃ for 1h; 100μl of different concentrations of fusion protein and its control sample were added to each well and incubated at 37℃ for 1h; anti-IFNα2 rabbit pAb was then added and the mixture was incubated at 37℃ for 1h, followed by anti-rabbit IgG HRP, followed by incubation at 37℃ for 1h. 100μl of TMB was added to each well and allowed to develop for 5min before adding 50μl of termination solution and reading OD450 with a microplate reader.

[0103] The results are shown in Figure 2. The binding ability of IL-2-Fc and IL-2-Fc-IFNα to the receptor IL-2Rα protein was significantly improved compared to wild-type IL-2 (11848-HNAH1-E, Sino Biological Co., Ltd.).

[0104] Example 3: Binding ability to receptor IL-2Rβ protein by ELISA IL-2Rβ protein (10696-H05H, Sino Biological) was coated at 100μl / well at a concentration of 2μg / ml and incubated overnight at 4℃; 3% skim milk 300μl / well was used for blocking at 37℃ for 1h; 100μl of different concentrations of fusion protein and its control sample were added to each well and incubated at 37℃ for 1h; anti-IFNα2 rabbit pAb was then added and the mixture was incubated at 37℃ for 1h, followed by anti-rabbit IgG HRP, followed by incubation at 37℃ for 1h. 100μl of TMB was added to each well and allowed to develop for 5min before adding 50μl of termination solution and OD450 was read on a microplate reader.

[0105] The results are shown in Figure 3. The binding ability of IL-2-Fc and IL-2-Fc-IFNα to the receptor IL-2Rβ protein was improved by 24-fold or more compared to wild-type IL-2.

[0106] Example 4: Binding ability to receptor IFNαR2 protein by ELISA IFNαR2 protein (10359-H02H, Sino Biological) was coated at a concentration of 2 μg / ml, 100 μl / well and incubated overnight at 4°C; 300 μl / well of 3% skim milk was used for blocking at 37°C for 1 h; 100 μl of different concentrations of fusion protein and its control sample were added to each well and incubated at 37°C for 1 h; anti-IL-2 rabbit pAb was then added and the mixture was incubated at 37°C for 1 h, followed by anti-rabbit IgG HRP and subsequent incubation at 37°C for 1 h. 100 μl of TMB was added to each well and allowed to develop for 5 min before adding 50 μl of termination solution and the OD450 was read on a microplate reader. The results are shown in Figure 4. Compared with wild-type IFNα, the binding ability of Fc-IFNα to the receptor IFNαR2 protein was significantly reduced, and the binding ability of IL-2-Fc-IFNα to the receptor IFNαR2 protein was significantly increased.

[0107] Example 5: Binding ability to receptor IL-2Rα protein among different species by ELISA Human, mouse and cynomolgus IL-2Rα proteins were coated separately at 2 μg / ml, 100 μl / well and incubated overnight at 4°C. 300 μl / well of 3% skim milk was used for blocking at 37°C for 1 h, and 100 μl of different concentrations of fusion protein and its control sample were added to each well and incubated at 37°C for 1 h; anti-IFNα2 rabbit pAb was then added and the mixture was incubated at 37°C for 1 h, followed by anti-rabbit IgG HRP, followed by incubation at 37°C for 1 h. 100 μl of TMB was added to each well and allowed to develop for 5 min before adding 50 μl of termination solution and OD450 was read on a microplate reader.

[0108] The results are shown in Figure 5. It was confirmed that IL-2-Fc-IFNα has similar binding ability to the receptor IL-2Rα protein of humans, mice, and cynomolgus monkeys, and exhibits cross-species binding between mice and cynomolgus monkeys.

[0109] Example 6: Binding ability to receptor IL-2Rβ protein among different species by ELISA Human, mouse and cynomolgus IL-2Rβ proteins were coated separately at 2 μg / ml, 100 μl / well and incubated overnight at 4°C. 300 μl / well of 3% skim milk was used for blocking at 37°C for 1 h, and 100 μl of different concentrations of fusion protein and its control sample were added to each well and incubated at 37°C for 1 h; anti-IFNα2 rabbit pAb was then added and the mixture was incubated at 37°C for 1 h, followed by anti-rabbit IgG HRP, followed by incubation at 37°C for 1 h. 100 μl of TMB was added to each well and allowed to develop for 5 min before adding 50 μl of termination solution and OD450 was read on a microplate reader.

[0110] The results are shown in Figure 6. The experiment confirmed that IL-2-Fc-IFNα binds to the receptor IL-2Rβ proteins of humans, mice, and cynomolgus monkeys across species. The binding affinity to cynomolgus monkey IL-2Rβ protein was similar to that to human IL-2Rβ protein, and the binding affinity to mouse IL-2Rβ protein was lower than that to human IL-2Rβ protein.

[0111] Example 7: Binding ability to receptor IFNαR2 protein among different species by ELISA Human, mouse and cynomolgus IFNαR2 proteins were separately coated at 2μg / ml in 100μl / well and incubated overnight at 4℃; 3% skim milk 300μl / well was used for blocking at 37℃ for 1h; 100μl of different concentrations of fusion protein and its control sample were added to each well and incubated at 37℃ for 1h; anti-IL-2 rabbit pAb was then added and the mixture was incubated at 37℃ for 1h, followed by anti-rabbit IgG HRP and further incubation at 37℃ for 1h. 100μl of TMB was added to each well and allowed to develop for 5min before adding 50μl of termination solution and OD450 was read on a microplate reader.

[0112] The results are shown in Figure 7. IL-2-Fc-IFNα was confirmed to bind to the receptor IFNαR2 protein of humans, mice, and cynomolgus monkeys, demonstrating binding across species.

[0113] Example 8: Detection of binding ability to FcRn protein 8.1 Binding ability to FcRn protein by ELISA method FcRn protein (10359-H02H, Sino Biological) was coated at a concentration of 2 μg / ml, 100 μl / well, and incubated overnight at 4°C. 3% skim milk, 300 μl / well, was used for blocking at 37°C for 1 h, and 100 μl of different concentrations of fusion protein and its control sample were added to each well and incubated at 37°C for 1 h; anti-IFNα2 rabbit pAb was then added and the mixture was incubated at 37°C for 1 h, followed by anti-rabbit IgG HRP, followed by incubation at 37°C for 1 h. 100 μl of TMB was added to each well and allowed to develop for 5 min before adding 50 μl of termination solution, and OD450 was read on a microplate reader.

[0114] The results are shown in Figure 8 A. The experiment confirmed that IL-2-Fc-IFNα had a significantly higher binding affinity than the Herceptin antibody (trastuzumab).

[0115] 8.2 Detection of FcRn protein binding ability using Fortebio The test protein was loaded with ProA and HIS probes at a concentration of 4 μg / ml, and bound to and dissociated from different concentrations of FcRn (4 μg / ml, 1 μg / ml, 0.25 μg / ml, 0), and the affinity of the test molecule to FcRn was calculated. The results are shown in Figure 8B. The experiment confirmed that IL-2-Fc-IFNα has a significantly higher affinity for FcRn protein compared to the Herceptin antibody (trastuzumab).

[0116] Example 9: Detection of immune cell subsets after stimulation of human PBMC cells Fresh human PBMCs were plated at 150,000 cells / well in a 96-well plate, and 500 nM of protein was added according to the experimental design, followed by 72 h of culture. After the culture was completed, staining was performed for flow cytometry detection, with anti-Foxp3 (FITC), anti-CD4 (PE), anti-CD56 (PE), anti-CD8 (PE), and anti-Granzyme B (FITC) labeled antibodies, respectively, and flow cytometry data was collected, summarized, and analyzed.

[0117] The results are shown in Figure 9. This experiment confirmed that the ability of IL-2-Fc-IFNα to stimulate activation of NK cells and CD8+ T cells in PBMCs, and expression of granzyme B, maintained the same activity as wild-type IL-2. Treg cells (FOXP3 + The ability of IL-2 to stimulate proliferation of IL-1 cells was significantly reduced compared to wild-type IL-2.

[0118] Example 10: Detection of the ability of CTLL-2 to stimulate proliferation of mouse T lymphocytes Mouse CTLL-2 cells were cultured and expanded to the required cell number, then plated in a 96-well plate at 100,000 cells / well. Proteins were added at different concentrations (100 nM, 4 nM, 800 pM, 400 pM, 200 pM, 100 pM, 50 pM, 25 pM, 0) according to the experimental design, and the cells were cultured for 72 hours. The expression level of formazan in live cells was measured using the CCK-8 method to evaluate cell viability. The specific operation was performed according to the instructions. Briefly, 10% CCK-8 solution was added after the end of the culture, mixed well, and then cultured for 2 to 4 hours. The absorbance data was collected by a microplate reader. The data was analyzed and processed in Excel, and the pharmacodynamic dose curve was fitted based on the absorbance data using GraphPad Prism 7 software, and the IC50 was calculated.

[0119] The results are shown in Figure 10. This experiment confirmed that the ability of IL-2-Fc-IFNα to stimulate the proliferation of CTLL-2 cells was 10-fold higher than that of wild-type IL-2.

[0120] Example 11: Detection of direct killing ability against tumor cells in vitro Direct killing ability is the measurement of the ability of IFN-a to directly kill tumor cells in the absence of immune cells.

[0121] 11.1 Detection of direct killing activity against human gastric cancer cell line NCI-N87 NCI-N87 human gastric cancer cells were cultured and grown to the required cell number, then plated in a 96-well plate at 4000 cells / well, and proteins at different concentrations (4000 nM, 1000 nM, 250 nM, 100 nM, 62.5 nM, 15.625 nM, 10 nM, 4 nM, 1.5625 nM, 1 nM, 244.14 pM, 156.25 pM, 100 pM, 100 pM, 10 pM, 1 pM, 0) were added according to the experimental design, and the cells were then cultured for 5 days. The expression level of formazan in live cells was measured using the CCK-8 method to evaluate cell viability. The specific operation was performed according to the instructions. Briefly, 10% CCK-8 solution was added after the end of the culture, mixed well, and then cultured for 2 to 4 hours. The absorbance data was collected by a microplate reader. Data were analyzed and processed in Excel, and GraphPad Prism 7 software was used to fit pharmacodynamic dose curves and calculate IC50s based on absorbance data.

[0122] Cell viability The results are shown in Figure 11A. Wild-type IFNα exhibited a significant killing effect on NCI-N87 cells, whereas IL-2-Fc-IFNα exhibited a killing effect on NCI-N87 cells only at high concentrations. The negative control TTI-622 (SIRPa-Fc) did not exhibit killing function on NCI-87 cells.

[0123] 11.2 Detection of direct killing activity against human breast cancer cell line MDA-MB-231 MDA-MB-231 human breast cancer cells were cultured and grown to the required cell number, then plated in a 96-well plate at 2,000 cells / well, and proteins were added at different concentrations (4000nM, 1000nM, 250nM, 62.5nM, 15.625nM, 4nM, 1nM, 244.14pM, 61pM, 15.26pM, 0) according to the experimental design, and the culture was continued for 5 days. The expression level of formazan in live cells was measured using the CCK-8 method to evaluate cell viability. The specific operation was performed according to the instructions. Briefly, 10% CCK-8 solution was added after the end of the culture, mixed well, and then cultured for 2 to 4 hours. The absorbance data was collected by a microplate reader. The data was analyzed and processed in Excel, and the pharmacodynamic dose curve was fitted based on the absorbance data using GraphPad Prism 7 software, and the IC50 was calculated.

[0124] The results are shown in Figure 11B. Wild-type IFNα had significant killing function against MDA-MB-231 cells; however, IL-2-Fc-IFNα had killing effect against MDA-MB-231 cells only at high concentrations. The negative control TTI-622 (SIRPa-Fc) did not show killing function against MDA-MB-231 cells.

[0125] 11.3 Detection of direct killing ability against human melanoma cell line A375 After culturing and growing to the required cell number, A375 human melanoma cells were plated at 1000 cells / well in a 96-well plate, and different concentrations of proteins (16000nM, 1600nM, 160nM, 16nM, 1.6nM, 160pM, 16pM, 1.6pM, 0.16pM, 0) were added according to the experimental design, and then the culture was continued for 5 days. The expression level of formazan in live cells was measured using the CCK-8 method to evaluate the cell viability. The specific operation was performed according to the instructions. In brief, 10% CCK-8 solution was added after the end of the culture, mixed well, and then cultured for 2 to 4 hours. The absorbance data were collected by a microplate reader. The data were analyzed and processed in Excel, and the pharmacodynamic dose curve was fitted based on the absorbance data using GraphPad Prism 7 software to calculate the IC50.

[0126] The results are shown in Figure 11C. Wild-type IFNα had significant killing function against A375 cells; however, IL-2-Fc-IFNα had killing effect against A375 cells only at high concentrations. The negative control TTI-622 (SIRPa-Fc) did not show killing function against A375 cells.

[0127] 11.4 Detection of direct killing ability against human Burkitt lymphoma cell line Daudi B After culturing and expanding to the required cell number, Daudi B human Burkitt's lymphoma cells were plated at 10,000cells / well in a 96-well plate, and different concentrations of proteins (62.5nM, 12.5nM, 2.5nM, 500pM, 250pM, 125pM, 62.5pM, 31.25pM, 15.625pM, 7.813pM, 4pM, 2pM, 0) were added according to the experimental design, and then the culture was continued for 5 days. The expression amount of formazan in live cells was measured using the CCK-8 method to evaluate the cell viability. The specific operation was performed according to the instruction manual. Briefly, 10% CCK-8 solution was added after the end of the culture, and after mixing well, the cells were cultured for 2 to 4 hours. The absorbance data were collected by a microplate reader. Data were analyzed and processed in Excel, and GraphPad Prism 7 software was used to fit pharmacodynamic dose curves and calculate IC50s based on absorbance data.

[0128] The results are shown in Figure 11D. Wild-type IFNα had significant killing function against Daudi B cells; however, IL-2-Fc-IFNα had killing effect against Daudi B cells only at high concentrations. The negative control TTI-622 (SIRPa-Fc) did not show killing function against Daudi B cells.

[0129] This experiment confirmed that the direct killing function of the fusion protein herein was weaker than wild-type IFNα2, yet retained the direct killing function of the IFNα arm.

[0130] Example 12: Detection of comprehensive killing ability against tumor cells in vitro Global killing function refers to the sum of direct and indirect killing capabilities, which is achieved in the presence of immune cells such as PBMCs.

[0131] 12.1 Detection of comprehensive killing activity against human gastric cancer cell line NCI-N87 After culturing and growing to the required cell number, NCI-N87 human gastric cancer cells were plated in 96-well plates at 4,000 cells / well and allowed to adhere for 6 h. After procuring fresh human PBMCs, the cells were plated according to an effector-target ratio of 10:1, and different concentrations of proteins (800nM, 80nM, 8nM, 800pM, 80pM, 8pM, 0.8pM, 0) were added according to the experimental design, and then culture was continued for 5 days. The plates were washed three times with PBS to remove PBMCs, and the expression level of formazan in live cells was measured using the CCK-8 method to evaluate the cell viability. The specific operation was performed according to the instructions. Briefly, 10% CCK-8 solution was added after the end of the culture, mixed well, and then incubated for 2 to 4 h. The absorbance data were collected by a microplate reader. Data were analyzed and processed in Excel, and GraphPad Prism 7 software was used to fit pharmacodynamic dose curves and calculate IC50s based on absorbance data.

[0132] The results are shown in Figure 12 A. The comprehensive killing ability of IL-2-Fc-IFNα against NCI-N87 cells was stronger than that of wild-type IL-2.

[0133] 12.2 Detection of comprehensive killing activity against human breast cancer cell line MDA-MB-231 MDA-MB-231 human breast cancer cells were cultured and grown to the required cell number, then plated in a 96-well plate at 4,000 cells / well and allowed to adhere for 6 h. After procuring fresh human PBMCs, the cells were plated according to an effector-target ratio of 10:1, and different concentrations of proteins (800nM, 80nM, 8nM, 800pM, 80pM, 8pM, 0.8pM, 0) were added according to the experimental design, and then culture was continued for 5 days. The plate was washed three times with PBS to remove PBMCs, and the expression level of formazan in live cells was measured using the CCK-8 method to evaluate the cell viability. The specific operation was performed according to the instructions. Briefly, 10% CCK-8 solution was added after the end of the culture, mixed well, and then incubated for 2 to 4 h. The absorbance data was collected by a microplate reader. Data were analyzed and processed in Excel, and GraphPad Prism 7 software was used to fit pharmacodynamic dose curves and calculate IC50s based on absorbance data.

[0134] The results are shown in Figure 12B. The comprehensive killing ability of IL-2-Fc-IFN-a against MDA-MB-231 cells was similar to that of wild-type IL-2.

[0135] 12.3 Detection of comprehensive killing ability against human melanoma cell line A375 After culturing and expanding to the required cell number, A375 human melanoma cells were plated in 96-well plates at 4,000 cells / well and allowed to adhere for 6 h. After procuring fresh human PBMCs, the cells were plated according to an effector-target ratio of 10:1, and different concentrations of proteins (800nM, 80nM, 8nM, 800pM, 80pM, 8pM, 0.8pM, 0) were added according to the experimental design, and then the culture was continued for 5 days. The plates were washed three times with PBS to remove PBMCs, and the expression level of formazan in live cells was measured using the CCK-8 method to evaluate the cell viability. The specific operation was performed according to the instructions. Briefly, 10% CCK-8 solution was added after the end of the culture, mixed well, and then incubated for 2 to 4 h. The absorbance data were collected by a microplate reader. Data were analyzed and processed in Excel, and GraphPad Prism 7 software was used to fit pharmacodynamic dose curves and calculate IC50s based on absorbance data.

[0136] The results are shown in Figure 12C. The comprehensive killing ability of IL-2-Fc-IFN-a against A375 cells was significantly stronger than that of wild-type IL-2.

[0137] 12.4 Comprehensive killing of human Burkitt lymphoma cell line Daudi B Daudi B human Burkitt lymphoma cells were cultured and expanded to the required cell number. Fresh PBMCs were collected and isolated from donors. Daudi B cells were plated in 96-well plates at 10,000 cells / well, two replicate wells, and PBMCs (effector-target ratios were set at 10:1 and 5:1) and different concentrations of IAMA005 (i.e., IL-2-Fc-IFN-a, 16 nM or 160 nM) were added to the corresponding well plates according to the experimental layout design, and a total of three plates were harvested and used separately according to different detection times. After 24 h, 48 h, and 72 h of co-culture, the culture supernatants were sequentially collected and kept in LDH detection storage solution and stored in a -80 °C freezer. The cells in each well were resuspended separately, followed by staining and detection according to the requirements of flow cytometry, flow cytometry data were collected, and data were tabulated and analyzed. The results were shown in Figure 12D.

[0138] After culturing and expanding to the required cell number, Daudi B human Burkitt's lymphoma cells were stained with CFSE, and Daudi B cells were plated at 20,000 cells / well in 96-well plates. After procuring fresh human PBMCs, cells were plated according to an effector-target ratio of 10:1, and different concentrations of proteins (800nM, 80nM, 8nM, 800pM, 80pM, 8pM, 0.8pM, 0) were added according to the experimental design, and then culture was continued for 5 days. After washing the plates three times with PBS and centrifuging at low speed to remove cell debris, a fluorescent microplate reader was employed to measure the fluorescence intensity of live cells and evaluate cell viability. Excel software was used for data analysis and processing, and GraphPad Prism 7 software was used for fitting pharmacodynamic dose curves based on absorbance value data to calculate IC50.

[0139] The results are shown in Figure 12E. The comprehensive killing capacity of IL-2-Fc-IFN-a against Daudi B cells maintained activity comparable to that of wild-type IL-2.

[0140] Example 13: Pharmacodynamic studies in an in vitro organoid system Pleural fluid samples (identified as HER2 2+ positive) were collected from breast cancer patients, and cells, including tumor cells and immune cells, were isolated from the samples. Candidate and reference drugs were co-cultured with tumor cell-immune cell complexes in vitro. After 4-5 days of administration, the culture medium and cells were collected and analyzed for tumor cell, granzyme B, IFN-γ, etc.

[0141] The results are shown in Figure 13. In the ex vivo evaluation system of this sample, the antitumor activity of pleural effusion immune cells stimulated with IL-2-Fc-IFN-a was significantly increased, and the antitumor cytokines granzyme B and IFN-γ secreted by the pleural effusion immune cells were also significantly increased.

[0142] Exemplary embodiments of the present disclosure have been described above in conjunction with the accompanying drawings. However, those skilled in the art should understand that the present disclosure is not limited to the specific structures disclosed. Various changes and modifications can be made to the exemplary embodiments of the present disclosure without departing from the spirit and scope of the present disclosure. All of these changes and modifications are included in the scope of protection defined by the claims of the present disclosure.

[0143] References [1]Yang Y, Lundqvist A. Immunomodulatory effects of IL-2 and IL-15; implications for cancer immunotherapy[J]. Cancers, 2020, 12(12): 3586. [2] Choudhry H, Helmi N, Abdulaal WH, et al. Prospects of IL-2 in cancer immunotherapy[J]. BioMed research international, 2018, 2018. [3] Levin A M, Bates D L, Ring A M, et al. Exploiting a natural conformational switch to engineer an interleukin-2 ‘superkine’[J]. Nature, 2012, 484(7395): 529-533. [4] Khailaie S, Montaseri G, Meyer-Hermann M. An adaptive control scheme for Interleukin-2 therapy[J]. Iscience, 2020, 23(11): 101663. [5] Cauwels A, Van Lint S, Garcin G, et al. A safe and highly efficient tumor-targeted type I interferon immunotherapy depends on the tumor microenvironment[J]. Oncoimmunology, 2018, 7(3): e1398876. [6] Garcin G, Paul F, Staufenbiel M, et al. High efficiency cell-specific targeting of cytokine activity[J]. Nature communications, 2014, 5(1): 1-9. [7] Piehler J, Roisman L C, Schreiber G. New structural and functional aspects of the type I interferon-receptor interaction revealed by comprehensive mutational analysis of the binding interface[J]. Journal of biological chemistry, 2000, 275(51): 40425-40433. [8] Sim G C, Liu C, Wang E, et al. IL2 variant circumvents ICOS+ regulatory T-cell expansion and promotes NK cell activation[J]. Cancer Immunology Research, 2016, 4(11): 983-994. [9] Heaton K M, Ju G, Morris D K, et al. Characterization of lymphokine-activated killing by human peripheral blood mononuclear cells stimulated with interleukin 2 (IL-2) analogs specific for the intermediate affinity IL-2 receptor[J]. Cellular immunology, 1993, 147(1): 167-179.

[10] Fukushima K, Yamashita K. Interleukin-2 carbohydrate recognition modulates CTLL-2 cell proliferation[J]. Journal of Biological Chemistry, 2001, 276(10): 7351-7356.

[11] Luo F, Qian J, Yang J, et al. Bifunctional αHER2 / CD3 RNA-engineered CART-like human T cells specifically eliminate HER2+ gastric cancer[J]. Cell Research, 2016, 26(7): 850-853.

[12] Chen X, Ai X, Wu C, et al. A novel human IL-2 mutein with minimal systemic toxicity exerts greater antitumor efficacy than wild-type IL-2[J]. Cell death & disease, 2018, 9(10): 989.

[13] Carmenate T, Pacios A, Enamorado M, et al. Human IL-2 mutein with higher antitumor efficacy than wild type IL-2[J]. The Journal of Immunology, 2013, 190(12): 6230-6238.

Claims

1. A fusion protein comprising an IL-2 portion and an Fc portion, wherein the IL-2 portion comprises an amino acid sequence having one or more mutations compared to a wild-type IL-2 protein, and the amino acid sequence has at least 90% identity with SEQ ID NO:

2.

2. The mutations are as follows, with reference to the amino acid positions of SEQ ID NO:1: R38A, L80F, R81D, L85V, I86V and I91F The fusion protein of claim 1, comprising one or more substitutions selected from:

3. 3. The fusion protein of claim 1 or 2, wherein the Fc portion comprises a human IgG Fc, such as human IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, or a variant thereof.

4. The Fc variant comprises: L234A and L235A mutations, as well as M252Y, S254T and T256E mutations The fusion protein of claim 3, comprising one or more mutations selected from: L234A

5. The fusion protein of any one of claims 1 to 4, wherein the fusion protein further comprises an IFNα portion.

6. The fusion protein of claim 5 , wherein the IFNα portion comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 6 or 5.

7. The IFNα portion has the following structure, with reference to the amino acid positions of SEQ ID NO:5: R144A and R149A The fusion protein of claim 6, comprising one or more substitutions selected from:

8. (a) the IL-2 moiety operably linked to the Fc moiety; (b) the IL-2 moiety operably linked to the Fc moiety, and the Fc moiety operably linked to the IFNα moiety; (c) the IL-2 portion operably linked to the IFNα portion, and the IFNα portion operably linked to the Fc portion; or (d) the IFNα portion operably linked to the IL-2 portion, and the IL-2 portion operably linked to the Fc portion. The fusion protein according to any one of claims 1 to 7, comprising:

9. From the N-terminus to the C-terminus, (a) the IL-2 moiety operably linked to the Fc moiety; (b) the IL-2 moiety operably linked to the Fc moiety, and the Fc moiety operably linked to the IFNα moiety; (c) the Fc portion operably linked to the IL-2 portion; or (d) the IFNα portion operably linked to the Fc portion, and the Fc portion operably linked to the IL-2 portion. The fusion protein of claim 8 .

10. The fusion protein of claim 8 or 9, wherein the operable linkage is a direct linkage or a linkage via a peptide linker, optionally wherein the peptide linker is a GS series linker, such as the peptide linker set forth in SEQ ID NO:

11.

11. The fusion protein according to any one of claims 1 to 10, comprising the amino acid sequence set forth in SEQ ID NO: 3, 4, 9 or 10.

12. A fusion protein comprising the IFNα portion and the Fc portion, wherein the IFNα portion comprises an amino acid sequence having one or more mutations compared to a wild-type IFNα protein, and the amino acid sequence has at least 90% identity to SEQ ID NO:

6.

13. The IFNα portion has the following structure, with reference to the amino acid positions of SEQ ID NO:5: R144A and R149A The fusion protein of claim 12, comprising one or more substitutions selected from:

14. The fusion protein of claim 12 or 13, wherein the Fc portion comprises a human IgG Fc, such as human IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, or a variant thereof.

15. The Fc variant comprises: L234A and L235A mutations, as well as M252Y, S254T and T256E mutations The fusion protein of claim 14, comprising one or more mutations selected from:

16. From the N-terminus to the C-terminus, (a) the IFNα portion operably linked to the Fc portion; or (b) the Fc portion operably linked to the IFNα portion. The fusion protein according to any one of claims 12 to 15, comprising:

17. The fusion protein of claim 16, comprising the amino acid sequence set forth in SEQ ID NO: 7 or 8.

18. A nucleic acid molecule comprising a nucleic acid sequence encoding the fusion protein according to any one of claims 1 to 16.

19. A vector comprising the nucleic acid molecule of claim 18.

20. 20. A host cell comprising the nucleic acid molecule of claim 18 or the vector of claim 19.

21. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 16 or a nucleic acid molecule encoding the same, and a pharma- ceutically acceptable carrier.

22. A method for producing the fusion protein according to any one of claims 1 to 16, comprising the steps of: - expressing said fusion protein in a host cell comprising a vector encoding said fusion protein; and - isolating said fusion protein from the host cell culture.

23. A method for modulating an immune response in a subject, comprising administering to the subject a fusion protein according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 21.

24. A method for treating or preventing cancer or an infectious disease in a subject, comprising administering to the subject an effective amount of a fusion protein according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 21.

25. 25. The method of claim 24, wherein the cancer is selected from breast cancer, gastric cancer, melanoma, lymphoma, lung cancer, colon cancer, ovarian cancer, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, pancreatic cancer and leukemia.

26. 20. Use of a fusion protein according to any one of claims 1 to 16 in the manufacture of a medicament for the prevention, treatment and / or management of cancer or an infectious disease.

27. The fusion protein according to any one of claims 1 to 16 for use in the treatment or prevention of cancer or an infectious disease.

28. A kit comprising a container containing the fusion protein of any one of claims 1 to 16 or the pharmaceutical composition of claim 21.

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