New fusion protein and its use

Abstract

The modification of human interleukin-2 (IL-2) and interferon α (IFNα); the fusion protein formed by the modified IL-2, IFNα or IL-2 / IFNα dual factor and Fc; and the design, preparation and use of these fusion proteins. IFNα changes the binding ability of IFNα to its receptor through gene mutation to obtain a better fusion protein, which solves the shortcomings of weak specificity and large side effects of existing drugs.

Description

[0001] Cross-references

[0002] This application claims priority to Chinese patent application 202210521837.5 filed on May 13, 2022, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a Sequence Listing, the entire contents of which are incorporated herein by reference. Technical Field

[0005] The present invention belongs to the field of biomedicine technology, and specifically relates to the design, preparation and use of a novel IL-2, IFNα and Fc fusion protein. Background Art

[0006] IL-2, also known as T cell growth factor, has a gene located on chromosome 4, including a 7kb sequence, composed of 133 amino acids, and a molecular weight of about 15kD. IL-2 acts through IL-2R, which includes three subunits, IL-2Rα (i.e. CD25), IL-2Rβ (i.e. CD122), and IL-2Rγ (i.e. CD132). The three subunits can form three receptor forms: high binding receptors contain all three subunits IL-2Rα / β / γ, medium binding receptors contain two subunits IL-2Rβ / γ, and low binding receptor IL-2Rα. Among them, IL-2Rβ and IL-2Rγ are necessary for IL-2 to activate the downstream signaling pathway. When IL-2 binds to IL-2Rβ and IL-2Rγ at the same time, the two receptor subunits form a heterodimer, phosphorylate STAT5 in the cell, enter the cell nucleus and lead to the transcription and expression of the corresponding genes; IL-2Rα is not necessary for the signal, but it can promote the binding of IL-2 with 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 the expression level will increase after T cells are activated; IL-2Rα is continuously highly expressed in regulatory T cells, and is briefly expressed in activated CD8+T cells, and then the expression level is downregulated [1,2]. IL-2 was the second approved immunotherapy, used to treat metastatic melanoma (1988) and renal cell carcinoma (1992); IFNα was the first approved immunotherapy (1986), used to treat hairy cell leukemia, and was later approved to treat metastatic melanoma, renal cell carcinoma, non-Hodgkin's lymphoma, and Kaposi's sarcoma.

[0007] IL-2 drugs have a short half-life, so approved IL-2 drugs for the treatment of tumors require higher doses to be effective, but high doses of IL-2 can cause significant toxic side effects and cannot be widely used. They have low selectivity for NK cells and CD8+T cells that express IL-2Rβ and IL-2Rγ, so they cannot fully exert their ability to kill tumors [3,4].

[0008] On the other hand, IFNα is a cytokine produced by the body's immune cells. It is a group of low-molecular glycoproteins with similar structures and functions that are produced by immune cells through antiviral responses when the body is infected by a virus. Interferon plays a very important role in the body's immune system [5,6]. Recombinant human interferon α has been approved for the treatment of viral infections and tumors. Since interferon α receptors are widely distributed and expressed in many normal cells, they are prone to cause significant toxic side effects.

[0009] There is a need in the art to generate new cytokine constructs that provide more effective therapeutic effects and at the same time fewer side effects. Summary of the invention

[0010] To solve the above problems, the present invention discloses the design, preparation and use of a novel fusion protein of IL-2, IFNα and Fc. By means of gene mutation, the binding ability of IL-2 to its receptor and IFNα to its receptor is changed, thereby solving the shortcomings of existing drugs such as weak specificity, short half-life and large side effects.

[0011] In some aspects, the present disclosure provides 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, the amino acid sequence being an amino acid sequence having at least 90% identity with SEQ ID NO:2.

[0012] In some embodiments, the mutation comprises one or more substitutions selected from the group consisting of R38A, L80F, R81D, L85V, I86V, and I91F with reference to an amino acid position in SEQ ID NO: 1.

[0013] In some embodiments, the Fc portion comprises human IgG Fc, such as human IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc or variants thereof. For example, the Fc variant comprises one or more mutations selected from the group consisting of L234A and L235A mutations, and M252Y, S254T and T256E mutations.

[0014] 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 the amino acid positions in SEQ ID NO: 5: R144A and R149A.

[0015] In some embodiments, the fusion protein comprises:

[0016] (a) the IL-2 portion is operably linked to the Fc portion;

[0017] (b) the IL-2 portion is operably linked to the Fc portion and the Fc portion is operably linked to the IFNα portion;

[0018] (c) the IL-2 portion is operably linked to the IFNα portion and the IFNα portion is operably linked to the Fc portion; or

[0019] (d) the IFNα portion is operably linked to the IL-2 portion and the IL-2 portion is operably linked to the Fc portion.

[0020] In some further embodiments, the fusion protein comprises:

[0021] (a) the IL-2 portion is operably linked to the Fc portion;

[0022] (b) the IL-2 portion is operably linked to the Fc portion and the Fc portion is operably linked to the IFNα portion;

[0023] (c) the Fc portion is operably linked to the IL-2 portion; or

[0024] (d) the IFNα portion is operably linked to the Fc portion and the Fc portion is operably linked to the IL-2 portion.

[0025] In some embodiments, the operably linked is directly linked or linked via a peptide linker, optionally the peptide linker is a GS series linker, such as (GS)n, wherein n = 1-5. In some embodiments, the linker is as shown in SEQ ID NO:11.

[0026] In some embodiments, the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 3, 4, 9 or 10.

[0027] In some aspects, the present 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, the amino acid sequence being an amino acid sequence having at least 90% identity to SEQ ID NO:6.

[0028] In some embodiments, the IFNα portion comprises one or more substitutions selected from the group consisting of: R144A and R149A with reference to an amino acid position in SEQ ID NO:5.

[0029] In some embodiments, the Fc portion comprises human IgG Fc, such as human IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc or variants thereof. For example, the Fc variant comprises one or more mutations selected from the group consisting of L234A and L235A mutations, and M252Y, S254T and T256E mutations.

[0030] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus:

[0031] (a) the IFNα portion is operably linked to the Fc portion; or

[0032] (b) The Fc portion is operably linked to the IFNα portion.

[0033] In some embodiments, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:7 or 8.

[0034] In some aspects, the present disclosure provides a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein disclosed herein.

[0035] In some aspects, the present disclosure provides vectors comprising the nucleic acid molecules disclosed herein.

[0036] In some aspects, the disclosure provides host cells comprising a nucleic acid molecule or a vector disclosed herein.

[0037] In some aspects, the present disclosure provides a pharmaceutical composition comprising the fusion protein disclosed herein or a nucleic acid molecule encoding the same, and a pharmaceutically acceptable carrier.

[0038] In some aspects, the present disclosure provides a method for producing a fusion protein disclosed herein, comprising the following steps:

[0039] - expressing the fusion protein in a host cell comprising a vector encoding the fusion protein; and

[0040] - isolating the fusion protein from the host cell culture.

[0041] In some aspects, the present disclosure provides a method of modulating an immune response in a subject, comprising administering to the subject a fusion protein or a pharmaceutical composition disclosed herein.

[0042] In some aspects, 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 a pharmaceutical composition disclosed herein.

[0043] In some embodiments, 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.

[0044] In some aspects, the present disclosure provides use of the fusion protein disclosed herein in the preparation of a medicament for preventing, treating and / or managing cancer or infectious diseases.

[0045] In some aspects, the present disclosure provides the fusion proteins disclosed herein for use in treating or preventing cancer or infectious diseases.

[0046] In some aspects, the present disclosure provides a kit comprising a container comprising a fusion protein or a pharmaceutical composition disclosed herein.

[0047] The present invention also relates to the following embodiments:

[0048] 1. A novel IL-2, IFNα and Fc fusion protein, characterized in that it comprises: IL-2 and IFNα dual factor Fc fusion protein, IL-2 and Fc fusion protein, IFNα and Fc fusion protein;

[0049] IL-2 and IFNα dual-factor Fc fusion protein: mutant IL-2 and mutant IFNα are fused to the N-terminus or C-terminus of Fc, respectively, to obtain polypeptide chains IL-2-Fc-IFNα or IFNα-Fc-IL-2;

[0050] IL-2 and Fc fusion protein: The mutant IL-2 is fused to the N-terminus or C-terminus of Fc to obtain the polypeptide chain Fc-IL-2 or IL-2-Fc;

[0051] IFNα and Fc fusion protein: The mutant IFNα is fused to the N-terminus or C-terminus of Fc to obtain a polypeptide chain Fc-

[0052] IFNα or IFNα-Fc.

[0053] 2. According to the novel IL-2, IFNα and Fc fusion protein described in Embodiment 1, it is characterized in that in the IL-2 and IFNα dual-factor Fc fusion protein, the Fc fragment includes 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.

[0054] 3. According to the novel IL-2, IFNα and Fc fusion protein described in Embodiment 1, it is characterized in that in the IL-2 and Fc fusion protein, the Fc fragment includes 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.

[0055] 4. According to the novel IL-2, IFNα and Fc fusion protein described in Embodiment 1, it is characterized in that the IFNα and Fc fusion protein, the Fc fragment includes 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.

[0056] 5. The novel IL-2, IFNα and Fc fusion protein according to any one of embodiments 1-2 is characterized in that, in the IL-2 and IFNα dual-factor Fc fusion protein, the Fc fragment also introduces L234A and L235A mutations, and simultaneously introduces M252Y, S254T and T256E mutations.

[0057] 6. The novel IL-2, IFNα and Fc fusion protein according to any one of embodiments 1 or 3 is characterized in that, in the IL-2 and Fc fusion protein, the Fc fragment also introduces L234A and L235A mutations, and simultaneously introduces M252Y, S254T and T256E mutations.

[0058] 7. The novel IL-2, IFNα and Fc fusion protein according to any one of embodiments 1 or 4 is characterized in that, in the IFNα and Fc fusion protein, the Fc fragment also introduces L234A and L235A mutations, and simultaneously introduces M252Y, S254T and T256E mutations.

[0059] 8. According to the novel IL-2, IFNα and Fc fusion protein described in Embodiment 1, it is characterized in that the IL-2 and IFNα dual-factor Fc fusion protein, the IL-2 mutant is obtained by introducing mutations on the basis of 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.

[0060] 9. According to the novel IL-2, IFNα and Fc fusion protein described in Embodiment 3, it is characterized in that the IL-2 and Fc fusion protein, the IL-2 mutant is obtained by introducing mutations on the basis of 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.

[0061] 10. According to the novel IL-2, IFNα and Fc fusion protein described in Embodiment 1, it is characterized in that the IL-2 and IFNα dual-factor Fc fusion protein, the IFNα derivative is obtained by introducing mutations on the basis of wild-type IFNα by molecular biological means, and the introduced mutations include but are not limited to R144A or R149A.

[0062] 11. According to the novel IL-2, IFNα and Fc fusion protein described in Embodiment 4, it is characterized in that the IFNα and Fc fusion protein, the IFNα derivative is obtained by introducing mutations on the basis of wild-type IFNα by molecular biological means, and the introduced mutations include but are not limited to R144A or R149A.

[0063] 12. A method for preparing a novel IL-2, IFNα and Fc fusion protein as described in any one of embodiments 1-2, characterized in that the method for preparing the IL-2 and IFNα dual factor Fc fusion protein comprises the following steps:

[0064] (1) The IL-2 mutant is connected to the N-terminus or C-terminus of the Fc fragment through a flexible linker, and the IFNα derivative is connected to the C-terminus or N-terminus of the Fc fragment through a flexible linker to obtain a polypeptide chain IL-2-Fc-IFNα or IFNα-Fc-

[0065] IL-2; introduced mutations L234A, L235A, M252Y, S254T and T256E in the Fc fragment;

[0066] (2) cloning the DNA fragment obtained in step (1) into a pcDNA series vector or other vectors used in mammalian cell expression systems to obtain a recombinant vector;

[0067] (3) Transfecting the recombinant vector obtained in step (2) into mammalian cells to express the fusion protein, and obtaining the IL-2 and IFNα dual factor Fc fusion protein after purification.

[0068] 13. A method for preparing a novel IL-2, IFNα and Fc fusion protein as described in any one of embodiments 1 or 3, characterized in that the method for preparing the IL-2 and Fc fusion protein comprises the following steps:

[0069] (1) Connecting the IL-2 mutant to the N-terminus or C-terminus of the Fc fragment via a flexible linker to obtain a polypeptide chain IL-2-

[0070] Fc or Fc-IL-2; introducing mutations L234A, L235A, M252Y, S254T and T256E in the Fc fragment;

[0071] (2) cloning the DNA fragment obtained in step (1) into a pcDNA series vector or other vectors used in mammalian cell expression systems to obtain a recombinant vector;

[0072] (3) Transfecting the recombinant vector obtained in step (2) into mammalian cells to express the fusion protein, and obtaining the IL-2 and Fc fusion protein after purification.

[0073] 14. A method for preparing a novel IL-2, IFNα and Fc fusion protein as described in any one of embodiments 1 or 4, characterized in that the method for preparing the IFNα and Fc fusion protein comprises the following steps:

[0074] (1) Connecting the IFNα derivative to the C-terminus or N-terminus of the Fc fragment via a flexible linker to obtain a polypeptide chain Fc-

[0075] IFNα or IFNα-Fc; introducing mutations L234A, L235A, M252Y, S254T and T256E in the Fc fragment;

[0076] (2) cloning the DNA fragment obtained in step (1) into a pcDNA series vector or other vectors used in mammalian cell expression systems to obtain a recombinant vector;

[0077] (3) Transfecting the recombinant vector obtained in step (2) into mammalian cells to express the fusion protein, and obtaining the IFNα and Fc fusion protein after purification.

[0078] 15. The method for preparing a novel fusion protein of IL-2, IFNα and Fc according to any one of embodiments 12-14, characterized in that the mammalian cells in step (3) include HEK293 cells, CHO cells or cells derived from the above cells.

[0079] 16. The method for preparing a novel IL-2, IFNα and Fc fusion protein according to any one of embodiments 12-14 is characterized in that the introduction of mutations into the Fc fragment in step (1) is specifically: introducing L234A and L235A mutations into the Fc fragment, and simultaneously introducing M252Y, S254T and T256E mutations into the Fc fragment.

[0080] 17. The use of the novel IL-2, IFNα and Fc fusion protein described in any one of Embodiments 1-11 in the preparation of broad-spectrum anti-tumor drugs includes monotherapy for the treatment of tumor indications approved by IL-2 and IFN-a and combination with other tumor treatment methods for broad-spectrum tumor treatment.

[0081] 18. The use of the novel IL-2, IFNα and Fc fusion protein prepared by the method described in any one of Embodiments 12-16 in the preparation of Fc fusion protein drugs includes monotherapy for the treatment of tumor indications approved by IL-2 and IFN-a and combination with other tumor treatment methods for broad-spectrum tumor treatment.

[0082] The above is an overview, and therefore contains simplifications, generalizations and omissions of details when necessary; therefore, those skilled in the art will recognize that the overview is illustrative only and is not intended to be limiting in any way. Other aspects, features and advantages of the methods, compositions and uses and / or other subjects described herein will become apparent in the teachings shown herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1A The schematic diagrams of various Fc fusion protein structures covered by the present invention are illustrated. From the top to the bottom of each small figure is from the N-terminus to the C-terminus, and the trapezoid represents the hinge region.

[0084] Figure 1B-1C The SDS PAGE expression chart and HPLC analysis chart of the Fc fusion protein of the present invention are respectively illustrated.

[0085] Figure 2 The diagram illustrates the detection of the binding ability between the Fc fusion protein of the present invention and the receptor IL-2Rα protein.

[0086] Figure 3 The diagram illustrates the detection of the binding ability between the Fc fusion protein of the present invention and the receptor IL-2Rβ protein.

[0087] Figure 4 The diagram illustrates the detection of the binding ability between the Fc fusion protein of the present invention and the receptor IFNαR2 protein.

[0088] Figure 5 The diagram illustrates the binding ability test of the Fc fusion protein of the present invention to the receptor IL-2Rα protein of different species.

[0089] Figure 6 The diagram illustrates the binding ability test of the Fc fusion protein of the present invention to the receptor IL-2Rβ protein of different species.

[0090] Figure 7 The diagram illustrates the binding ability test of the Fc fusion protein of the present invention to the IFNαR2 protein of different species.

[0091] Fig. 8A and Figure 8B The figures respectively illustrate the results of detecting the binding ability of the Fc fusion protein of the present invention to the FcRn protein by ELISA and Fortebio.

[0092] Fig. 9 The diagram exemplifies the detection of the distribution of specific cell subpopulations of PBMC stimulated by the Fc fusion protein of the present invention.

[0093] Fig.10 The detection of the ability of the Fc fusion protein of the present invention to stimulate the proliferation of CTLL-2 mouse T lymphocytes is exemplified.

[0094] FIG. 11A to FIG. 11D The in vitro direct killing ability test of the Fc fusion protein of the present invention on tumor cells (A: NCI-N87; B: MDA-MB-231; C: A375; D: Burkitt's lymphoma cells) is illustrated.

[0095] FIG. 12A to FIG. 12E The in vitro comprehensive killing ability test of the Fc fusion protein of the present invention on tumor cells (A: NCI-N87; B: MDA-MB-231; C: A375; D and E: Burkitt's lymphoma cells) is illustrated.

[0096] Fig.13 The pharmacodynamic activity of the Fc fusion proteins of the present invention in the ExVivo organoid system is exemplified. DETAILED DESCRIPTION OF THE INVENTION

[0098] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. All patents, patent applications and other publications cited herein are incorporated by reference in their entirety. If a definition set forth herein conflicts with a definition set forth in a patent, patent application and other publication incorporated herein by reference, the definition set forth herein shall prevail.

[0099] As used herein, the term "IL-2" refers to interleukin-2, and is intended to include any form of IL-2, for example, 1) natural unprocessed IL-2 molecules, full-length IL-2 proteins, or naturally occurring IL-2 variants; 2) any form of IL-2 produced by intracellular processing; or 3) full-length or modified forms. In the present disclosure, the term "IL-2" or "IL-2 domain" includes wild-type IL-2 and IL-2 variants.

[0100] As used herein, the term "IFNα" refers to interferon α, and is intended to include any form of IFNα, for example, 1) natural unprocessed IFNα molecules, full-length IFNα proteins, or naturally occurring IFNα variants; 2) any form of IFNα produced by intracellular processing; or 3) full-length or modified forms. In the present disclosure, the term "IFNα" or "IFNα domain" includes wild-type IFNα and IFNα variants.

[0101] The term "variant", with respect to a polypeptide or protein, refers to a biologically active polypeptide comprising one or more amino acid mutations to a native protein sequence. Optionally, one or more amino acid mutations include amino acid substitutions and / or insertions at certain positions in the amino acid sequence. Preferably, the variant has at least about 80% amino acid sequence identity with the corresponding native sequence polypeptide. Such variants include, for example, polypeptides to which one or more amino acid (naturally occurring amino acids and / or non-naturally occurring amino acids) residues are added at the N-terminus and / or C-terminus of the polypeptide. Variants for use in the present disclosure can be prepared by various methods well known in the art, such as site-directed mutagenesis or phage display technology of nucleotides in DNA encoding a natural protein, thereby generating DNA encoding a variant, which is then expressed in recombinant cell culture. In certain embodiments disclosed herein, the IL-2 variant includes one or more substitutions compared to the wild-type IL-2 protein. In certain embodiments disclosed herein, the IFNα variant includes one or more substitutions compared to the wild-type IFNα protein.

[0102] The term "Fc" as used herein has the same meaning as used with respect to antibodies, and refers to the following portion of an antibody, which includes the second (CH2) and third (CH3) constant regions of the first heavy chain bound to the second and third constant regions of the 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 the present disclosure, the term "Fc" includes wild-type Fc and Fc variants.

[0103] As used herein, the term "operably linked" refers to the juxtaposition of two or more biological sequences of interest (with or without a spacer or linker or intervening sequence) so that they are in a relationship that allows them to function in the intended manner. When used in relation to polypeptides, it refers to the connection of polypeptide sequences in a manner that allows the connected product to have the intended biological function. For example, a fusion protein can be operably linked to an immunoglobulin constant region to provide a stable product with ligand binding activity. For another example, a fusion protein can be operably linked to an immunoglobulin constant region through an intervening sequence therebetween, and such an intervening sequence can be a spacer or can contain a longer sequence.

[0104] As used herein, when used with respect to amino acid sequences (e.g., peptides, polypeptides, or proteins), the term "fusion" or "fused" refers to the combination of two or more amino acid sequences, for example, by chemical bonding or recombination, to form a single amino acid sequence that does not exist in nature. The fused amino acid sequence can be produced by genetic recombination of two encoding polynucleotide sequences, and can be expressed by introducing a construct comprising the recombinant polynucleotide into a host cell.

[0105] The term "fusion protein" as used herein refers to a polypeptide having two (or more) parts operably linked together, wherein each part is a polypeptide with different properties. 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, etc. The two parts can be directly connected by a single peptide bond, or connected by a peptide linker containing one or more amino acid residues. In general, the two parts and the linker will be in the reading frame with each other. In certain embodiments, the fusion protein is a fusion protein of an IL-2 part, an Fc part, and an IFNα part. In certain embodiments, the fusion protein is a fusion protein of an IL-2 part and an Fc part. In certain embodiments, the fusion protein is a fusion protein of an Fc part and an IFNα part.

[0106] As used herein, the term "vector" refers to a nucleic acid vector into which a polynucleotide can be inserted. When a vector allows the expression of a protein encoded by a polynucleotide inserted therein, the vector is referred to as an expression vector. The vector can express the genetic material elements carried in a host cell by transformation, transduction or transfection into a host cell. Vectors are well known to those skilled in the art, including but not limited to plasmids, bacteriophages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phages or M13 phages and animal viruses. 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 virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). 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. In addition, the vector may contain an origin of replication.

[0107] As used herein, the term "host cell" refers to a cell system that can be engineered to produce a protein, protein fragment or peptide of interest. Host cells include, but are not limited to, cultured cells, such as mammalian cultured cells derived from rodents (rat, mouse, guinea pig or hamster), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; or human tissue or hybridoma cells, yeast cells and insect cells, as well as cells contained in transgenic animals or cultured tissues. The term covers not only specific test cells, but also the offspring of such cells. Due to mutations or environmental influences, certain modifications may occur in offspring, so such offspring may be different from parental cells, but are still included in the scope of the term "host cell".

[0108] As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules determined by alignment and comparison of the sequences. "Percent identity" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecules being compared. For these calculations, gaps in the alignment, if any, are preferably addressed by a specific mathematical model or computer program (i.e., an "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: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0109] The term "subject" includes any human or non-human animal, preferably a human.

[0110] As used herein, the term "cancer" refers to any tumor or malignant cell growth that causes a medical condition, whether solid or non-solid tumors that mediate proliferation or metastasis.

[0111] The terms "treatment" and "treating" as used herein in the context of treating a condition generally refer to treatment and therapy of a human or animal in which some desired therapeutic effect is achieved, for example, inhibition of disease progression, including a decrease in the rate of progression, stagnation of the rate of progression, regression of the disease, improvement of the disease, and cure of the disease. Treatment as a preventive measure (i.e., prevention, protection) is also included. For cancer, "treatment" may refer to inhibiting or slowing tumor or malignant cell growth, proliferation, or metastasis, or some combination thereof. For tumors, "treatment" includes removing all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.

[0112] As used herein, the term "effective amount" relates to an amount of an active compound, or an amount of a material, composition or dosage comprising an active compound, which, when administered according to the desired treatment regimen, is effective for producing some desired therapeutic effect commensurate with a reasonable benefit / risk ratio.

[0113] IL-2 variants and IFNα variants

[0114] In certain aspects, the present disclosure provides IL-2 variants, which include one or more modifications, such as insertions, deletions and / or substitutions, compared to wild-type IL-2 proteins, such as human wild-type IL-2 proteins. The present disclosure further provides fusion proteins, including IL-2 protein portions fused to Fc portions. The IL2 protein portion may include wild-type IL-2 proteins, or include IL-2 variants comprising one or more modifications (such as insertions and / or substitutions) compared to wild-type IL-2 proteins. In some embodiments, compared to wild-type IL-2 proteins, IL-2 variants include one or more substitutions selected from, but not limited to, R38A, L80F, R81D, L85V, I86V, and I91F. For example, compared to wild-type IL-2 proteins, the 38th amino acid in the IL-2 variant is modified. In some embodiments, the IL-2 variants also include conservative substitutions compared to wild-type IL-2 proteins. In some embodiments, the IL-2 variants include or consist of the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the IL-2 variant comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to SEQ ID NO:2.

[0115] In certain aspects, the present disclosure provides IFNα variants, which include one or more modifications, such as insertions, deletions and / or substitutions, compared to wild-type IFNα proteins, such as human wild-type IFNα proteins. The present disclosure further provides fusion proteins, including IFNα protein portions fused to Fc portions. The IFNα protein portion may include wild-type IFNα proteins, or include IFNα variants comprising one or more modifications (such as insertions and / or substitutions) compared to wild-type IFNα proteins. In some embodiments, compared to wild-type IFNα proteins, the R144 and / or R149 amino acids in the IFNα variants are modified. In some embodiments, the IFNα variants also include conservative substitutions compared to wild-type IFNα proteins. In some embodiments, the IFNα variants include or consist of the amino acid sequence shown in SEQ ID NO:6. In some embodiments, the IFNα variant comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to SEQ ID NO:6.

[0116] The fusion protein of the present invention

[0117] 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 can be single-chain or multi-chain. In some embodiments, the fusion protein is a double-chain homodimer or heterodimer. Each IL-2 portion, Fc portion and IFNα portion in each chain can be the same or different.

[0118] In some embodiments, the fusion protein of the invention comprises an IL-2 portion operably linked to an Fc portion from the N-terminus to the C-terminus (also referred to as an IL-2-Fc fusion protein).

[0119] In some embodiments, the fusion protein of the invention comprises an Fc portion operably linked to an IL-2 portion from the N-terminus to the C-terminus (also referred to as an Fc-IL-2 fusion protein).

[0120] In some embodiments, the fusion protein of the present invention comprises an IFNα portion operably linked to an Fc portion from N-terminus to C-terminus (also referred to as an IFNα-Fc fusion protein).

[0121] In some embodiments, the fusion protein of the invention comprises an Fc portion operably linked to an IFNα portion from the N-terminus to the C-terminus (also referred to as an Fc-IFNα fusion protein).

[0122] In some embodiments, the fusion protein of the invention comprises, from N-terminus to C-terminus, an IL-2 portion operably linked to an Fc portion and the Fc portion operably linked to an IFNα portion (also referred to as an IL-2-Fc-IFNα fusion protein).

[0123] In some embodiments, the fusion protein of the invention comprises, from N-terminus to C-terminus, an IFNα portion operably linked to an Fc portion and the Fc portion operably linked to an IL-2 portion (also referred to as an IFNα-Fc-IL-2 fusion protein).

[0124] The IL-2 portion contained in the fusion protein may contain a wild-type IL-2 protein or an IL-2 variant. The IFNα portion contained in the fusion protein may contain a wild-type IFNα protein or an IFNα variant. The Fc portion contained in the fusion protein may contain a wild-type Fc or an Fc variant.

[0125] In some embodiments, the amino acid at position 38 in the IL-2 portion included in the fusion protein is modified. It has been found that the region near the amino acid at position 38 plays a key role in IL-2 causing increased vascular permeability, so the modification of the amino acid at position 38 can significantly reduce vascular permeability.

[0126] In some embodiments, the fusion protein contains a modified interferon alpha portion, which has a significantly reduced affinity for the interferon alpha receptor compared to natural interferon alpha. This reduction can cause a significant reduction in the toxic side effects caused by interferon alpha while retaining its anti-tumor activity. In some embodiments, the R144 or R149 amino acid in the interferon alpha portion contained in the fusion protein is modified. These amino acids play a key role in the binding of interferon alpha to its receptor, and mutations can cause a significant reduction in affinity without affecting its anti-tumor activity.

[0127] The operably linked (or indicated as "-") can be a direct link between the two parts, or a linker such as a peptide linker. In some embodiments, the IL-2 part is operably linked to the Fc part via a peptide linker. In some embodiments, IFN The α part is operably linked to the Fc part via a peptide linker. The peptide linker can be any peptide linker conventionally used in the art, such as a GS series linker, as shown in SEQ ID NO:11.

[0128] The fusion protein provided by the present invention can improve the therapeutic effect, reduce toxic side effects, and increase the therapeutic safety window.

[0129] Fusion proteins containing Fc regions

[0130] In some embodiments, the fusion protein provided by the present invention further comprises an immunoglobulin constant domain sequence, such as a constant domain sequence of human IgG, and more specifically may comprise a hinge region and an Fc region, such as an Fc region sequence of IgG1, IgG2, IgG3, and IgG4. As known in the art, Fc refers to the part of an antibody consisting of the second and third constant regions of the first heavy chain of an antibody bound to the second and third constant regions of the second heavy chain via disulfide bonding, and optionally the Fc region also comprises all or part of the hinge region. The Fc region herein includes both wild-type Fc regions and variants thereof, with different mutations for a variety of purposes. The variant may comprise one or more amino acid residue modifications, such as substitutions, in the Fc region.

[0131] In certain embodiments, the Fc region variant comprises one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn). Such a variant can have an extended pharmacokinetic half-life because it binds to FcRn at acidic pH, thereby enabling it to escape degradation in the lysosome and then be transported and released from the cell. Methods for engineering antibody molecules to improve binding affinity to FcRn are well known in the art, see, e.g., Vaughn, D. et al., Structure, 6(1):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).

[0132] In certain embodiments, the fusion protein provided by the present invention comprises L234A / L235A substitutions to reduce the binding ability to receptor FcgRIIIa. In certain embodiments, the fusion protein provided by the present invention comprises one or more amino acid substitutions in the interface of the Fc region to assist and / or promote heterodimerization. These modifications include introducing a protrusion into the first Fc polypeptide and introducing a cavity into the second Fc polypeptide, wherein the protrusion can be located 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 these modifications are known in the art, for example, as described in U.S. Patent No. 5,731,168.

[0133] In certain embodiments, the Fc domain further comprises the triple mutation M252Y / S254T / T256E ("YTE"). It is reported that this triple mutation results in an approximately 10-fold increase in binding to the human neonatal Fc receptor (FcRn), and that the serum half-life of human IgG containing YTE in cynomolgus monkeys is increased by nearly 4-fold (Oganesyan V. et al., Mol Immunol. 2009 May; 46(8-9): 1750-5). In some further embodiments, the Fc domain comprises additional mutations to enhance the interaction between Fc and human FcRn.

[0134] In some embodiments, the Fc variant comprises 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.

[0135] Features of the fusion protein of the present invention

[0136] 1. By fusing with Fc, the half-life of the drug can be extended, the dosage of cytokines can be reduced, and the toxic side effects can be reduced.

[0137] 2. By modifying the structure of IL-2, the affinity of cytokines to IL-2Rβ and IL-2Rγ on the surface of NK cells and CD8+T cells is increased, thereby enhancing the tumor-killing effect of these cells.

[0138] 3. Modification of the 38th amino acid of IL-2 can significantly reduce the toxic side effect of increased vascular permeability caused by IL-2.

[0139] 4. The modification of R144 and R149 can reduce their binding ability to the receptor IFNαR2, reduce toxic side effects, while retaining their anti-tumor activity.

[0140] 5. Based on the above characteristics, the modified IL-2, IFNα and Fc fusion protein can overcome the shortcomings of current IL-2 and IFNα drugs, improve the therapeutic effect, reduce toxic side effects, and increase the therapeutic safety window.

[0141] Polynucleotides, vectors and host cells

[0142] In some aspects, the present invention also provides polynucleotides encoding such fusion proteins. The polynucleotides can be used to express the fusion protein. In some embodiments, the polynucleotides can also be used as therapeutic agents in active form for achieving in vivo expression of the polypeptide fusion protein.

[0143] The polynucleotide encoding the fusion protein is 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.

[0144] Using known recombinant techniques, the polynucleotide encoding the fusion protein can be inserted into a vector for further cloning (amplification of the DNA) or expression. The vector components typically include, but are not limited to, one or more of the following: 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.

[0145] 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 selection marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), λ phages, and M13 phages, liposomes, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEM EX, 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.

[0146] The vector that comprises the polynucleotide sequence of encoding fusion protein can be imported into host cell for cloning or gene expression. Suitable host cells for cloning or expressing DNA in the vector herein are prokaryotes, yeast or higher eukaryotic cells. Suitable prokaryotes for this purpose include true bacteria, for example gram-negative or gram-positive bacteria, such as Escherichia coli. Except prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable clones or expression hosts for the vector provided. Saccharomyces cerevisiae or common baker's yeast are the most commonly used in lower eukaryotic host microorganisms. However, many other genera, species and bacterial strains are generally available and useful in this article.

[0147] Suitable host cells for expressing fusion proteins provided herein can also be derived from multicellular organisms. The example of invertebrate cells includes plant 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 other mammalian cell lines, such as human cell lines.

[0148] Host cells are transformed with the above-described expression or cloning vectors for production of fusion proteins and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants or amplifying genes encoding the desired sequences.

[0149] Host cells for producing fusion proteins provided herein can be cultivated in a variety of culture media. Any of these culture media can be supplemented with hormones and / or other growth factors (such as insulin, transferrins or epidermal growth factor) as required, salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ medicines), trace elements (defined as inorganic compounds usually present in the ultimate concentration of micromolar range), and glucose or equivalent energy sources. Any other necessary supplements can also be included with appropriate concentrations well known to those skilled in the art. Culture conditions, such as temperature, pH, etc., are those conditions previously used together with the host cells selected for expression, and are apparent to those of ordinary skill in the art.

[0150] Fusion proteins produced by cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being a preferred purification technique.

[0151] Pharmaceutical composition

[0152] In some aspects, the present invention provides a composition, such as a pharmaceutical composition, comprising a fusion protein of the present invention formulated with a pharmaceutically acceptable carrier. Such a composition comprises at least one fusion protein of the present invention or a polynucleotide encoding the fusion protein.

[0153] As used herein, "pharmaceutically acceptable carrier" includes any and all pharmaceutically acceptable liquid, gel or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, isolating agents or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances that are physiologically compatible, other components known in the art or various combinations thereof, etc. The carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, transdermal, intravitreal injection or implantation administration, etc. Depending on the route of administration, the active compound, i.e., the ingredient of the present invention, can be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0154] Pharmaceutical composition of the present invention may also include a pharmaceutically acceptable antioxidant. Suitable aqueous carriers and non-aqueous carrier examples that may be used in pharmaceutical composition of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. For example, suitable fluidity may be maintained by utilizing coating materials (such as lecithin), by maintaining required particle size and by utilizing surfactants in the case of dispersants.

[0155] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersants.The pharmaceutical composition may be in the form of a solid, paste, ointment, gel, liquid, aerosol, spray, polymer, film, emulsion or suspension.

[0156] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, such as liquid solutions, suspensions, emulsions or solid forms suitable for producing liquid solutions, suspensions or emulsions. The preparation for injection can include sterile and / or non-pyrolysis solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be mixed with solvents before use, including subcutaneous tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with vehicles before use, and sterile and / or non-pyrolysis emulsions. The solution can be aqueous or non-aqueous. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injection solutions or dispersions.

[0157] The actual dosage level of active ingredients and small molecules in the pharmaceutical compositions of the present invention may be varied in order to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration without being toxic to the patient. The selected dosage level depends on various pharmacokinetic factors, including the activity of the specific combination of the present invention used, or its ester, salt or amide, the route of administration, the time of administration, the excretion rate of the specific compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific combination used, the age, sex, weight, condition, general health and previous medical history of the patient to be treated, and similar factors well known in the medical field.

[0158] Applications of the present disclosure

[0159] The fusion proteins, pharmaceutical compositions and methods disclosed herein have many in vitro and in vivo uses, including, for example, the enhancement of immune responses. For example, these molecules can be administered to cultured cells in vitro or ex vivo, or, for example, administered to human subjects in vivo, to enhance immunity in various situations. The immune response can be modulated, for example, enhanced, stimulated or upregulated.

[0160] For example, the subject includes a human patient who needs to regulate an immune response. In a specific embodiment, the method is particularly suitable for treating cancer in vivo. In order to achieve the enhancement of immunity, 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 in any order or simultaneously.

[0161] In some aspects, the disclosure provides a method for treating a disorder or disease in a mammal, comprising administering a therapeutically effective amount of a fusion protein as disclosed herein to a subject (e.g., a human) in need of treatment. The disorder or disease may be a cancer, such as a cancer treatable by IL-2 and IFNα. The disclosure provides a method for treating or preventing a variety of cancers, whether malignant or benign, and whether primary or secondary. Cancer may be a solid tumor or a hematological malignancy.

[0162] The fusion proteins disclosed herein can be used alone as a monotherapy, or can be used in combination with cellular immunotherapy, targeted therapy, chemotherapy or radiotherapy, etc.

[0163] Summary of sequences involved in the present invention

[0164] SEQ ID NO: 1 (wild-type IL-2 amino acid sequence)

[0165] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR

[0166] WITFCQSIISTLT

[0167] SEQ ID NO:2 (Amino acid sequence of IL-2 derivative)

[0168] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT

[0169] SEQ ID NO:3 (Amino acid sequence of IL-2-Fc fusion protein)

[0170] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPK

[0171] SEQ ID NO:4 (Amino acid sequence of Fc-IL-2 fusion protein)

[0172] EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPAGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0173] SEQ ID NO:5 (Wild-type IFNα Amino Acid Sequence)

[0174] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMNEDSILAVR

[0175] KYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE

[0176] SEQ ID NO:6 (IFNα Derivative Amino Acid Sequence)

[0177] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMNEDSILAVRKYFQRITLYLKEKKYSPCAWEVVAAEIMRSFSLSTNLQESLRSKE

[0178] SEQ ID NO:7 (IFNα-Fc Fusion Protein Amino Acid Sequence)

[0179] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMNEDSILAVRKYFQRITLYLKEKKYSPCAWEVVAAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPK

[0180] SEQ ID NO:8 (Amino acid sequence of Fc-IFNα fusion protein)

[0181] EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPAGGGGSGGGGSGGGGSCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMNEDSILAVRKYFQRITLYLKEKKYSPCAWEVVAAEIMRSFSLSTNLQESLRSKE

[0182] SEQ ID NO:9 (Amino acid sequence of IL-2-Fc-IFNα fusion protein)

[0183] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPAGGGGSGGGGSGGGGSCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMNEDSILAVRKYFQRITLYLKEKKYSPCAWEVVAAEIMRSFSLSTNLQESLRSKE

[0184] SEQ ID NO:10 (Amino acid sequence of IFNα-Fc-IL-2 fusion protein)

[0185] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMNEDSILAVRKYFQRITLYLKEKKYSPCAWEVVAAEIMRSFSLSTNLQESLRSKEGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPAGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT

[0186] SEQ ID NO:11 (Flexible linker amino acid sequence)

[0187] GGGGSGGGGSGGGGS

[0188] SEQ ID NO:12 (Fc region amino acid sequence)

[0189] EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPK

[0190] The present invention will be further explained below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Example

[0191] Example 1: Molecular construction and production of fusion protein

[0192] An IL-2-Fc-IFNα fusion protein was constructed, which comprises an IL-2 portion operably linked to an immunoglobulin Fc portion from the N-terminus to the C-terminus, and the immunoglobulin Fc portion operably linked to an IFNα portion, the fusion protein is hereinafter referred to as IL-2-Fc-IFNα, and its amino acid sequence is shown in SEQ ID NO: 9. An IL-2-Fc fusion protein was also constructed, which comprises an IL-2 portion operably linked to an immunoglobulin Fc portion from the N-terminus to the C-terminus, the fusion protein is hereinafter referred to as IL-2-Fc, and its amino acid sequence is shown in SEQ ID NO: 3. An Fc-IFNα fusion protein was also constructed, which comprises an immunoglobulin Fc portion operably linked to an IFNα portion from the N-terminus to the C-terminus, the fusion protein is hereinafter referred to as Fc-IFNα, and its amino acid sequence is shown in SEQ ID NO: 8.

[0193] The antibody light and heavy chain genes were amplified by PCR using conventional molecular biology techniques, and the genes encoding the amino acid sequences were connected to the pcDNA3.4 vector using homologous recombination techniques. The plasmids of the positive clones after sequencing were extracted and transfected into Expi293F cells, which were cultured for 7 days in a shaker at 37°C / 5% CO2 / 125rpm. After the culture was completed, the supernatant was collected and purified by protein A affinity chromatography to obtain the purified fusion protein molecules, and the antibody concentration was determined by UV280 combined with the theoretical extinction coefficient.

[0194] Figure 1B The SDS PAGE gel image of IL-2-Fc-IFNα fusion protein is shown. Figure 1C An analytical chart of its HPLC purity is shown.

[0195] Example 2: ELISA detection of binding ability to receptor IL-2Rα protein

[0196] Coat with IL-2Rα protein (10165-H08H, Sino Biological) at a coating concentration of 2μg / ml, 100ul / well at 4℃ overnight; block with 300ul / well 3% skim milk powder at 37℃ for 1h; add 100ul of different concentrations of fusion protein and its control sample to each well and incubate at 37℃ for 1h; then add Anti-IFNα2Rabbit pAb and incubate at 37℃ for 1h, then add Anti-Rabbit IgG HRP and incubate at 37℃ for 1h; add 100ul TMB to each well for color development for 5min, then add 50ul stop solution, and read OD450 on a microplate reader.

[0197] Results Figure 2 Compared with wild-type IL-2 (11848-HNAH1-E, Sino Biological), the binding ability of IL-2-Fc and IL-2-Fc-IFNα to the receptor IL-2Rα protein was significantly improved.

[0198] Example 3: ELISA to detect the binding ability to the receptor IL-2Rβ protein

[0199] Coat with IL-2Rβ protein (10696-H05H, Sino Biological) at a coating concentration of 2μg / ml, 100ul / well at 4℃ overnight; block with 300ul / well 3% skim milk powder at 37℃ for 1h; add 100ul of different concentrations of fusion protein and its control sample to each well and incubate at 37℃ for 1h; then add Anti-IFNα2Rabbit pAb and incubate at 37℃ for 1h, then add Anti-Rabbit IgG HRP and incubate at 37℃ for 1h; add 100ul TMB to each well for color development for 5min, then add 50ul stop solution, and read OD450 on a microplate reader.

[0200] Results Figure 3 Compared with wild-type IL-2, the binding ability of IL-2-Fc and IL-2-Fc-IFNα to the receptor IL-2Rβ protein was increased by more than 24 times.

[0201] Example 4: ELISA detection of binding ability to receptor IFNαR2 protein

[0202] Coat with IFNαR2 protein (10359-H02H, Sino Biological) at a coating concentration of 2μg / ml, 100ul / well at 4℃ overnight; block with 300ul / well 3% skim milk powder at 37℃ for 1h; add 100ul of different concentrations of fusion protein and its control sample to each well and incubate at 37℃ for 1h; then add Anti-IL-2Rabbit pAb and incubate at 37℃ for 1h, then add Anti-Rabbit IgG HRP and incubate at 37℃ for 1h; add 100ul TMB to each well for color development for 5min, then add 50ul stop solution, and read OD450 on a microplate reader.

[0203] Results Figure 4 Compared with wild-type IFNα, the binding ability of Fc-IFNα to the receptor IFNαR2 protein was significantly reduced, while the binding ability of IL-2-Fc-IFNα to the receptor IFNαR2 protein was significantly increased.

[0204] Example 5: ELISA detection of binding ability to IL-2Rα protein of different species

[0205] Human, mouse and cynomolgus monkey IL-2Rα proteins were coated separately at a coating concentration of 2μg / ml, 100ul / well, overnight at 4℃; blocked with 300ul / well 3% skim milk powder at 37℃ for 1h; 100ul of different concentrations of fusion protein and its control sample were added to each well and incubated at 37℃ for 1h; then Anti-IFNα2Rabbit pAb was added and incubated at 37℃ for 1h, and then Anti-Rabbit IgG HRP was added and incubated at 37℃ for 1h; 100ul TMB was added to each well for color development for 5min, then 50ul stop solution was added, and OD450 was read on a microplate reader.

[0206] Results Figure 5 Experiments have confirmed that IL-2-Fc-IFNα has similar binding abilities to the IL-2Rα protein of human, mouse, and cynomolgus monkey receptors, and therefore has species crossover with mice and cynomolgus monkeys.

[0207] Example 6: ELISA to detect the binding ability to the IL-2Rβ protein of different species

[0208] Human, mouse and cynomolgus monkey IL-2Rβ proteins were coated at a coating concentration of 2μg / ml, 100ul / well, overnight at 4℃; blocked with 300ul / well 3% skim milk powder at 37℃ for 1h; 100ul of different concentrations of fusion protein and its control sample were added to each well and incubated at 37℃ for 1h; then Anti-IFNα2Rabbit pAb was added and incubated at 37℃ for 1h, and then Anti-Rabbit IgG HRP was added and incubated at 37℃ for 1h; 100ul TMB was added to each well for color development for 5min, and then 50ul stop solution was added, and OD450 was read on a microplate reader.

[0209] Results Figure 6 The experiment confirmed that IL-2-Fc-IFNα has species crossover with human, mouse, and cynomolgus monkey receptor IL-2Rβ proteins. The binding ability with cynomolgus monkey IL-2Rβ protein is similar to that of human. The binding with mouse IL-2Rβ protein is lower than that with human.

[0210] Example 7: ELISA to detect the binding ability to the IFNαR2 protein of different species

[0211] Human, mouse and cynomolgus monkey IFNαR2 proteins were coated at a coating concentration of 2μg / ml, 100ul / well, overnight at 4℃; blocked with 300ul / well 3% skim milk powder at 37℃ for 1h; 100ul of different concentrations of fusion protein and its control sample were added to each well and incubated at 37℃ for 1h; then Anti-IL-2Rabbit pAb was added and incubated at 37℃ for 1h, and then Anti-Rabbit IgG HRP was added and incubated at 37℃ for 1h; 100ul TMB was added to each well for color development for 5min, and then 50ul stop solution was added, and OD450 was read on a microplate reader.

[0212] Results Figure 7 The experiment confirmed that IL-2-Fc-IFNα binds to the IFNαR2 protein of human, mouse and cynomolgus monkey receptors, demonstrating species cross-reactivity.

[0213] Example 8: Detection of binding ability to FcRn protein

[0214] 8.1ELISA to detect binding ability to FcRn protein

[0215] FcRn protein (10359-H02H, Sino Biological) was coated at a concentration of 2 μg / ml, 100 ul / well, overnight at 4°C; 300 ul / well of 3% skim milk powder was used for blocking at 37°C for 1 h; 100 ul of fusion protein of different concentrations and its control sample were added to each well and incubated at 37°C for 1 h; then Anti-IFNα2 Rabbit pAb was added and incubated at 37°C for 1 h, and then Anti-Rabbit IgG HRP was added and incubated at 37°C for 1 h; 100 ul of TMB was added to each well for color development for 5 min, and then 50 ul of stop solution was added, and OD450 was read on an ELISA reader.

[0216] Results Fig. 8A Experiments have shown that compared with Herceptin antibody, IL-2-Fc-IFNα has significantly higher binding affinity with FcRn protein

[0217] 8.2 Fortebio detection of binding ability to FcRn protein

[0218] The protein to be tested was loaded with ProA and HIS probes (concentration was 4 μg / ml), and then bound and dissociated with different concentrations of FcRn (4ug / ml, 1ug / ml, 0.25ug / ml and 0), and the affinity between the molecule to be tested and FcRn was calculated.

[0219] Results Figure 8B The experiment confirmed that compared with Herceptin antibody, IL-2-Fc-IFNα has a significantly higher affinity with FcRn protein.

[0220] Example 9: Detection of immune cell subsets after stimulation of human PBMC cells

[0221] Fresh human PBMCs were plated into 96-well plates at 150,000 cells / well, and 500 nM of protein was added according to the experimental design, and culture was continued for 72 hours. After the incubation, Anti-Foxp3 antibody (FITC), Anti-CD4 antibody (PE), Anti-CD56 antibody (PE), Anti-CD8 antibody (PE) and Anti-Granzyme B antibody (FITC) were stained according to the flow cytometry requirements, and the flow cytometry data were collected and analyzed, and the data were summarized and analyzed.

[0222] Results Fig. 9 The experiment confirmed that IL-2-Fc-IFNα stimulated the activation of NK and CD8 positive T cells in PBMC and the ability to express Granzyme B to maintain the same activity as wild-type IL-2; stimulated Treg cells (FOXP3 + The proliferation capacity of IL-2 cells was significantly lower than that of wild-type IL-2.

[0223] Example 10: Detection of the ability of CTLL-2 mouse T lymphocytes to stimulate proliferation

[0224] Mouse CTLL-2 cells were cultured and expanded to the required cell mass, and mouse CTLL-2 cells were plated into 96-well plates at 100,000 cells / well. Different concentrations (100nM, 4nM, 800pM, 400pM, 200pM, 100pM, 50pM, 25pM, 0) of protein were added according to the experimental design, and the culture was continued for 72h; the expression of Formazan in living cells was determined by CCK-8 method, and the cell viability was evaluated. The specific operation was carried out according to the instructions. After the culture was completed, 10% CCK-8 solution was added, mixed, and incubated for 2-4 hours, and the absorbance value data was collected in the microplate reader. The data were analyzed and processed using Excel software, and the GraphPad Prism 7 software was used to fit the drug efficacy dose curve according to the absorbance value data and calculate IC50.

[0225] Results Fig.10 The experiment confirmed that the ability of IL-2-Fc-IFNα to stimulate the proliferation of CTLL-2 cells was 10 times higher than that of wild-type IL-2.

[0226] Example 11: In vitro direct killing ability test of tumor cells

[0227] The direct killing ability is to measure the direct killing of tumor cells by IFN-a in the absence of immune cells.

[0228] 11.1 Detection of direct killing ability against human gastric cancer cell line NCI-N87

[0229] Human gastric cancer cell NCI-N87 was cultured and expanded to the required cell amount, and NCI-N87 cells were plated into 96-well plates at 4000 cells / well. Different concentrations of protein (4000nM, 1000nM, 250nM, 100nM, 62.5nM, 15.625nM, 10nM, 4nM, 1.5625nM, 1nM, 244.14pM, 156.25pM, 100pM, 100pM, 10pM, 1pM, 0) were added according to the experimental design, and the culture was continued for 5 days; the expression of Formazan in living cells was determined by CCK-8 method, and then the cell viability was evaluated. The specific operation was carried out according to the instructions. After the culture was completed, 10% CCK-8 solution was added, mixed, and incubated for 2-4 hours, and the absorbance value data was collected in the microplate reader. Excel software was used to analyze and process the data, and GraphPad Prism 7 software was used to fit the drug efficacy dose curve according to the absorbance data to calculate the IC50.

[0230] Results Fig.11AWild-type IFNα has a significant killing effect on NCI-N87 cells; while IL-2-Fc-IFNα has a killing effect on NCI-N87 cells only at high concentrations. The negative control TTI-622 (SIRPa-Fc) has no killing effect on NCI-87 cells.

[0231] 11.2 Detection of direct killing ability against human breast cancer cell line MDA-MB-231

[0232] The human breast cancer cell MDA-MB-231 was cultured and expanded to the required cell amount, and the MDA-MB-231 cells were plated into 96-well plates at 2000 cells / well. Different concentrations (4000nM, 1000nM, 250nM, 62.5nM, 15.625nM, 4nM, 1nM, 244.14pM, 61pM, 15.26pM, 0) of protein were added according to the experimental design, and the culture was continued for 5 days; the expression of Formazan in living cells was determined by CCK-8 method, and then the cell viability was evaluated. The specific operation was carried out according to the instructions. After the culture was completed, 10% CCK-8 solution was added, mixed, and incubated for 2-4 hours, and the absorbance value data was collected in the microplate reader. The data were analyzed and processed using Excel software, and the GraphPad Prism 7 software was used to fit the drug efficacy dose curve according to the absorbance value data and calculate IC50.

[0233] Results Fig. 11B Wild-type IFNα has a significant killing effect on MDA-MB-231 cells; while IL-2-Fc-IFNα has a killing effect on MDA-MB-231 cells only at high concentrations. The negative control TTI-622 (SIRPa-Fc) has no killing effect on MDA-MB-231 cells.

[0234] 11.3 Detection of direct killing ability against human melanoma cell line A375

[0235] Human breast cancer cells A375 were cultured and expanded to the required cell mass, and A375 cells were plated in 96-well plates at 1000 cells / well. Different concentrations of protein (16000nM, 1600nM, 160nM, 16nM, 1.6nM, 160pM, 16pM, 1.6pM, 0.16pM, 0) were added according to the experimental design, and the culture was continued for 5 days; the expression of Formazan in living cells was determined by CCK-8 method, and the cell viability was evaluated. The specific operation was carried out according to the instructions. After the culture was completed, 10% CCK-8 solution was added, mixed, and incubated for 2-4 hours, and the absorbance data was collected in the microplate reader. The data were analyzed and processed using Excel software, and the GraphPad Prism 7 software was used to fit the drug efficacy dose curve according to the absorbance data and calculate IC50.

[0236] Results Fig. 11C Wild-type IFNα has a significant killing effect on A375 cells; while IL-2-Fc-IFNα has a killing effect on A375 cells only at high concentrations. The negative control TTI-622 (SIRPa-Fc) has no killing effect on A375 cells.

[0237] 11.4 Detection of direct killing ability of human Burkitt's lymphoma cell line Daudi B cells

[0238] The human Burkitt's lymphoma Daudi B cells were cultured and expanded to the required cell amount, and the Daudi B cells were plated into 96-well plates at 10,000 cells / well. Different concentrations (62.5nM, 12.5nM, 2.5nM, 500pM, 250pM, 125pM, 62.5pM, 31.25pM, 15.625pM, 7.813pM, 4pM, 2pM, 0) of protein were added according to the experimental design, and the culture was continued for 5 days; the expression of Formazan in living cells was determined by CCK-8 method, and then the cell viability was evaluated. The specific operation was carried out according to the instructions. After the culture was completed, 10% CCK-8 solution was added, mixed, and incubated for 2-4 hours, and the absorbance value data was collected in the microplate reader. The data were analyzed and processed using Excel software, and the GraphPad Prism 7 software was used to fit the drug efficacy dose curve according to the absorbance value data and calculate IC50.

[0239] Results Fig.11D Wild-type IFNα has a significant killing effect on Daudi B cells; while IL-2-Fc-IFNα has a killing effect on Daudi B cells only at higher concentrations. The negative control TTI-622 (SIRPa-Fc) has no killing effect on Daudi B cells.

[0240] Experiments have confirmed that the direct killing function of the fusion protein of the present invention is weaker than that of wild-type IFNα2, but still retains the direct killing function of the IFNα arm.

[0241] Example 12: In vitro comprehensive killing ability detection of tumor cells

[0242] The comprehensive killing function is the sum of direct killing ability and indirect killing ability. The comprehensive killing ability is obtained in the presence of immune cells PBMC.

[0243] 12.1 Comprehensive killing ability test on human gastric cancer cell line NCI-N87

[0244] Human gastric cancer cells NCI-N87 were cultured and expanded to the required cell volume, and NCI-N87 cells were plated into 96-well plates at 4000 cells / well, and the cells were allowed to adhere for 6 hours; fresh human PBMCs were purchased and plated at an effect-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 culture was continued for 5 days; the plates were washed three times with PBS, PBMCs were removed, and the expression of Formazan in living cells was determined by CCK-8 method to evaluate cell viability. The specific operation was carried out according to the instructions. After the culture was completed, 10% CCK-8 solution was added, mixed, and incubated for 2-4 hours, and the absorbance data was collected in the microplate reader. The data were analyzed and processed using Excel software, and the drug efficacy dose curve was fitted according to the absorbance data using GraphPad Prism 7 software to calculate IC50.

[0245] Results Fig. 12A The comprehensive killing ability of IL-2-Fc-IFNα on NCI-N87 cells is stronger than that of wild-type IL-2.

[0246] 12.2 Comprehensive killing ability test on human breast cancer cell line MDA-MB-231

[0247] Culture and expand human breast cancer cells MDA-MB-231 to the required cell volume, plate MDA-MB-231 cells in 96-well plates at 4000 cells / well, and wait for 6 hours for cells to adhere to the wall; purchase fresh human PBMCs, plate them according to the effect-target ratio of 10:1, add different concentrations (800nM, 80nM, 8nM, 800pM, 80pM, 8pM, 0.8pM, 0) of protein according to the experimental design, and continue to culture for 5 days; wash the plate three times with PBS, remove PBMCs, and use CCK-8 method to determine the expression of Formazan in living cells, and then evaluate cell viability. The specific operation is carried out according to the instructions. After the culture is completed, 10% CCK-8 solution is added, mixed, and incubated for 2-4 hours, and the absorbance value data is collected in the microplate reader. The data are analyzed and processed using Excel software, and the drug efficacy dose curve is fitted according to the absorbance value data using GraphPad Prism 7 software to calculate IC50.

[0248] Results Fig. 12B The comprehensive killing ability of IL-2-Fc-IFN-a on MDA-MB-231 cells was similar to that of wild-type IL-2.

[0249] 12.3 Comprehensive killing ability test on human melanoma cell line A375

[0250] Human melanoma cells A375 were cultured and expanded to the required cell volume, and A375 cells were plated into 96-well plates at 4000 cells / well, and the cells were allowed to adhere for 6 hours; fresh human PBMCs were purchased and plated at 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 culture was continued for 5 days; the plates were washed three times with PBS, PBMCs were removed, and the expression of Formazan in living cells was determined by CCK-8 method to evaluate cell viability. The specific operation was carried out according to the instructions. After the culture was completed, 10% CCK-8 solution was added, mixed, and incubated for 2-4 hours, and the absorbance data was collected in the microplate reader. The data were analyzed and processed using Excel software, and the GraphPad Prism 7 software was used to fit the drug efficacy dose curve according to the absorbance data and calculate IC50.

[0251] Results Fig. 12C The comprehensive killing ability of IL-2-Fc-IFN-a on A375 cells was significantly stronger than that of wild-type IL-2.

[0252] 12.4 Detection of the comprehensive killing ability of Daudi B cells in human Burkitt's lymphoma cell line

[0253] Human Burkitt's lymphoma Daudi B cells were cultured and expanded to the required cell amount. Fresh PBMCs were collected and separated from the donors. Daudi B cells were plated into 96-well plates at 10,000 cells / well. Two replicate wells were set up. PBMCs (target ratios were set to 10:1 and 5:1) and different concentrations of IAMA005 (16nM, 160nM) were added to the corresponding wells according to the experimental layout design. There were 3 plates in total. They were taken according to different detection times. After Co-Culture culture for 24h, 48h, and 72h, the culture supernatant was distributed into the LDH detection preservation solution and stored in a -80 degree refrigerator. After resuspending the cells in each well, they were stained and tested according to the flow cytometry requirements. The flow cytometry data were collected and analyzed, and the data were summarized and analyzed. The results are shown in Fig.12D .

[0254] Human Burkitt's lymphoma Daudi B cells were cultured and expanded to the required cell mass, CFSE stained Daudi B cells, and then plated into 96-well plates at 20,000 cells / well; fresh human PBMCs were purchased and plated at an effector-target ratio of 10:1, and different concentrations of protein (800nM, 80nM, 8nM, 800pM, 80pM, 8pM, 0.8pM, 0) were added according to the experimental design, and cultured for 5 days; the plates were washed three times with PBS and centrifuged at low speed to remove cell debris, and the fluorescence intensity of live cells was measured by fluorescence microplate reader to evaluate cell viability. The data were analyzed and processed using Excel software, and the drug efficacy dose curve was fitted according to the absorbance data using GraphPad Prism 7 software to calculate IC50.

[0255] Results Fig.12E The comprehensive killing ability of IL-2-Fc-IFN-a on Daudi B cells maintained an activity comparable to that of wild-type IL-2.

[0256] Example 13: Drug efficacy study in the ExVivo organoid system

[0257] Pleural effusion samples from breast cancer patients (identified as HER2 2+ positive) were collected and cells in the samples, including tumor cells and immune cells, were separated. Candidate drugs and reference drugs were co-incubated with tumor cell immune cell complexes in vitro. After 4-5 days of treatment, culture medium and cells were collected for analysis, including tumor cells, Granzyme B, IFN-γ, etc.

[0258] Results Fig.13 In the ExVivo evaluation system of this sample, the anti-tumor activity of pleural effusion immune cells stimulated by IL-2-Fc-IFN-a was significantly enhanced, and the anti-tumor active cytokines Granzyme B and IFN-γ secreted by them were also significantly increased.

[0259] The exemplary embodiments of the present disclosure are described above in conjunction with the accompanying drawings. However, it should be understood by those skilled in the art 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 these changes and modifications are included in the scope of protection defined by the claims of the present disclosure.

[0260] References

[0261] [1]Yang Y,Lundqvist A.Immunomodulatory effects of IL-2and IL-15;implications for cancer immunotherapy[J].Cancers,2020,12(12):3586.

[0262] [2]Choudhry H,Helmi N,Abdulaal W H,et al.Prospects of IL-2in cancerimmunotherapy[J].BioMed research international,2018,2018.

[0263] [3]Levin A M,Bates D L,Ring A M,et al.Exploiting a naturalconformational switch to engineer an interleukin-2‘superkine’[J].Nature,2012,484(7395):529-533.

[0264] [4]Khailaie S,Montaseri G,Meyer-Hermann M.An adaptive control schemefor Interleukin-2therapy[J].Iscience,2020,23(11):101663.

[0265] [5]Cauwels A,Van Lint S,Garcin G,et al.Asafe and highly efficienttumor-targeted type I interferon immunotherapy depends on the tumormicroenvironment[J].Oncoimmunology,2018,7(3):e1398876.

[0266] [6]Garcin G,Paul F,Staufenbiel M,et al.High efficiency cell-specifictargeting of cytokine activity[J].Nature communications,2014,5(1):1-9.

[0267] [7]Piehler J,Roisman L C,Schreiber G.New structural and functionalaspects of the type I interferon-receptor interaction revealed bycomprehensive mutational analysis of the binding interface[J].Journal ofbiological chemistry,2000,275(51):40425-40433.

[0268] [8]Sim G C,Liu C,Wang E,et al.IL2 variant circumvents ICOS+regulatoryT-cell expansion and promotes NK cell activation[J].Cancer ImmunologyResearch,2016,4(11):983-994.

[0269] [9]Heaton K M,Ju G,Morris D K,et al.Characterization of lymphokine-activated killing by human peripheral blood mononuclear cells stimulated withinterleukin 2(IL-2)analogs specific for the intermediate affinity IL-2receptor[J].Cellular immunology,1993,147(1):167-179.

[0270]

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

[0271]

[11] Luo F,Qian J,Yang J,et al.BifunctionalαHER2 / CD3 RNA-engineeredCART-like human T cells specifically eliminate HER2+gastric cancer[J].CellResearch,2016,26(7):850-853.

[0272]

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

[0273]

[13] Carmenate T,Pacios A,Enamorado M,et al.Human IL-2mutein withhigher 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 operably linked to a human immunoglobulin Fc portion, wherein the IL-2 portion consists of the amino acid sequence shown in SEQ ID NO:

2.

2. A fusion protein, which consists of an IL-2 portion operably linked to a human immunoglobulin Fc portion from the N-terminus to the C-terminus and the Fc portion operably linked to an IFNα portion, wherein the IL-2 portion consists of the amino acid sequence shown in SEQ ID NO: 2, and the IFNα portion consists of the amino acid sequence shown in SEQ ID NO:

6.

3. The fusion protein of claim 1 or 2, wherein the Fc portion consists of the amino acid sequence shown in SEQ ID NO:

12.

4. The fusion protein of claim 1 or 2, wherein the operably linked is via a peptide linker.

5. The fusion protein of claim 1, which consists of the amino acid sequence shown in SEQ ID NO:

3. The fusion protein of claim 2 , which consists of the amino acid sequence shown in SEQ ID NO:

9.

7. A nucleic acid molecule comprising a nucleic acid sequence encoding the fusion protein of any one of claims 1-6.

8. A vector comprising the nucleic acid molecule of claim 7.

9. A host cell comprising the nucleic acid molecule of claim 7 or the vector of claim 8.

10. A method for producing the fusion protein according to any one of claims 1 to 6, comprising the following steps: expressing the fusion protein in a host cell comprising a vector encoding the fusion protein; and The fusion protein is isolated from the host cell culture.

11. Use of the fusion protein according to any one of claims 2 or 6 in the preparation of a medicament for preventing and / or treating cancer, wherein the cancer is selected from melanoma, lymphoma, renal cell carcinoma, gastric cancer and breast cancer.

Images