WO2023217288A1 - 新融合蛋白及其用途 - Google Patents

新融合蛋白及其用途 Download PDF

Info

Publication number
WO2023217288A1
WO2023217288A1 PCT/CN2023/094205 CN2023094205W WO2023217288A1 WO 2023217288 A1 WO2023217288 A1 WO 2023217288A1 CN 2023094205 W CN2023094205 W CN 2023094205W WO 2023217288 A1 WO2023217288 A1 WO 2023217288A1
Authority
WO
WIPO (PCT)
Prior art keywords
fusion protein
ifnα
operably linked
amino acid
cells
Prior art date
Application number
PCT/CN2023/094205
Other languages
English (en)
French (fr)
Inventor
刘立明
韩镇
康平
Original Assignee
南京吉盛澳玛生物医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 南京吉盛澳玛生物医药有限公司 filed Critical 南京吉盛澳玛生物医药有限公司
Priority to CN202380012626.1A priority Critical patent/CN117881704A/zh
Publication of WO2023217288A1 publication Critical patent/WO2023217288A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the invention belongs to the field of biomedical technology, and specifically relates to the design, preparation and use of novel IL-2, IFN ⁇ and Fc fusion proteins.
  • IL-2 also known as T cell growth factor, has a gene located on chromosome 4, including a 7kb sequence, consisting of 133 amino acids, and a molecular weight of approximately 15kD.
  • IL-2 acts through IL-2R, which includes three subunits, IL-2R ⁇ (CD25), IL-2R ⁇ (CD122) and IL-2R ⁇ (CD132).
  • Three subunits can form three receptor forms: high-binding receptors contain all three subunits IL-2R ⁇ / ⁇ / ⁇ , medium-binding receptors contain two subunits IL-2R ⁇ / ⁇ and low-binding Receptor IL-2R ⁇ .
  • IL-2R ⁇ and IL-2R ⁇ are necessary for IL-2 to activate downstream signaling pathways.
  • IL-2 When IL-2 binds to IL-2R ⁇ and IL-2R ⁇ at the same time, the two receptor subunits form a heterodimer and are phosphorylated.
  • STAT5 in cells enters the nucleus and leads to corresponding gene transcription and expression; IL-2R ⁇ is not necessary for signaling, but can promote the binding of IL-2 to IL-2R ⁇ and IL-2R ⁇ .
  • IL-2R ⁇ is expressed in all immune cells; IL-2R ⁇ is expressed in CD8+ T cells, NK cells, and regulatory T cells; and the expression level increases after T cells are activated; IL-2R ⁇ plays a role in regulating CD8+ T cells continue to be highly expressed, and will be briefly expressed in activated CD8+ T cells, and then the expression level will be downregulated [1,2].
  • IL-2 was the second immunotherapy approved for the treatment of metastatic melanoma (1988) and renal cell carcinoma (1992); IFN ⁇ was the first immunotherapy approved (1986) for the treatment of hairy cell leukemia. It was later approved to treat metastatic melanoma, renal cell carcinoma, non-Hodgkin lymphoma, and Kaposi's sarcoma.
  • IL-2 drugs that have been approved to treat tumors require higher doses to be effective. High doses of IL-2 will cause obvious toxic side effects and cannot be widely used.
  • the selectivity for NK cells and CD8 + T cells expressing IL-2R ⁇ and IL-2R ⁇ is low, so the tumor-killing ability of NK cells and CD8 + T cells cannot be fully exerted [3,4].
  • IFN ⁇ is a cytokine produced by the body's immune cells. It is a group of low-molecular glycoproteins with similar structures and similar functions produced by immune cells through anti-viral 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 alfa approved to treat viral infection treatment of infections and tumors. Since interferon alpha receptors are widely distributed and expressed in many normal cells, they can easily cause obvious toxic side effects.
  • the present invention discloses the design, preparation and use of a new IL-2, IFN ⁇ and Fc fusion protein.
  • IL-2 IL-2
  • IFN ⁇ IFN ⁇
  • Fc fusion protein Fc fusion protein
  • 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 with one or more mutations compared to a wild-type IL-2 protein, said The amino acid sequence is an amino acid sequence that is at least 90% identical to SEQ ID NO:2.
  • the mutations include one or more substitutions selected from the group consisting of: R38A, L80F, R81D, L85V, I86V, and I91F with reference to the amino acid position in SEQ ID NO:1.
  • the Fc portion comprises a human IgG Fc, such as human IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, or a variant thereof.
  • the Fc variant comprises one or more mutations selected from the group consisting of the L234A and L235A mutations, and the M252Y, S254T and T256E mutations.
  • the fusion protein further comprises an IFNa moiety.
  • the IFNa portion may comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 6 or 5.
  • the IFNa portion includes one or more substitutions selected from the group consisting of R144A and R149A with reference to the amino acid position in SEQ ID NO:5.
  • the fusion protein comprises:
  • the IL-2 portion is operably linked to the Fc portion and the Fc portion is operably linked to the IFN ⁇ portion;
  • the IL-2 portion is operably linked to the IFN ⁇ portion and the IFN ⁇ portion is operably linked to the Fc portion;
  • the IFNa portion is operably linked to the IL-2 portion and the IL-2 portion is operably linked to the Fc portion.
  • the fusion protein comprises:
  • the IL-2 portion is operably linked to the Fc portion and the Fc portion is operably linked to the IFN ⁇ portion;
  • the Fc portion is operably linked to the IL-2 portion;
  • the IFNa portion is operably linked to the Fc portion and the Fc portion is operably linked to the IL-2 portion.
  • the linker is set forth in SEQ ID NO: 11.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 3, 4, 9 or 10 List.
  • the present disclosure provides a fusion protein comprising an IFN ⁇ portion and an Fc portion, wherein the IFN ⁇ portion includes an amino acid sequence having one or more mutations compared to a wild-type IFN ⁇ protein, the amino acid sequence being the same as SEQ ID NO:6 Amino acid sequence with at least 90% identity.
  • the IFNa portion includes one or more substitutions selected from the group consisting of R144A and R149A with reference to the amino acid position in SEQ ID NO:5.
  • the Fc portion comprises a human IgG Fc, such as human IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, or a variant thereof.
  • the Fc variant comprises one or more mutations selected from the group consisting of the L234A and L235A mutations, and the M252Y, S254T and T256E mutations.
  • the fusion protein includes from the N-terminus to the C-terminus:
  • the IFN ⁇ portion is operably linked to the Fc portion;
  • the Fc portion is operably linked to the IFNa portion.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 7 or 8.
  • the present disclosure provides a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein disclosed herein.
  • the present disclosure provides vectors comprising the nucleic acid molecules disclosed herein.
  • the present disclosure provides host cells comprising a nucleic acid molecule or vector disclosed herein.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a fusion protein disclosed herein, or a nucleic acid molecule encoding the same, and a pharmaceutically acceptable carrier.
  • the present disclosure provides a method for producing a fusion protein disclosed herein, comprising the steps of:
  • the present disclosure provides a method of modulating an immune response in a subject, comprising administering to the subject a fusion protein or pharmaceutical composition disclosed herein.
  • the present disclosure provides a method for treating or preventing cancer or infectious disease in a subject, comprising administering to the subject an effective amount of a fusion protein or pharmaceutical composition disclosed herein.
  • 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 cancer, liver cancer, prostate cancer, pancreatic cancer, and leukemia.
  • the present disclosure provides use of a fusion protein disclosed herein in the manufacture of a medicament for the prevention, treatment, and/or management of cancer or infectious disease.
  • the present disclosure provides fusion proteins disclosed herein for use in the treatment or prevention of cancer or infectious diseases.
  • the present disclosure provides a kit including a container comprising a fusion protein or pharmaceutical composition disclosed herein.
  • New IL-2, IFN ⁇ and Fc fusion proteins characterized by including: IL-2 and IFN ⁇ dual factor Fc fusion protein, IL-2 and Fc fusion protein, IFN ⁇ and Fc fusion protein;
  • IL-2 and IFN ⁇ dual-factor Fc fusion protein fuse mutant IL-2 and mutant IFN ⁇ to the N-terminal or C-terminal of Fc respectively to obtain the polypeptide chain IL-2-Fc-IFN ⁇ or IFN ⁇ -Fc-IL-2 ;
  • IL-2 and Fc fusion protein fuse mutant IL-2 to the N-terminus or C-terminus of Fc to obtain the polypeptide chain Fc-IL-2 or IL-2-Fc;
  • IFN ⁇ and Fc fusion protein The mutant IFN ⁇ is fused to the N-terminal or C-terminal of Fc, respectively, to obtain the polypeptide chain Fc-IFN ⁇ or IFN ⁇ -Fc.
  • novel IL-2 and IFN ⁇ and Fc fusion protein according to any one of embodiments 1-2, characterized in that the IL-2 and IFN ⁇ dual factor Fc fusion protein, the Fc fragment also introduces L234A and L235A mutations , while introducing M252Y, S254T and T256E mutations.
  • novel IL-2 and IFN ⁇ and Fc fusion protein according to any one of embodiments 1 or 3, characterized in that the IL-2 and Fc fusion protein and Fc fragment also introduce L234A and L235A mutations, and at the same time M252Y, S254T and T256E mutations were introduced.
  • novel IL-2, IFN ⁇ and Fc fusion protein according to any one of embodiments 1 or 4, characterized in that the IFN ⁇ and Fc fusion protein, Fc fragment also introduces L234A and L235A mutations, and simultaneously introduces M252Y , S254T and T256E mutations.
  • Type IL-2 is obtained by introducing mutations. The introduced mutations include but are not limited to R38A, L80F, R81D, L85V, I86V and I91F.
  • novel IL-2, IFN ⁇ and Fc fusion protein according to embodiment 3 characterized in that, the IL-2 and Fc fusion protein, IL-2 mutant is obtained by molecular biology means in wild-type IL- 2, the introduced mutations include but are not limited to R38A, L80F, R81D, L85V, I86V and I91F.
  • the mutations introduced include but are not limited to R144A or R149A.
  • the entered mutations include but are not limited to R144A or R149A.
  • step (2) Clone the DNA fragment obtained in step (1) into the pcDNA series vector or other vectors used in mammalian cell expression systems to obtain a recombinant vector;
  • step (3) Transfect the recombinant vector obtained in step (2) into mammalian cells for expression of the fusion protein, and obtain the IL-2 and IFN ⁇ double factor Fc fusion protein after purification.
  • step (2) Clone the DNA fragment obtained in step (1) into the pcDNA series vector or other vectors used in mammalian cell expression systems to obtain a recombinant vector;
  • step (3) Transfect the recombinant vector obtained in step (2) into mammalian cells to express the fusion protein, and obtain the IL-2 and Fc fusion protein after purification.
  • step (2) Clone the DNA fragment obtained in step (1) into the pcDNA series vector or other vectors used in mammalian cell expression systems to obtain a recombinant vector;
  • step (3) Transfect the recombinant vector obtained in step (2) into mammalian cells to express the fusion protein, and obtain the IFN ⁇ and Fc fusion protein after purification.
  • step (3) the mammalian cells include HEK293 cells, CHO cells or cells of the above cells. Derived cells.
  • the method for preparing novel IL-2, IFN ⁇ and Fc fusion proteins according to any one of embodiments 12-14, It is characterized in that the introduction of mutations into the Fc fragment in step (1) is specifically: the introduction of L234A and L235A mutations into the Fc fragment, and the introduction of M252Y, S254T and T256E mutations into the Fc fragment at the same time.
  • novel IL-2, IFN ⁇ 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 IL-2 and IFN-a. Approved tumor treatment indications and broad-spectrum treatment of tumors in combination with other tumor treatment methods.
  • Figure 1A illustrates a schematic structural diagram of various Fc fusion proteins encompassed by the present invention.
  • the top to bottom of each small figure is from the N end to the C end, and the trapezoid represents the hinge area.
  • Figures 1B-1C respectively illustrate the SDS PAGE expression diagram and HPLC analysis diagram of the Fc fusion protein of the present invention.
  • Figure 2 illustrates the detection chart of the binding ability of the Fc fusion protein of the present invention and the receptor IL-2R ⁇ protein.
  • Figure 3 illustrates the detection chart of the binding ability of the Fc fusion protein of the present invention and the receptor IL-2R ⁇ protein.
  • Figure 4 illustrates the detection chart of the binding ability of the Fc fusion protein of the present invention and the receptor IFN ⁇ R2 protein.
  • Figure 5 illustrates the detection chart of the binding ability of the Fc fusion protein of the present invention to the interspecies receptor IL-2R ⁇ protein of different species.
  • Figure 6 illustrates the detection chart of the binding ability of the Fc fusion protein of the present invention and the interspecies receptor IL-2R ⁇ protein of different species.
  • Figure 7 illustrates the detection chart of the binding ability of the Fc fusion protein of the present invention and the interspecies receptor IFN ⁇ R2 protein of different species.
  • Figures 8A and 8B illustrate the results of detecting the binding ability of the Fc fusion protein of the present invention and FcRn protein through ELISA and Fortebio respectively.
  • Figure 9 illustrates a distribution detection chart of specific cell subpopulations of PBMC stimulated by the Fc fusion protein of the present invention.
  • Figure 10 illustrates the detection of the ability of the Fc fusion protein of the present invention to stimulate the proliferation of CTLL-2 mouse T lymphocytes.
  • Figures 11A to 11D illustrate the in vitro direct killing ability 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).
  • Figures 12A to 12E illustrate the in vitro comprehensive killing ability 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).
  • Figure 13 illustrates the pharmacodynamic activity of the Fc fusion protein of the present invention in the ExVivo organoid system.
  • IL-2 refers to interleukin-2 and is intended to include any form of IL-2, e.g., 1) native unprocessed IL-2 molecule, full-length IL-2 protein, or native Variants of IL-2 that exist; 2) any form of IL-2 produced by intracellular processing; or 3) full-length or modified forms.
  • IL-2 or "IL-2 domain” includes wild-type IL-2 and IL-2 variants.
  • IFN ⁇ refers to interferon ⁇ and is intended to include any form of IFN ⁇ , e.g., 1) a natural unprocessed IFN ⁇ molecule, a full-length IFN ⁇ protein, or a naturally occurring IFN ⁇ variant; 2) in a cell Any form of IFNa produced by endogenous processing; or 3) full-length or modified form.
  • IFNa or “IFNa domain” includes wild-type IFNa and IFNa variants.
  • variant refers to a biologically active polypeptide that includes one or more amino acid mutations to the native protein sequence.
  • one or more amino acid mutations include amino acid substitutions and/or insertions at certain positions in the amino acid sequence.
  • the variant has at least about 80% amino acid sequence identity with the corresponding native sequence polypeptide.
  • variants include, for example, polypeptides in which one or more amino acid (naturally occurring amino acids and/or non-naturally occurring amino acids) residues are 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 of nucleotides in the DNA encoding the native protein, thereby generating DNA encoding the variant, which is then The DNA is expressed in recombinant cell culture.
  • an IL-2 variant includes one or more substitutions compared to wild-type IL-2 protein.
  • an IFNa variant includes one or more substitutions compared to a wild-type IFNa protein.
  • Fc as used herein has the same meaning as used with respect to an antibody and refers to the portion of the antibody that includes the second (CH2) and third (CH3) constant regions of the first heavy chain linked to the first heavy chain by disulfide bonds.
  • the second and third constant regions of the duplex chain bind.
  • the Fc region may also include part or all of the hinge region.
  • the Fc region of antibodies is responsible for various effector functions such as ADCC and CDC, but does not have antigen-binding functions.
  • the term "Fc” includes wild-type Fc and Fc variants.
  • the term "operably linked” refers to the juxtaposition (with or without spacers or linkers or insert sequences) of two or more biological sequences of interest such that they are in a position that permits the intended relationship.
  • the polypeptide sequences are joined in a manner that allows the product of the joining to have the intended biological function.
  • the fusion protein can be operably linked to an immunoglobulin constant region to provide a stable product with ligand binding activity.
  • the fusion protein may be operably linked to an immunoglobulin constant region through an intervening sequence therebetween, and such intervening sequence may be a spacer or may comprise a longer sequence.
  • fusion when used with respect to an amino acid sequence (eg, a peptide, polypeptide, or protein) refers to the combination of two or more amino acid sequences, such as by chemical bonding or recombination. , forming a single amino acid sequence that does not occur naturally. Fusion amino acid sequences can be produced by genetic recombination of two encoding polynucleotide sequences and can be expressed by introducing a construct containing the recombinant polynucleotide into a host cell.
  • fusion protein refers to a polypeptide having two (or more) moieties operably linked together, where each moiety is a polypeptide with different properties.
  • the property may 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 moieties can be linked directly by a single peptide bond or by a peptide linker containing one or more amino acid residues. Generally, the two moieties and the linker will be in reading frame with each other.
  • the fusion protein is a fusion protein of an IL-2 portion, an Fc portion, and an IFNa portion.
  • the fusion protein is a fusion protein of an IL-2 portion and an Fc portion.
  • the fusion protein is a fusion protein of an Fc portion and an IFNa portion.
  • vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector is called an expression vector when it permits the expression of a protein encoded by a polynucleotide inserted therein.
  • the vector can be transformed, transduced or transfected into the host cell so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); phages such as lambda phage or M13 bacteriophage and animal viruses.
  • artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); phages such as lambda phage or M13 bacteriophage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, and papillomaviruses.
  • Viruses (such as SV40).
  • Vectors 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.
  • host cell refers to a cellular 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 the specific subject cell, but also the progeny of such cells. Such progeny may differ from the parent cell due to mutations or environmental influences that may undergo certain modifications in the progeny, but still be included within the scope of the term "host cell”.
  • identity 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 percentage of identical residues between amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecule 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 are included in Computational Molecular Biology, (ed.
  • subject includes any human or non-human animal, preferably humans.
  • cancer refers to any tumor or malignant cell growth causing a medical condition, which may be both solid and non-solid tumors mediated by proliferation or metastasis.
  • treatment in the context of treating a condition generally relate to treatment and therapy of humans or animals in which some desired therapeutic effect is achieved, e.g., inhibition of progression of the condition, including a reduction in the rate of progression, arrest in the rate of progression , the condition subsides, the condition improves and the condition is cured.
  • Treatment as a preventive measure i.e. prophylaxis, protection
  • treatment may mean inhibiting or slowing tumor or malignant cell growth, proliferation or metastasis, or some combination thereof.
  • treatment includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination thereof.
  • the term "effective amount” refers to an amount of active compound, or an amount of material, composition, or dosage containing an active compound, which, when administered in accordance with the desired treatment regimen, is effective to produce a benefit/risk ratio commensurate with the reasonable certain desired therapeutic effects.
  • the present disclosure provides IL-2 variants that include one or more modifications, such as insertions, deletions, and/or substitutions compared to wild-type IL-2 protein, such as human wild-type IL-2 protein.
  • the present disclosure further provides fusion proteins comprising an IL-2 protein portion fused to an Fc portion.
  • the IL2 protein portion may comprise wild-type IL-2 protein, or an IL-2 variant comprising one or more modifications (eg, insertions and/or substitutions) compared to wild-type IL-2 protein.
  • the IL-2 variant comprises one or more substitutions selected from, but not limited to, R38A, L80F, R81D, L85V, I86V, and I91F compared to wild-type IL-2 protein.
  • amino acid 38 is modified in the IL-2 variant compared to the wild-type IL-2 protein.
  • IL-2 variants further comprise conservative substitutions compared to wild-type IL-2 protein.
  • an IL-2 variant comprises or consists of the amino acid sequence set forth in SEQ ID NO:2.
  • the IL-2 variant comprises or consists of at least 85%, at least 90%, at least 95%, or at least 99% (e.g., at least 91%, 92%, 93%, 94%) of SEQ ID NO:2 , 95%, 96%, 97%, 98%, 99%) identical amino acid sequences.
  • the present disclosure provides IFNa variants that include one or more modifications, such as insertions, deletions, and/or substitutions compared to wild-type IFNa protein, such as human wild-type IFNa protein.
  • the present disclosure further provides fusion proteins comprising an IFNa protein portion fused to an Fc portion.
  • the IFN[alpha] protein portion may comprise wild-type IFN[alpha] protein, or an IFN[alpha] variant comprising one or more modifications (eg, insertions and/or substitutions) compared to wild-type IFN[alpha] protein.
  • amino acid position R144 and/or R149 in the IFN ⁇ variant is modified compared to wild-type IFN ⁇ protein.
  • the IFNa variant further comprises conservative substitutions compared to wild-type IFNa protein.
  • the IFNa variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6.
  • the IFNa variant comprises or consists of at least 85%, at least 90%, at least 95, or at least 99% (e.g., at least 91%, 92%, 93%, 94%, 95%) of SEQ ID NO:6 , 96%, 97%, 98%, 99%) identical amino acid sequences.
  • the present disclosure provides fusion proteins including an IL-2 portion and an Fc portion, fusion proteins including an IFN ⁇ portion and an Fc portion, and fusion proteins including an IL-2 portion, an Fc portion, and an IFN ⁇ portion.
  • the fusion protein can be single-chain or multiple-chain. In some embodiments, the fusion protein is a double-stranded homodimer or heterodimer.
  • the individual IL-2 portions, Fc portions and IFNa portions in each chain can be the same or different.
  • fusion proteins of the invention comprise an IL-2 moiety operably linked to an Fc moiety from the N-terminus to the C-terminus (also known as an IL-2-Fc fusion protein).
  • fusion proteins of the invention comprise an Fc portion operably linked to an IL-2 portion from the N-terminus to the C-terminus (also known as an Fc-IL-2 fusion protein).
  • fusion proteins of the invention comprise an IFNa moiety operably linked to an Fc moiety from the N-terminus to the C-terminus (also known as an IFNa-Fc fusion protein).
  • fusion proteins of the invention comprise an Fc portion operably linked to an IFN ⁇ portion from the N-terminus to the C-terminus (also known as an Fc-IFN ⁇ fusion protein).
  • fusion proteins of the invention comprise an IL-2 portion operably linked to an Fc portion from the N-terminus to the C-terminus and the Fc portion is operably linked to an IFNa portion (also known as IL-2-Fc- IFN ⁇ fusion protein).
  • fusion proteins of the invention comprise an IFNa moiety from the N-terminus to the C-terminus operably linked to an Fc moiety and the Fc moiety is operably linked to an IL-2 moiety (also known as IFNa-Fc-IL- 2 fusion protein).
  • the IL-2 portion included in the fusion protein may comprise wild-type IL-2 protein or an IL-2 variant.
  • the IFN ⁇ portion included in the fusion protein may comprise wild-type IFN ⁇ protein or an IFN ⁇ variant.
  • the Fc portion included in the fusion protein may comprise wild-type Fc or an Fc variant.
  • amino acid 38 in the IL-2 portion included in the fusion protein is modified. It has been found that the region near amino acid 38 plays a key role in the increase in vascular permeability caused by IL-2, so modification of amino acid 38 can significantly reduce vascular permeability.
  • the fusion protein contains a modified portion of interferon alpha that has significantly reduced affinity for the interferon alpha receptor compared to native interferon alpha. This reduction can lead to a significant reduction in the toxic side effects caused by interferon ⁇ while retaining its anti-tumor activity.
  • the R144 or R149 amino acid in the interferon alpha portion included in the fusion protein is modified. These amino acids play a key role in the binding of interferon ⁇ to its receptor. Mutation can cause a significant reduction in affinity without affecting its anti-tumor activity.
  • the operative connection can be a direct connection of the two parts, or a connection through a linker such as a peptide linker.
  • the IL-2 portion is operably linked to the Fc portion via a peptide linker.
  • the IFN alpha portion is operably linked to the Fc portion via a peptide linker.
  • the peptide linker can be any peptide linker commonly used in the art, such as GS series linkers, as shown in SEQ ID NO: 11.
  • the fusion protein provided by the invention can improve the therapeutic effect, reduce toxic and side effects, and increase the therapeutic safety window.
  • the fusion protein provided by the invention also includes an immunoglobulin constant domain sequence, such as a human IgG constant domain sequence, and more specifically may include a hinge region and an Fc region, such as IgG1, IgG2, IgG3, IgG4 Fc region sequence.
  • Fc refers to the portion of an antibody consisting of the second and third constant regions of the first heavy chain of the antibody bound via disulfide bonds to the second and third constant regions of the second heavy chain,
  • the Fc region also contains all or part of the hinge region.
  • An Fc region as used herein includes both wild-type Fc regions and variants thereof, with different mutations for various purposes.
  • the variants may comprise one or more amino acid residue modifications, such as substitutions, in the Fc region.
  • Fc region variants comprise one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn).
  • FcRn neonatal Fc receptor
  • Such a variant may have an extended pharmacokinetic half-life because it binds FcRn at acidic pH allowing it to escape degradation in lysosomes and then be transported and released out of the cell.
  • Methods of engineering antibody molecules to improve binding affinity to FcRn are well known in the art, see, for example, Vaughn, D. et al., Structure, 6(1):63-73, 1998; Kontermann, R. et al. Human, 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).
  • the fusion protein provided by the invention contains L234A/L235A substitutions to reduce the binding ability to the receptor FcgRIIIa.
  • the fusion proteins provided by the invention contain one or more amino acid substitutions in the interface of the Fc region to assist and/or promote heterodimerization.
  • These modifications include the introduction of protrusions into the first Fc polypeptide and the introduction of cavities into the second Fc polypeptide, where the protrusions can be located in the cavities to facilitate the interaction of the first and second Fc polypeptides to form heterodimers or complexes. Methods of generating protein molecules with these modifications are known in the art, for example, as described in U.S. Patent No. 5,731,168.
  • the Fc domain also includes the triple mutation M252Y/S254T/T256E ("YTE"). This triple mutation was reported to result in an approximately 10-fold increase in binding to the human neonatal Fc receptor (FcRn) and an almost 4-fold increase in the serum half-life of YTE-containing human IgG in cynomolgus monkeys (Oganesyan V. et al. Human, Mol Immunol. 2009 May;46(8-9):1750-5). In some further embodiments, the Fc domain includes additional mutations to enhance the interaction between Fc and human FcRn.
  • YTE triple mutation M252Y/S254T/T256E
  • 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.
  • IL-2R ⁇ and IL-2R ⁇ By modifying the structure of IL-2, the affinity of cytokines to IL-2R ⁇ and IL-2R ⁇ on the surfaces of NK cells and CD8+ T cells is increased, and the killing effect of these cells on tumors is enhanced.
  • Modification of the 38th amino acid of IL-2 can significantly reduce the toxic side effects of increased vascular permeability caused by IL-2.
  • R144 and R149 can reduce their binding ability to the receptor IFN ⁇ R2, reduce toxic side effects, and at the same time retain its antitumor activity.
  • the reconstructed IL-2, IFN ⁇ and Fc fusion proteins can overcome the shortcomings of current IL-2 and IFN ⁇ drugs, improve the therapeutic effect, reduce toxic and side effects, and increase the safety window of treatment.
  • the invention also provides polynucleotides encoding such fusion proteins.
  • the polynucleotides can be used to express fusion proteins.
  • the polynucleotides may also be used as therapeutic agents in active forms for achieving in vivo expression of polypeptide fusion proteins.
  • polynucleotides encoding the fusion proteins are readily isolated and sequenced using routine procedures known in the art.
  • the polynucleotides can also be obtained synthetically.
  • the polynucleotide is codon optimized for expression in eukaryotic host cells, particularly mammalian cells.
  • the polynucleotide encoding the fusion protein can be inserted into a vector for further cloning (amplification of DNA) or expression.
  • 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, enhancer elements, a promoter (eg, SV40, CMV, EF-1 ⁇ ), and a transcription termination sequence.
  • 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-1 ⁇ ), and at least one selectable marker.
  • a vector e.g., an expression vector
  • at least one promoter e.g., SV40, CMV, EF-1 ⁇
  • at least one selectable marker e.g., SV40, CMV, EF-1 ⁇
  • vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40 ), lambda phage, and M13 phage, liposomes, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, p
  • a vector containing a polynucleotide sequence encoding a fusion protein can be introduced into a host cell for cloning or gene expression.
  • Suitable host cells for cloning or expressing DNA in the vectors herein are prokaryotes, yeast or higher eukaryotic cells.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive bacteria, such as E. coli.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for the provided vectors. Saccharomyces cerevisiae or common baker's yeast is the most commonly used of the lower eukaryotic host microorganisms. However, many other genera, species, and strains are commonly available and useful herein.
  • Suitable host cells for expression of the fusion proteins provided herein can also be derived from multicellular organisms.
  • invertebrate cells include plant and insect cells.
  • the host cells are Chinese Hamster Ovary (CHO) cells.
  • the host cell is another mammalian cell line, such as human Cell lines.
  • Host cells are transformed with the above-mentioned expression or cloning vectors for producing fusion proteins and cultured in appropriately modified conventional nutrient media for inducing promoters, selecting transformants or amplifying genes encoding desired sequences.
  • Host cells used to produce the fusion proteins provided herein can be cultured in a variety of media. Any of these media can be supplemented as needed 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 the drug GENTAMYCINTM), trace elements (defined as inorganic compounds typically present in final concentrations in the micromolar range), and glucose or equivalent energy sources. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., are those previously used with host cells selected for expression and will be apparent to the ordinarily skilled person.
  • 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
  • Fusion proteins prepared from 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 the preferred purification technique. .
  • compositions such as pharmaceutical compositions, comprising a fusion protein of the invention formulated with a pharmaceutically acceptable carrier.
  • Such compositions comprise at least one fusion protein of the invention or a polynucleotide encoding the fusion protein.
  • pharmaceutically acceptable carrier includes any and all pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, etc. that are physiologically compatible Penetrating agents, buffers, antioxidants, anesthetics, suspension/dispersing agents, isolating agents or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art or their respective combinations and so on.
  • the carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, transepidermal, intravitreal injection or implantation administration, and the like.
  • the active compound ie, the ingredient of the present invention, may be coated in a material to protect the compound from acids and other natural conditions that may inactivate the compound.
  • compositions of the present invention may also include pharmaceutically acceptable antioxidants.
  • suitable aqueous and non-aqueous carriers useful in the pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and appropriate mixtures thereof, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate.
  • polyols e.g., glycerol, propylene glycol, polyethylene glycol, etc.
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper flow properties can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersants, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Pharmaceutical compositions may be in the form of solid, paste, ointment, gel, liquid, aerosol, spray, polymer, film, emulsion or suspension.
  • compositions are formulated as injectable compositions.
  • injectable pharmaceutical compositions may be prepared in any conventional form, such as liquid solutions, suspensions, emulsions, or solid forms suitable for producing liquid solutions, suspensions, or emulsions.
  • Preparations for injection may include sterile and/or nonpyrolytic solutions prepared for injection, sterile dry soluble products, Such as lyophilized powders, prepared for mixing with solvents before use, including subcutaneous tablets, sterile suspensions prepared for injection, sterile dry insoluble products prepared for combination with vehicles before use, and sterile and/or non- Thermal Lotion. Solutions can be aqueous or non-aqueous.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the actual dosage levels of active ingredients and small molecules in the pharmaceutical compositions of the invention may vary in order to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition and mode of administration without being toxic to said patient.
  • the dosage level selected will depend on various pharmacokinetic factors, including the activity of the particular combination of the invention employed, or its ester, salt or amide, the route of administration, the time of administration, the excretion rate of the particular compounds employed, Duration of treatment, other drugs, compounds and/or materials used in conjunction with the specific combination used, age, sex, weight, condition, general health and preexisting medical history of the patient to be treated, and similar factors well known in the medical field .
  • the fusion proteins, pharmaceutical compositions and methods of the present disclosure have many in vitro and in vivo applications, including, for example, the enhancement of immune responses.
  • these molecules can be administered to cultured cells in vitro or ex vivo, or, for example, in vivo to human subjects to enhance immunity in various situations.
  • the immune response can be modulated, such as enhanced, stimulated or up-regulated.
  • subjects include human patients in need of modulation of immune responses.
  • the method is particularly suitable for treating cancer in vivo.
  • the fusion protein can be administered alone or in combination with another therapy.
  • the two may be administered in any order or simultaneously.
  • the present disclosure provides methods of treating a condition or disease in a mammal, comprising administering to a subject (eg, a human) in need of treatment a therapeutically effective amount of a fusion protein as disclosed herein.
  • the condition or disease may be a cancer, such as a cancer treatable by IL-2 and IFNa.
  • a variety of cancers, whether malignant or benign, and primary or secondary, can be treated or prevented using the methods provided by this disclosure.
  • the cancer can be a solid tumor or a hematological malignancy.
  • fusion proteins disclosed herein can be used alone as a single therapy or in combination with cellular immunotherapy, targeted therapy, chemotherapy or radiotherapy.
  • SEQ ID NO: 1 wild-type IL-2 amino acid sequence
  • SEQ ID NO:3 (IL-2-Fc fusion protein amino acid sequence)
  • SEQ ID NO:4 Fc-IL-2 fusion protein amino acid sequence
  • SEQ ID NO:7 IFN ⁇ -Fc fusion protein amino acid sequence
  • SEQ ID NO:10 IFN ⁇ -Fc-IL-2 fusion protein amino acid sequence
  • Example 1 Molecular construction and production of fusion proteins
  • An IL-2-Fc-IFN ⁇ fusion protein was constructed, which includes the IL-2 part from the N terminus to the C terminus operably linked to the immunoglobulin Fc part, and the immunoglobulin Fc part operably linked to the IFN ⁇ part. , this fusion protein is hereinafter referred to as IL-2-Fc-IFN ⁇ , and its amino acid sequence is shown in SEQ ID NO: 9.
  • An IL-2-Fc fusion protein was also constructed, which contains the IL-2 part from the N terminus to the C terminus operably linked to the immunoglobulin Fc part. This fusion protein is hereinafter referred to as IL-2-Fc, and its amino acid sequence is shown in SEQ ID NO: 3.
  • Fc-IFN ⁇ fusion protein
  • Fc-IFN ⁇ immunoglobulin Fc part from the N terminus to the C terminus operably linked to the IFN ⁇ part.
  • This fusion protein is hereinafter referred to as Fc-IFN ⁇ , and its amino acid sequence is as SEQ ID NO: 8 shown.
  • the antibody light and heavy chain genes are amplified by PCR using conventional molecular biology techniques, and the genes encoding amino acid sequences are connected to the pcDNA3.4 vector through homologous recombination technology. Extract plasmids from the sequenced positive clones and transfect them into Expi293F cells. Continue culturing for 7 days in a shaker at 37°C/5% CO 2 /125 rpm. After the culture, collect the supernatant and perform proteinA affinity chromatography to obtain purified cells. Fusion protein molecules, and determine the antibody concentration through UV280 binding theoretical extinction coefficient.
  • Figure 1B shows the SDS PAGE gel image of IL-2-Fc-IFN ⁇ fusion protein
  • Figure 1C shows its HPLC purity analysis chart.
  • Example 2 ELISA detection of binding ability to receptor IL-2R ⁇ protein
  • the coating concentration is 2 ⁇ g/ml, 100ul/well overnight at 4°C; blocked with 300ul/well 3% skimmed milk powder at 37°C for 1 hour; different concentrations of fusion were added to each well
  • Example 3 ELISA detection of binding ability to receptor IL-2R ⁇ protein
  • the coating concentration is 2 ⁇ g/ml, 100ul/well overnight at 4°C; blocked with 300ul/well 3% skimmed milk powder at 37°C for 1 hour; different concentrations of fusion were added to each well
  • the coating concentration was 2 ⁇ g/ml, 100ul/well overnight at 4°C; blocked with 300ul/well 3% skimmed milk powder at 37°C for 1 hour; different concentrations of fusion protein and 100ul of each control sample was incubated at 37°C for 1 hour; then Anti-IL-2 Rabbit pAb was added, incubated at 37°C for 1 hour, and then Anti-Rabbit IgG HRP was added and incubated at 37°C for 1 hour; 100ul of TMB was added to each well for color development for 5 minutes, and then 50ul was added to terminate solution and read the OD450 on a microplate reader.
  • Example 5 ELISA detection of binding ability to different interspecies receptor IL-2R ⁇ proteins
  • the human, mouse and cynomolgus monkey IL-2R ⁇ proteins were respectively coated at a coating concentration of 2 ⁇ g/ml, 100 ul/well at 4°C overnight; blocked with 300 ul/well 3% skimmed milk powder at 37°C for 1 hour; different concentrations of fusion proteins were added to each well.
  • Example 6 ELISA detection of binding ability to different interspecies receptor IL-2R ⁇ proteins
  • the human, mouse and cynomolgus monkey IL-2R ⁇ proteins were respectively coated at a coating concentration of 2 ⁇ g/ml, 100 ul/well overnight at 4°C; blocked with 300 ul/well 3% skimmed milk powder at 37°C for 1 hour; different concentrations of fusion proteins were added to each well.
  • IL-2-Fc-IFN ⁇ has species crossover with human, mouse, and cynomolgus monkey receptor IL-2R ⁇ proteins.
  • the IL-2R ⁇ protein binding ability of cynomolgus monkey is similar to that of humans. Binding to mouse IL-2R ⁇ protein is lower than binding to human.
  • Example 7 ELISA detection of binding ability to different interspecies receptor IFN ⁇ R2 proteins
  • the coating concentration is 2 ⁇ g/ml, 100ul/well overnight at 4°C; blocked with 300ul/well 3% skimmed milk powder at 37°C for 1 hour; different concentrations of fusion protein and Add 100ul of each control sample and incubate at 37°C for 1 hour; then add Anti-IFN ⁇ 2 Rabbit pAb, incubate at 37°C for 1 hour, and then add Anti-Rabbit IgG Incubate HRP at 37°C for 1 hour; add 100ul TMB to each well for color development for 5 minutes, then add 50ul stop solution, and read OD450 on a microplate reader.
  • Example 9 Detection of immune cell subsets after stimulation of human PBMC cells
  • FITC Anti-Foxp3 antibody
  • PE Anti-CD4 antibody
  • PE Anti-CD56 antibody
  • PE Anti-CD8 antibody
  • FITC Anti-Granzyme B antibody
  • Example 11 Detection of direct killing ability of tumor cells in vitro
  • the direct killing ability is to measure the direct killing of tumor cells by IFN-a in the absence of immune cells.
  • NCI-N87 Culture and expand human gastric cancer cell NCI-N87 to the required cell volume. Plate NCI-N87 cells into a 96-well plate at 4000 cells/well. Add different concentrations (4000nM, 1000nM, 250nM, 100nM, 62.5nM) according to the experimental design. , 15.625nM, 10nM, 4nM, 1.5625nM, 1nM, 244.14pM, 156.25pM, 100pM, 100pM, 10pM, 1pM, 0) protein, continue to culture for 5 days; use CCK-8 method to measure the expression of Formazan in living cells, and then evaluate the cells vitality. The specific operation was performed according to the instructions.
  • Example 12 Detection of comprehensive killing ability of tumor cells in vitro
  • the comprehensive killing function is the sum of direct killing capability and indirect killing capability.
  • Comprehensive killing capacity is obtained in the presence of immune cells PBMC.
  • pleural fluid samples from breast cancer patients identified as HER2 2+ positive
  • Candidate drugs and reference drugs are incubated with tumor cell immune cell complexes in vitro. After 4-5 days of treatment, the culture medium and cells were collected for analysis, including tumor cells, Granzyme B, IFN- ⁇ , etc.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Toxicology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)

Abstract

针对人类白细胞介素-2(IL-2)和干扰素α(IFNα)的改构；改构后IL-2、IFNα或IL-2/IFNα双因子和Fc形成的融合蛋白；以及这些融合蛋白的设计、制备和用途。IFNα通过基因突变，改变IFNα与其受体的结合能力，得到更优的融合蛋白，解决现有药物特异性弱和副作用大的缺点。

Description

新融合蛋白及其用途
交叉引用
本申请要求2022年5月13日提交的中国专利申请202210521837.5的优先权，其全部内容通过提述并入本文。
序列表
本申请包含序列表，并且其全部内容通过引用并入本文。
技术领域
本发明属于生物医药技术领域，具体涉及新型IL-2与IFNα和Fc融合蛋白的设计、制备及用途。
背景技术
IL-2，也称为T细胞生长因子，基因位于4号染色体，包括7kb的序列，由133个氨基酸组成，分子量约15kD。IL-2通过IL-2R发生作用，IL-2R包括三个亚基，IL-2Rα(即CD25)、IL-2Rβ(即CD122)和IL-2Rγ(即CD132)。三个亚基可以形成三种受体形式：高结合力受体包含所有三个亚基IL-2Rα/β/γ，中结合力受体包含IL-2Rβ/γ两个亚基和低结合力受体IL-2Rα。其中IL-2Rβ和IL-2Rγ是IL-2激活下游信号通路所必需的，当IL-2同时结合IL-2Rβ和IL-2Rγ时，两个受体亚基形成异源二聚体，磷酸化细胞内的STAT5，进入细胞核导致相应的基因转录和表达；IL-2Rα并非信号所必需，但可以促进IL-2与IL-2Rβ和IL-2Rγ的结合。IL-2Rγ在所有免疫细胞中均有表达；IL-2Rβ在CD8+T细胞、NK细胞、调节性T细胞中均有表达；而且在T细胞被激活后会提升表达水平；IL-2Rα在调节性T细胞持续高表达，在被激活的CD8+T细胞中会有短暂表达，随即下调表达水平[1,2]。IL-2是第二个批准的免疫疗法，用于治疗转移黑色素瘤(1988)及肾细胞癌(1992)；IFNα是第一个批准的免疫疗法(1986)，用于治疗毛状细胞白血病,后被批准治疗转移黑色素瘤、肾细胞癌、非霍奇金淋巴瘤、卡波济氏肉瘤。
IL-2药物的半衰期较短，所以已批准治疗肿瘤的IL-2药物需要较高剂量才有效果，而高剂量的IL-2会引起明显的毒副作用而不能广泛得到应用。对表达IL-2Rβ和IL-2Rγ的NK细胞和CD8+T细胞选择性较低，因此不能充分发挥NK细胞和CD8+T细胞杀伤肿瘤的能力[3,4]。
在另一方面，IFNα是机体免疫细胞产生的一种细胞因子，是机体受到病毒感染时，免疫细胞通过抗病毒应答反应而产生的一组结构类似、功能接近的低分子糖蛋白。干扰素在机体的免疫系统中起着非常重要的作用[5,6]。重组人干扰素α已批准用于治疗病毒性感 染和肿瘤的治疗。由于干扰素α受体分布较广，在很多正常细胞中均有表达，所以容易引起明显的毒副作用。
本领域中需要产生新的细胞因子构建体，从而提供更有效的治疗效果和同时更小的副作用。
发明内容
为解决上述问题，本发明公开了新型IL-2与IFNα和Fc融合蛋白的设计、制备和用途，通过基因突变的方式，改变IL-2与其受体、IFNα与其受体的结合能力，解决现有药物特异性弱、半衰期短和副作用大的缺点。
在一些方面，本公开提供了融合蛋白，其包含IL-2部分和Fc部分，其中所述IL-2部分包含与野生型IL-2蛋白相比具有一个或多个突变的氨基酸序列，所述氨基酸序列为与SEQ ID NO:2具有至少90％同一性的氨基酸序列。
在一些实施方案中，所述突变包括参照SEQ ID NO:1中氨基酸位置的选自以下的一种或多种取代：R38A、L80F、R81D、L85V、I86V和I91F。
在一些实施方案中，所述Fc部分包含人IgG Fc，如人IgG1 Fc、IgG2 Fc、IgG3 Fc、IgG4 Fc或其变体。例如，所述Fc变体包含选自以下的一种或多种突变：L234A和L235A突变、以及M252Y、S254T和T256E突变。
在一些实施方案中，所述融合蛋白还包含IFNα部分。所述IFNα部分可包含与SEQ ID NO:6或5具有至少90％同一性的氨基酸序列。在一些实施方案中，所述IFNα部分包括参照SEQ ID NO:5中氨基酸位置的选自以下的一种或多种取代：R144A和R149A。
在一些实施方案中，所述融合蛋白包含：
(a)IL-2部分可操作地连接于Fc部分；
(b)IL-2部分可操作地连接于Fc部分且Fc部分可操作地连接于IFNα部分；
(c)IL-2部分可操作地连接于IFNα部分且IFNα部分可操作地连接于Fc部分；或
(d)IFNα部分可操作地连接于IL-2部分且IL-2部分可操作地连接于Fc部分。
在一些进一步的实施方案中，所述融合蛋白包含：
(a)IL-2部分可操作地连接于Fc部分；
(b)IL-2部分可操作地连接于Fc部分且Fc部分可操作地连接于IFNα部分；
(c)Fc部分可操作地连接于IL-2部分；或
(d)IFNα部分可操作地连接于Fc部分且Fc部分可操作地连接于IL-2部分。
在一些实施方案中，所述可操作地连接是直接连接或通过肽接头进行连接，任选地所述肽接头为GS系列接头，例如(GS)n，其中n＝1-5。在一些实施方案中，所述接头如SEQ ID NO:11所示。
在一些实施方案中，所述融合蛋白包含如SEQ ID NO:3、4、9或10所示的氨基酸序 列。
在一些方面，本公开提供了融合蛋白，其包含IFNα部分和Fc部分，其中所述IFNα部分包含与野生型IFNα蛋白相比具有一个或多个突变的氨基酸序列，所述氨基酸序列为与SEQ ID NO:6具有至少90％同一性的氨基酸序列。
在一些实施方案中，所述IFNα部分包括参照SEQ ID NO:5中氨基酸位置的选自以下的一种或多种取代：R144A和R149A。
在一些实施方案中，所述Fc部分包含人IgG Fc，如人IgG1 Fc、IgG2 Fc、IgG3 Fc、IgG4 Fc或其变体。例如，所述Fc变体包含选自以下的一种或多种突变：L234A和L235A突变、以及M252Y、S254T和T256E突变。
在一些实施方案中，所述融合蛋白从N末端到C末端包含：
(a)IFNα部分可操作地连接于Fc部分；或
(b)Fc部分可操作地连接于IFNα部分。
在一些实施方案中，融合蛋白包含如SEQ ID NO:7或8所示的氨基酸序列。
在一些方面，本公开提供了一种核酸分子，其包括编码本文公开的融合蛋白的核酸序列。
在一些方面，本公开提供了包含本文公开的核酸分子的载体。
在一些方面，本公开提供了包含本文公开的核酸分子或载体的宿主细胞。
在一些方面，本公开提供了一种药物组合物，其包含本文公开的融合蛋白或编码其的核酸分子，以及药学上可接受的载剂。
在一些方面，本公开提供了一种用于生产本文公开的融合蛋白的方法，其包括以下步骤：
-在包含编码所述融合蛋白的载体的宿主细胞中表达所述融合蛋白；和
-从宿主细胞培养物中分离出所述融合蛋白。
在一些方面，本公开提供了一种调节受试者中的免疫应答的方法，其包括向受试者施用本文公开的融合蛋白或药物组合物。
在一些方面，本公开提供了一种用于治疗或预防受试者中的癌症或感染性疾病的方法，其包括向受试者施用有效量的本文公开的融合蛋白或药物组合物。
在一些实施方案中，所述癌症选自乳腺癌、胃癌、黑色素瘤、淋巴瘤、肺癌、结肠癌、卵巢癌、膀胱癌、肾细胞癌、肝癌、前列腺癌、胰腺癌和白血病。
在一些方面，本公开提供了本文公开的融合蛋白在制备用于预防、治疗和/或管理癌症或感染性疾病的药物中的用途。
在一些方面，本公开提供了本文公开的融合蛋白用于治疗或预防癌症或感染性疾病。
在一些方面，本公开提供了一种试剂盒，包括包含本文公开的融合蛋白或药物组合物的容器。
本发明还涉及以下实施方案：
1.新型IL-2与IFNα和Fc融合蛋白，其特征在于，包括：IL-2与IFNα双因子Fc融合蛋白、IL-2与Fc融合蛋白、IFNα与Fc融合蛋白；
IL-2与IFNα双因子Fc融合蛋白：将突变型IL-2和突变型IFNα分别融合在Fc的N端或C端，得到多肽链IL-2-Fc-IFNα或IFNα-Fc-IL-2；
IL-2与Fc融合蛋白：将突变型IL-2融合在Fc的N端或C端，得到多肽链Fc-IL-2或IL-2-Fc；
IFNα与Fc融合蛋白：将突变型IFNα分别融合在Fc的N端或C端，得到多肽链Fc-IFNα或IFNα-Fc。
2.根据实施方案1所述的新型IL-2与IFNα和Fc融合蛋白，其特征在于，所述IL-2与IFNα双因子Fc融合蛋白中，Fc片段包括重链恒定区铰链区、重链恒定区第二个结构域CH2和重链恒定区第三个结构域CH3。
3.根据实施方案1所述的新型IL-2与IFNα和Fc融合蛋白，其特征在于，所述IL-2与Fc融合蛋白中，Fc片段包括重链恒定区铰链区、重链恒定区第二个结构域CH2和重链恒定区第三个结构域CH3。
4.根据实施方案1所述的新型IL-2与IFNα和Fc融合蛋白，其特征在于，所述IFNα与Fc融合蛋白，Fc片段包括重链恒定区铰链区、重链恒定区第二个结构域CH2和重链恒定区第三个结构域CH3。
5.根据实施方案1-2任一项所述的新型IL-2与IFNα和Fc融合蛋白，其特征在于，所述IL-2与IFNα双因子Fc融合蛋白，Fc片段还引入L234A和L235A突变，同时引入M252Y、S254T和T256E突变。
6.根据实施方案1或3任一项所述的新型IL-2与IFNα和Fc融合蛋白，其特征在于，所述的IL-2与Fc融合蛋白，Fc片段还引入L234A和L235A突变，同时引入M252Y、S254T和T256E突变。
7.根据实施方案1或4任一项所述的新型IL-2与IFNα和Fc融合蛋白，其特征在于，所述的IFNα与Fc融合蛋白，Fc片段还引入L234A和L235A突变，同时引入M252Y、S254T和T256E突变。
8.根据实施方案1所述的新型IL-2与IFNα和Fc融合蛋白，其特征在于，所述IL-2与IFNα双因子Fc融合蛋白，IL-2突变体为通过分子生物学手段在野生型IL-2基础上引入突变得到，所引入突变包括但不限于R38A、L80F、R81D、L85V、I86V和I91F。
9.根据实施方案3所述的新型IL-2与IFNα和Fc融合蛋白，其特征在于，所述IL-2与Fc融合蛋白，IL-2突变体为通过分子生物学手段在野生型IL-2基础上引入突变得到，所引入突变包括但不限于R38A、L80F、R81D、L85V、I86V和I91F。
10.根据实施方案1所述的新型IL-2与IFNα和Fc融合蛋白，其特征在于，所述IL- 2与IFNα双因子Fc融合蛋白，IFNα衍生物为通过分子生物学手段在野生型IFNα基础上引入突变得到，所进入突变包括但不限于R144A或R149A。
11.根据实施方案4所述的新型IL-2与IFNα和Fc融合蛋白，其特征在于，所述IFNα与Fc融合蛋白，IFNα衍生物为通过分子生物学手段在野生型IFNα基础上引入突变得到，所进入突变包括但不限于R144A或R149A。
12.制备如实施方案1-2中任一项所述的新型IL-2与IFNα和Fc融合蛋白的方法，其特征在于，IL-2与IFNα双因子Fc融合蛋白的制备方法，包括以下步骤：
(1)将IL-2突变体通过柔性连接子连接在Fc片段N端或C端，将IFNα衍生物通过柔性连接子连接在Fc片段C端或N端，得到多肽链IL-2-Fc-IFNα或IFNα-Fc-IL-2；在Fc片段引入突变L234A、L235A、M252Y、S254T和T256E；
(2)将步骤(1)得到的DNA片段克隆至pcDNA系列载体或其它用于哺乳动物细胞表达系统的载体，得到重组载体；
(3)将步骤(2)中获得的重组载体转染至哺乳动物细胞以进行融合蛋白的表达，纯化后得到IL-2与IFNα双因子Fc融合蛋白。
13.制备如实施方案1或3中任一项所述的新型IL-2与IFNα和Fc融合蛋白的方法，其特征在于，IL-2与Fc融合蛋白的制备方法，包括以下步骤：
(1)将IL-2突变体通过柔性连接子连接在Fc片段N端或C端，得到多肽链IL-2-Fc或Fc-IL-2；在Fc片段引入突变L234A、L235A、M252Y、S254T和T256E；
(2)将步骤(1)得到的DNA片段克隆至pcDNA系列载体或其它用于哺乳动物细胞表达系统的载体，得到重组载体；
(3)将步骤(2)中获得的重组载体转染至哺乳动物细胞以进行融合蛋白的表达，纯化后得到IL-2与Fc融合蛋白。
14.制备如实施方案1或4中任一项所述的新型IL-2与IFNα和Fc融合蛋白的方法，其特征在于，IFNα与Fc融合蛋白的制备方法，包括以下步骤：
(1)将IFNα衍生物通过柔性连接子连接在Fc片段C端或N端，得到多肽链Fc-IFNα或IFNα-Fc；在Fc片段引入突变L234A、L235A、M252Y、S254T和T256E；
(2)将步骤(1)得到的DNA片段克隆至pcDNA系列载体或其它用于哺乳动物细胞表达系统的载体，得到重组载体；
(3)将步骤(2)中获得的重组载体转染至哺乳动物细胞以进行融合蛋白的表达，纯化后得到IFNα与Fc融合蛋白。
15.根据实施方案12-14任一项所述的制备新型IL-2与IFNα和Fc融合蛋白的方法，其特征在于，步骤(3)中哺乳动物细胞包括HEK293细胞、CHO细胞或上述细胞的衍生细胞。
16.根据实施方案12-14任一项所述的制备新型IL-2与IFNα和Fc融合蛋白的方法， 其特征在于，步骤(1)中Fc片段引入突变具体为：在Fc片段引入L234A和L235A突变，同时在Fc片段引入M252Y、S254T和T256E突变。
17.所述实施方案1-11中任一项所述的新型IL-2与IFNα和Fc融合蛋白在制备广谱抗肿瘤药物中的用途包括单药疗法治疗IL-2和IFN-a已批准的肿瘤治疗适应症以及和其他肿瘤治疗方法联合广谱治疗肿瘤。
18.实施方案12-16中任一项所述的方法制备的新型IL-2与IFNα和Fc融合蛋白在制备Fc融合蛋白药物中的用途包括单药疗法治疗IL-2和IFN-a已批准的肿瘤治疗适应症以及和其他肿瘤治疗方法联合广谱治疗肿瘤。
以上内容是一个概述，因此必要时包含细节的简化、概括和省略；因此，本领域技术人员将认识到，该概述仅是举例说明性的，并不意图以任何方式进行限制。本文所述的方法、组合物和用途和/或其他主题的其它方面、特征和优势将在本文所示的教导中变得明显。
附图说明
图1A例示了本发明涵盖的多种Fc融合蛋白结构示意图。每个小图上方到下方为从N端到C端，梯形表示铰链区。
图1B-1C分别例示了本发明Fc融合蛋白的SDS PAGE表达图和HPLC分析图。
图2例示了本发明Fc融合蛋白与受体IL-2Rα蛋白的结合能力检测图。
图3例示了本发明Fc融合蛋白与受体IL-2Rβ蛋白的结合能力检测图。
图4例示了本发明Fc融合蛋白与受体IFNαR2蛋白的结合能力检测图。
图5例示了本发明Fc融合蛋白与不同种属间受体IL-2Rα蛋白的结合能力检测图。
图6例示了本发明Fc融合蛋白与不同种属间受体IL-2Rβ蛋白的结合能力检测图。
图7例示了本发明Fc融合蛋白与不同种属间受体IFNαR2蛋白的结合能力检测图。
图8A和图8B分别例示了通过ELISA和Fortebio检测本发明Fc融合蛋白与FcRn蛋白的结合能力的结果图。
图9例示了本发明Fc融合蛋白刺激PBMC特定细胞亚群分布检测图。
图10例示了本发明Fc融合蛋白对CTLL-2小鼠T淋巴细胞刺激增殖能力检测。
图11A至图11D例示了本发明Fc融合蛋白对肿瘤细胞(A：NCI-N87；B：MDA-MB-231；C：A375；D：Burkitt's淋巴瘤细胞)的体外直接杀伤能力检测。
图12A至图12E例示了本发明Fc融合蛋白对肿瘤细胞(A：NCI-N87；B：MDA-MB-231；C：A375；D和E：Burkitt's淋巴瘤细胞)的体外综合杀伤能力检测。
图13例示了本发明Fc融合蛋白在ExVivo类器官系统中的药效活性。
发明详述
除非另有定义，否则本文使用的所有技术和科学术语具有与本公开所属领域的普通技 术人员通常所理解的相同含义。本文引用的所有专利、专利申请和其他出版物均通过引用全文并入。如果本文中提出的定义与通过引用并入本文的专利、专利申请和其他出版物中提出的定义相冲突，则以本文中提出的定义为准。
如本文所用的,术语“IL-2”是指白细胞介素-2，旨在包括IL-2的任何形式，例如，1)天然未加工的IL-2分子、全长IL-2蛋白或天然存在的IL-2变体；2)在细胞内加工产生的IL-2的任何形式；或3)全长或经修饰形式。在本公开中，术语“IL-2”或“IL-2结构域”包括野生型IL-2和IL-2变体。
如本文所用的,术语“IFNα”是指干扰素α，旨在包括IFNα的任何形式，例如，1)天然未加工的IFNα分子、全长IFNα蛋白或天然存在的IFNα变体；2)在细胞内加工产生的IFNα的任何形式；或3)全长或经修饰形式。在本公开中，术语“IFNα”或“IFNα结构域”包括野生型IFNα和IFNα变体。
术语“变体”，就多肽或蛋白质而言，是指包括对天然蛋白质序列的一个或多个氨基酸突变的生物活性多肽。任选地，一个或多个氨基酸突变包括在氨基酸序列中某些位置的氨基酸取代和/或插入。优选地，变体与相应的天然序列多肽具有至少约80％的氨基酸序列同一性。这样的变体包括，例如，在多肽的N端和/或C端添加一个或多个氨基酸(天然存在的氨基酸和/或非天然存在的氨基酸)残基的多肽。用于本公开的变体可以通过本领域众所周知的各种方法来制备，例如对编码天然蛋白的DNA中的核苷酸进行定点诱变或噬菌体展示技术，从而产生编码变体的DNA，随后在重组细胞培养中表达该DNA。在本文公开的某些实施方案中，IL-2变体与野生型IL-2蛋白相比包括一个或多个取代。在本文公开的某些实施方案中，IFNα变体与野生型IFNα蛋白相比包括一个或多个取代。
如本文所用的术语“Fc”与关于抗体使用的含义相同，指的是抗体的以下部分，其包括第一重链的第二(CH2)和第三(CH3)恒定区通过二硫键与第二重链的第二和第三恒定区结合。Fc区也可以包括部分或全部的铰链区。抗体的Fc区负责各种效应器功能如ADCC和CDC，但不具有抗原结合功能。在本公开中，术语“Fc”包括野生型Fc和Fc变体。
如本文所用的，术语“可操作地连接”是指两个或更多个感兴趣的生物序列的并置(带有或不带有间隔区或接头或插入序列)以使得它们处于允许以意图方式起作用的关系。当就多肽使用时，是指以允许连接的产物具有意图的生物学功能的方式连接多肽序列。例如，融合蛋白可以可操作地连接于免疫球蛋白恒定区，以便提供具有配体结合活性的稳定产物。又例如，融合蛋白可以与免疫球蛋白恒定区通过其间的插入序列可操作地连接，并且该类插入序列可以是间隔区或可以包含更长的序列。
如本文所用的，当就氨基酸序列(例如肽、多肽或蛋白质)使用时，术语“融合”或“融合的”是指两个或更多个氨基酸序列的组合，例如通过化学键合或重组的方式，形成天然不存在的单个氨基酸序列。融合氨基酸序列可以通过两个编码多核苷酸序列的遗传重组产生，并且可以通过将包含重组多核苷酸的构建体引入宿主细胞的方法来表达。
本文所用的术语“融合蛋白”是指具有两个(或更多个)部分可操作地连接在一起的多肽，其中每个部分是具有不同性质的多肽。该特性可以是一种生物特性，如体外或体内的活性。该特性也可以是简单的化学或物理特性，如与靶抗原的结合、对反应的催化作用等。两部分可以通过单一肽键直接连接，或通过含有一个或多个氨基酸残基的肽接头连接。一般来说，这两个部分和接头将彼此处于读码框内。在某些实施方案中，融合蛋白是IL-2部分、Fc部分和IFNα部分的融合蛋白。在某些实施方案中，融合蛋白是IL-2部分和Fc部分的融合蛋白。在某些实施方案中，融合蛋白是Fc部分和IFNα部分的融合蛋白。
如本文所用，术语“载体”是指可以在其中插入多核苷酸的核酸媒介物。当载体允许插入其中的多核苷酸编码的蛋白质的表达时，该载体称为表达载体。载体可以通过转化、转导或转染入宿主细胞而使携带的遗传物质元件在宿主细胞中表达。载体是本领域技术人员所熟知的，包括但不限于质粒，噬菌体，粘粒，人工染色体如酵母人工染色体(YAC)，细菌人工染色体(BAC)或P1衍生人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体和动物病毒。可用作载体的动物病毒包括但不限于逆转录病毒(包括慢病毒)，腺病毒，腺相关病毒，疱疹病毒(如单纯疱疹病毒)，痘病毒，杆状病毒，乳头瘤病毒，乳多空病毒(如SV40)。载体可以包含用于控制表达的多个元件，包括但不限于启动子序列，转录起始序列，增强子序列，选择元件和报告基因。另外，载体可以包含复制起点。
如本文所用，术语“宿主细胞”是指可被工程化以产生感兴趣的蛋白质、蛋白质片段或肽的细胞系统。宿主细胞包括但不限于培养的细胞，例如来源于啮齿动物(大鼠，小鼠，豚鼠或仓鼠)的哺乳动物培养细胞，如CHO，BHK，NSO，SP2/0，YB2/0；或人组织或杂交瘤细胞，酵母细胞和昆虫细胞，以及包含在转基因动物或培养组织内的细胞。该术语不仅涵盖特定的受试细胞，还涵盖这种细胞的后代。由于突变或环境影响可能在后代中发生某些修饰，因此这样的后代可能与亲本细胞不同，但仍包括在术语“宿主细胞”的范围内。
如本文所用，术语“同一性”是指通过比对和比较序列确定的两个或更多个多肽分子或两个或更多个核酸分子的序列之间的关系。“百分比同一性”是指比较分子中氨基酸或核苷酸之间相同残基的百分比，并基于被比较的最小分子的大小计算。对于这些计算，比对中的间隙(如果有的话)优选通过特定的数学模型或计算机程序(即“算法”)来寻址。可用于计算比对的核酸或多肽的同一性的方法包括在Computational Molecular Biology,(Lesk,A.M.编),1988,New York:Oxford University Press；Biocomputing Informatics and Genome Projects,(Smith,D.W.编),1993,New York:Academic Press；Computer Analysis of Sequence Data,第I部分,(Griffin,A.M.和Griffin,H.G.编),1994,New Jersey:Humana Press；von Heinje,G.,1987,Sequence Analysis in Molecular Biology,New York:Academic Press；Sequence Analysis Primer,(Gribskov,M.和Devereux,J.编),1991,New York:M.Stockton Press；和Carillo等人,1988,SIAMJ.Applied Math.48:1073中描述的那些。
术语“受试者”包括任何人或非人动物，优选人。
如本文所用，术语“癌症”是指引发医学病况的任何肿瘤或恶性细胞生长，可以是增殖或转移介导的实体瘤和非实体瘤。
本文在治疗病况的情况中使用的术语“治疗”和“医治”一般涉及人或动物的治疗和疗法，其中实现了一些期望的治疗效果，例如，抑制病情进展，包括进展速度下降，进展速度停滞，病情消退，病情改善和病情治愈。还包括了作为预防措施(即预防、防护)的治疗。对于癌症，“治疗”可能是指抑制或减缓肿瘤或恶性细胞生长、增殖或转移或其某种组合。对于肿瘤，“治疗”包括去除全部或部分肿瘤、抑制或减缓肿瘤生长和转移、预防或延迟肿瘤的发展或其某种组合。
如本文所用，术语“有效量”涉及活性化合物的量或包含活性化合物的材料、组合物或剂量的量，其在按照所需的治疗方案施用时有效用于产生与合理的益处/风险比相称的某些所需的治疗效果。
IL-2变体和IFNα变体
在某些方面，本公开提供了IL-2变体，其与野生型IL-2蛋白例如人类野生型IL-2蛋白相比包括一个或多个修饰，例如插入、缺失和/或取代。本公开进一步提供了融合蛋白，包括IL-2蛋白部分与Fc部分融合。IL2蛋白部分可包括野生型IL-2蛋白，或包括与野生型IL-2蛋白相比包含一个或多个修饰(如插入和/或取代)的IL-2变体。在一些实施方案中，相比于野生型IL-2蛋白，IL-2变体包含选自但不限于以下的一种或多种取代：R38A、L80F、R81D、L85V、I86V和I91F。例如，相比于野生型IL-2蛋白，IL-2变体中的第38位氨基酸被修饰。在一些实施方案中，IL-2变体还包含相比于野生型IL-2蛋白的保守性取代。在一些实施方案中，IL-2变体包含或组成为SEQ ID NO:2所示的氨基酸序列。在一些实施方案中，IL-2变体包含或组成为与SEQ ID NO:2至少85％、至少90％、至少95％或至少99％(例如至少91％、92％、93％、94％、95％、96％、97％、98％、99％)相同的氨基酸序列。
在某些方面，本公开提供了IFNα变体，其与野生型IFNα蛋白例如人类野生型IFNα蛋白相比包括一个或多个修饰，例如插入、缺失和/或取代。本公开进一步提供了融合蛋白，包括IFNα蛋白部分与Fc部分融合。IFNα蛋白部分可包括野生型IFNα蛋白，或包括与野生型IFNα蛋白相比包含一个或多个修饰(如插入和/或取代)的IFNα变体。在一些实施方案中，相比于野生型IFNα蛋白，IFNα变体中的第R144和/或R149位氨基酸被修饰。在一些实施方案中，IFNα变体还包含相比于野生型IFNα蛋白的保守性取代。在一些实施方案中，IFNα变体包含或组成为SEQ ID NO:6所示的氨基酸序列。在一些实施方案中，IFNα变体包含或组成为与SEQ ID NO:6至少85％、至少90％、至少95或至少99％(例如至少91％、92％、93％、94％、95％、96％、97％、98％、99％)相同的氨基酸序列。
本发明的融合蛋白
在某些方面，本公开提供了包括IL-2部分和Fc部分的融合蛋白，包括IFNα部分和Fc部分的融合蛋白，以及包括IL-2部分、Fc部分和IFNα部分的融合蛋白。所述融合蛋白可以是单链或多条链的。在一些实施方案中，所述融合蛋白为双链同二聚体或异二聚体。每条链中的各IL-2部分、Fc部分和IFNα部分可以相同或不同。
在一些实施方案中，本发明的融合蛋白从N末端到C末端包含IL-2部分可操作地连接于Fc部分(又称为IL-2-Fc融合蛋白)。
在一些实施方案中，本发明的融合蛋白从N末端到C末端包含Fc部分可操作地连接于IL-2部分(又称为Fc-IL-2融合蛋白)。
在一些实施方案中，本发明的融合蛋白从N末端到C末端包含IFNα部分可操作地连接于Fc部分(又称为IFNα-Fc融合蛋白)。
在一些实施方案中，本发明的融合蛋白从N末端到C末端包含Fc部分可操作地连接于IFNα部分(又称为Fc-IFNα融合蛋白)。
在一些实施方案中，本发明的融合蛋白从N末端到C末端包含IL-2部分可操作地连接于Fc部分且该Fc部分可操作地连接于IFNα部分(又称为IL-2-Fc-IFNα融合蛋白)。
在一些实施方案中，本发明的融合蛋白从N末端到C末端包含IFNα部分可操作地连接于Fc部分且该Fc部分可操作地连接于IL-2部分(又称为IFNα-Fc-IL-2融合蛋白)。
所述融合蛋白中包含的IL-2部分可包含野生型IL-2蛋白或IL-2变体。所述融合蛋白中包含的IFNα部分可包含野生型IFNα蛋白或IFNα变体。所述融合蛋白中包含的Fc部分可包含野生型Fc或Fc变体。
在一些实施方案中，融合蛋白中包含的IL-2部分中的第38位氨基酸被修饰。已发现第38位氨基酸附近的区域对IL-2引起血管通透性增加起关键作用，因此对第38位氨基酸的改造可显著降低血管通透性。
在一些实施方案中，融合蛋白中包含经过修饰的干扰素α部分，与天然干扰素α相比其与干扰素α受体的亲和力显著降低。这种降低可引起干扰素α引起的毒副作用显著降低，同时保留其抗肿瘤的活性。在一些实施方案中，融合蛋白中包含的干扰素α部分中的R144或R149氨基酸被修饰。这些氨基酸对干扰素α与其受体结合起关键作用，突变后可引起亲和力显著降低，而不影响其抗肿瘤活性。
所述可操作地连接(或者表示为“-”)可以是两部分直接连接，或者通过接头例如肽接头进行连接。在一些实施方案中，IL-2部分通过肽接头可操作地连接于Fc部分。在一些实施方案中，IFNα部分通过肽接头可操作地连接于Fc部分。所述肽接头可以是本领域中常规使用的任何肽接头，例如GS系列接头，如SEQ ID NO:11所示。
本发明提供的融合蛋白可以提高治疗效果，降低毒副作用，提高治疗安全窗。
包含Fc区的融合蛋白
在一些实施方案中，本发明提供的融合蛋白还包含免疫球蛋白恒定结构域序列，例如人IgG的恒定结构域序列，更具体地可包含铰链区和Fc区，例如IgG1、IgG2、IgG3、IgG4的Fc区序列。如本领域已知的，Fc是指由抗体的第一重链的第二和第三恒定区经由二硫键合结合至第二重链的第二和第三恒定区组成的抗体的部分，任选地Fc区还包含全部或部分的铰链区。本文中的Fc区既包括野生型Fc区还包括其变体，具有用于多种目的的不同突变。所述变体可在Fc区中包含一个或多个氨基酸残基修饰，例如取代。
在某些实施方案中，Fc区变体包含一个或多个氨基酸取代，其改善对新生儿Fc受体(FcRn)的pH依赖性结合。这样的变体可以具有延长的药代动力学半衰期，因为它在酸性pH结合FcRn从而使其能够逃离溶酶体中的降解并然后被转运并释放出细胞。工程化抗体分子以改善与FcRn的结合亲和力的方法在本领域中是众所周知的，参见，例如，Vaughn，D.等人，Structure，6(1):63-73，1998；Kontermann，R.等人，Antibody Engineering，Volume 1,Chapter 27:Engineering of the Fc region for improved PK,Springer,2010；Yeung,Y.等人,Cancer Research,70:3269-3277(2010)；和Hinton,P.等人,J.Immunology,176:346-356(2006)。
在某些实施方案中，本发明提供的融合蛋白包含L234A/L235A取代，以降低与受体FcgRIIIa的结合能力。在某些实施方案中，本发明提供的融合蛋白在Fc区的界面中包含一个或多个氨基酸取代，以协助和/或促进异二聚化。这些修饰包括将突起引入第一Fc多肽中和将腔引入第二Fc多肽中，其中突起可以位于腔中以促进第一和第二Fc多肽的相互作用而形成异二聚体或复合物。生成具有这些修饰的蛋白分子的方法在本领域中是已知的，例如，如在美国专利号5,731,168中所述。
在某些实施方案中，Fc结构域还包括三重突变M252Y/S254T/T256E(“YTE”)。据报道，这种三重突变导致与人新生儿Fc受体(FcRn)的结合增加了约10倍，并且含有YTE的人IgG在食蟹猴中的血清半衰期增加了近4倍(Oganesyan V.等人,Mol Immunol.2009年5月；46(8-9)：1750-5)。在一些进一步的实施方案中，Fc结构域包括另外的突变以增强Fc和人FcRn之间的相互作用。
在一些实施方案中，Fc变体包含或组成为与SEQ ID NO:12至少85％、至少90％或至少99％相同的氨基酸序列。
本发明的融合蛋白的特点
1.通过与Fc融合，延长药物的半衰期，减少细胞因子的用量，降低毒副作用。
2.通过对IL-2的改构，提高细胞因子对NK细胞和CD8+T细胞表面IL-2Rβ和IL-2Rγ的亲和力，增强这些细胞对肿瘤的杀伤作用。
3.对IL-2第38位氨基酸的改造可显著降低IL-2引起的血管通透性增加的毒副作用。
4.对R144和R149的改造可降低其与受体IFNαR2的结合能力，降低毒副作用，同时 保留其抗肿瘤活性。
5.综合以上特点，改构后的IL-2与IFNα和Fc融合蛋白可以克服目前IL-2与IFNα药物的缺点，改善治疗效果，降低毒副作用，提高治疗安全窗。
多核苷酸、载体和宿主细胞
在一些方面，本发明还提供编码此类融合蛋白的多核苷酸。所述多核苷酸可用于表达融合蛋白。在一些实施方案中，所述多核苷酸还可以用作用于实现多肽融合蛋白的体内表达的呈活性形式的治疗剂。
编码所述融合蛋白的多核苷酸易于使用本领域已知的常规程序进行分离和测序。所述多核苷酸也可以通过合成方法获得。优选地，所述多核苷酸经过密码子优化以便在真核宿主细胞，特别是哺乳动物细胞中表达。
使用已知的重组技术，可以将编码融合蛋白的多核苷酸插入载体中以进一步克隆(DNA的扩增)或表达。载体组分通常包括但不限于以下一种或多种：信号序列、复制起点、一个或多个标志物基因、增强子元件、启动子(例如SV40、CMV、EF-1α)以及转录终止序列。
在一些实施方案中，本公开提供了包含本文提供的编码融合蛋白的多核苷酸的载体(例如表达载体)，与所述多核苷酸可操作地连接的至少一个启动子(例如SV40、CMV、EF-1α)，和至少一个选择标志物。载体的实例包括但不限于逆转录病毒(包括慢病毒)，腺病毒，腺伴随病毒，疱疹病毒(例如单纯疱疹病毒)，痘病毒，杆状病毒，乳头瘤病毒，乳多空病毒(例如SV40)，λ噬菌体，和M13噬菌体，脂质体，质粒pcDNA3.3，pMD18-T，pOptivec，pCMV，pEGFP，pIRES，pQD-Hyg-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等。
可以将包含编码融合蛋白的多核苷酸序列的载体导入宿主细胞用于进行克隆或基因表达。用于在本文的载体中克隆或表达DNA的合适宿主细胞是原核生物、酵母或更高等的真核细胞。为此目的合适的原核生物包括真细菌，例如革兰氏阴性或革兰氏阳性细菌，如大肠杆菌。除了原核生物外，真核微生物如丝状真菌或酵母是用于提供的载体的合适克隆或表达宿主。酿酒酵母或普通的面包酵母是低等真核宿主微生物中最常用的。然而，许多其他属、种和菌株在本文中是普遍可获得的和有用的。
用于表达本文提供的融合蛋白的合适宿主细胞也可以衍生自多细胞生物。无脊椎动物细胞的实例包括植物和昆虫细胞。在一些优选的实施方案中，宿主细胞是中国仓鼠卵巢(CHO)细胞。在一些另外的优选的实施方案中，宿主细胞是其他哺乳动物细胞系，例如人 细胞系。
用上述表达或克隆载体转化宿主细胞用于产生融合蛋白并在经适当修饰的常规营养培养基中培养用于诱导启动子，选择转化子或扩增编码所需序列的基因。
用于产生本文提供的融合蛋白的宿主细胞可以在多种培养基中培养。这些培养基中的任何一种可以根据需要补充激素和/或其他生长因子(例如胰岛素、转铁蛋白或表皮生长因子)，盐(例如氯化钠、钙、镁和磷酸盐)，缓冲液(例如HEPES)，核苷酸(例如腺苷和胸苷)，抗生素(例如GENTAMYCINTM药物)，微量元素(定义为通常以微摩尔范围的最终浓度存在的无机化合物)，和葡萄糖或等效能量来源。也可以以本领域技术人员已知的适当浓度包括任何其他必要的补充剂。培养条件，例如温度、pH等是先前与选择用于表达的宿主细胞一起使用的那些条件，并且对于普通技术人员而言是显而易见的。
由细胞制备的融合蛋白可以使用例如羟磷灰石色谱、凝胶电泳、透析、DEAE-纤维素离子交换色谱、硫酸铵沉淀、盐析和亲和色谱来纯化，其中亲和色谱是优选的纯化技术。
药物组合物
在一些方面，本发明提供包含与药学上可接受的载体一起配制的本发明的融合蛋白的组合物，例如药物组合物。这类组合物包含至少一种本发明的融合蛋白或编码该融合蛋白的多核苷酸。
如本文所使用的，“药学上可接受的载体”包括生理上相容的任何和所有药学上可接受的液体、凝胶或固体载体、水性媒介物、非水媒介物、抗微生物剂、等渗剂、缓冲剂、抗氧化剂、麻醉剂、悬浮液/分散剂、隔绝剂或螯合剂、稀释剂、佐剂、赋形剂或无毒的辅助物质，本领域已知的其他组分或其各种组合等等。所述载体可以适于静脉内、肌内、皮下、胃肠外、脊髓、经表皮、玻璃体内注射或植入施用等。取决于给药途径，活性化合物，即本发明的成分，可以包被在材料中以防止所述化合物免受酸和其它可能灭活所述化合物的自然条件的作用。
本发明的药物组合物还可包括药学上可接受的抗氧化剂。可用于本发明的药物组合物中的适当的水性载体和非水性载体的实例包括水、乙醇、多元醇(例如甘油、丙二醇、聚乙二醇等等)和其适当的混合物、植物油如橄榄油及可注射的有机酯如油酸乙酯。例如，可以通过利用包衣材料(如卵磷脂)、在分散剂的情况下通过维持所需要的颗粒大小和通过利用表面活性剂维持适当的流动性。
这些组合物还可包含辅助剂如防腐剂、润湿剂、乳化剂和分散剂。药物组合物可以是固体、糊剂、软膏、凝胶、液体、气雾剂、喷雾剂、聚合物、薄膜、乳液或混悬液形式。
在某些实施方案中，药物组合物配制成可注射的组合物。可以以任何常规形式制备可注射药物组合物，例如液体溶液，悬浮剂，乳剂或适于产生液体溶液、悬浮剂或乳剂的固体形式。用于注射的制备物可以包括准备注射的无菌和/或非热解溶液，无菌干燥可溶产品， 如冻干粉，准备在使用前与溶剂混合，包括皮下注射片剂，准备注射的无菌悬浮液，准备在使用前与媒介物组合的无菌干燥的不溶性产品，以及无菌和/或非热解乳液。溶液可以是水性或非水性的。药学上可接受的载体包括无菌水溶液或分散液和用于临时制备无菌注射溶液或分散液的无菌粉末。
活性成分和小分子在本发明的药物组合物中的实际剂量水平可以有所变化，以便获得对特定患者、组成和施用模式有效实现期望治疗响应而对所述患者无毒性的活性成分的量。所选择的剂量水平取决于各种药代动力学因素，包括所使用的本发明的特定组合、或者其酯、盐或酰胺的活性、给药途径、施用时间、使用的特定化合物的排泄速率、治疗持续时间、与所使用的特定组合联合使用的其它药物、化合物和/或材料、待治疗患者的年龄、性别、体重、病症、总体健康状况和在先病史、以及医学领域内熟知的类似因素。
本公开的应用
本公开的融合蛋白、药物组合物和方法具有许多体外和体内用途，包括例如免疫应答的增强。例如，可以将这些分子体外或离体施用于培养的细胞，或例如体内施用于人受试者，以增强各种情况下的免疫力。免疫应答可以被调节，例如被增强、刺激或上调。
例如，受试者包括需要调节免疫应答的人类患者。在一个具体的实施方案中，所述方法特别适合于体内治疗癌症。为了实现免疫力的增强，可以将融合蛋白单独地或与另一疗法组合施用。当融合蛋白与另一种药剂一起施用时，两者可以以任何顺序施用或同时施用。
在一些方面，本公开提供了治疗哺乳动物中病症或疾病的方法，其包括向需要治疗的受试者(例如人)施用治疗有效量的如本文公开的融合蛋白。所述病症或疾病可以是癌症，例如IL-2和IFNα可治疗的癌症。可以使用本公开提供的方法治疗或预防多种癌症，无论是恶性的还是良性的，以及是原发性的还是继发性的。癌症可以是实体瘤或血液恶性癌。
本文公开的融合蛋白可以作为单一疗法单独使用，或者可以与细胞免疫疗法、靶向疗法、化疗或放疗等组合使用。
本发明涉及的序列汇总
SEQ ID NO:1(野生型IL-2氨基酸序列)
SEQ ID NO:2(IL-2衍生物氨基酸序列)
SEQ ID NO:3(IL-2-Fc融合蛋白氨基酸序列)
SEQ ID NO:4(Fc-IL-2融合蛋白氨基酸序列)
SEQ ID NO:5(野生型IFNα氨基酸序列)
SEQ ID NO:6(IFNα衍生物氨基酸序列)
SEQ ID NO:7(IFNα-Fc融合蛋白氨基酸序列)
SEQ ID NO:8(Fc-IFNα融合蛋白氨基酸序列)
SEQ ID NO:9(IL-2-Fc-IFNα融合蛋白氨基酸序列)
SEQ ID NO:10(IFNα-Fc-IL-2融合蛋白氨基酸序列)
SEQ ID NO:11(柔性连接子氨基酸序列)
SEQ ID NO:12(Fc部分氨基酸序列)
下面结合附图和具体实施方式，进一步阐明本发明，应理解下述具体实施方式仅用于说明本发明而不用于限制本发明的范围。
实施例
实施例1：融合蛋白的分子构建与生产
构建了一种IL-2-Fc-IFNα融合蛋白，从N末端到C末端分别包含IL-2部分可操作地连接于免疫球蛋白Fc部分且该免疫球蛋白Fc部分可操作地连接于IFNα部分，该融合蛋白在下文中称为IL-2-Fc-IFNα，其氨基酸序列如SEQ ID NO:9所示。还构建了IL-2-Fc融合蛋白，从N末端到C末端分别包含IL-2部分可操作地连接于免疫球蛋白Fc部分， 该融合蛋白在下文中称为IL-2-Fc，其氨基酸序列如SEQ ID NO:3所示。还构建了Fc-IFNα融合蛋白，从N末端到C末端分别包含免疫球蛋白Fc部分可操作地连接于IFNα部分，该融合蛋白在下文中称为Fc-IFNα，其氨基酸序列如SEQ ID NO:8所示。
通过常规的分子生物学技术PCR扩增抗体轻重链基因，并通过同源重组技术将编码氨基酸序列的基因连接到pcDNA3.4载体上。将测序后的阳性克隆提取质粒后转染到Expi293F细胞，在37℃/5％CO2/125rpm摇床中继续培养7天，培养结束后收集上清经proteinA亲和层析，获得纯化后的融合蛋白分子，并通过UV280结合理论消光系数确定抗体浓度。
图1B展示了IL-2-Fc-IFNα融合蛋白的SDS PAGE胶图，图1C显示了其HPLC纯度的分析图。
实施例2：ELISA检测与受体IL-2Rα蛋白的结合能力
包被IL-2Rα蛋白(10165-H08H，义翘神州)，包被浓度为2μg/ml，100ul/孔4℃过夜；用300ul/孔3％脱脂奶粉37℃封闭1h；每孔加入不同浓度融合蛋白及其对照样品各100ul，37℃孵育1h；然后加入Anti-IFNα2 Rabbit pAb，37℃孵育1h，再加入Anti-Rabbit IgG HRP 37℃孵育1h；每孔加入100ul TMB显色5min后加入50ul终止液，酶标仪上读取OD450。
结果见图2。与野生型IL-2(11848-HNAH1-E，义翘神州)相比，IL-2-Fc及IL-2-Fc-IFNα与受体IL-2Rα蛋白的结合能力明显提高。
实施例3：ELISA检测与受体IL-2Rβ蛋白的结合能力
包被IL-2Rβ蛋白(10696-H05H，义翘神州)，包被浓度为2μg/ml，100ul/孔4℃过夜；用300ul/孔3％脱脂奶粉37℃封闭1h；每孔加入不同浓度融合蛋白及其对照样品各100ul，37℃孵育1h；然后加入Anti-IFNα2 Rabbit pAb，37℃孵育1h，再加入Anti-Rabbit IgG HRP 37℃孵育1h；每孔加入100ul TMB显色5min后加入50ul终止液，酶标仪上读取OD450。
结果见图3。与野生型IL-2相比，IL-2-Fc及IL-2-Fc-IFNα与受体IL-2Rβ蛋白的结合能力有超24倍的提高。
实施例4：ELISA检测与受体IFNαR2蛋白的结合能力
包被IFNαR2蛋白(10359-H02H，义翘神州)，包被浓度为2μg/ml，100ul/孔4℃过夜；用300ul/孔3％脱脂奶粉37℃封闭1h；每孔加入不同浓度融合蛋白及其对照样品各100ul，37℃孵育1h；然后加入Anti-IL-2 Rabbit pAb，37℃孵育1h，再加入Anti-Rabbit IgG HRP 37℃孵育1h；每孔加入100ul TMB显色5min后加入50ul终止液，酶标仪上读取OD450。
结果见图4。与野生型IFNα相比，Fc-IFNα与受体IFNαR2蛋白的结合能力明显降低 而IL-2-Fc-IFNα与受体IFNαR2蛋白的结合能力明显提高。
实施例5：ELISA检测与不同种属间受体IL-2Rα蛋白的结合能力
分别包被人、小鼠和食蟹猴IL-2Rα蛋白，包被浓度为2μg/ml，100ul/孔4℃过夜；用300ul/孔3％脱脂奶粉37℃封闭1h；每孔加入不同浓度融合蛋白及其对照样品各100ul，37℃孵育1h；然后加入Anti-IFNα2 Rabbit pAb，37℃孵育1h，再加入Anti-Rabbit IgG HRP 37℃孵育1h；每孔加入100ul TMB显色5min后加入50ul终止液，酶标仪上读取OD450。
结果见图5。实验证实IL-2-Fc-IFNα与人、小鼠、食蟹猴受体IL-2Rα蛋白的结合能力相似，因此与小鼠及食蟹猴有种属交叉。
实施例6：ELISA检测与不同种属间受体IL-2Rβ蛋白的结合能力
分别包被人、小鼠和食蟹猴IL-2Rβ蛋白，包被浓度为2μg/ml，100ul/孔4℃过夜；用300ul/孔3％脱脂奶粉37℃封闭1h；每孔加入不同浓度融合蛋白及其对照样品各100ul，37℃孵育1h；然后加入Anti-IFNα2 Rabbit pAb，37℃孵育1h，再加入Anti-Rabbit IgG HRP 37℃孵育1h；每孔加入100ul TMB显色5min后加入50ul终止液，酶标仪上读取OD450。
结果见图6。实验证实IL-2-Fc-IFNα与人、小鼠、食蟹猴受体IL-2Rβ蛋白均有种属交叉。其中与食蟹猴的IL-2Rβ蛋白结合能力和人的相似。与小鼠的IL-2Rβ蛋白结合低于和人的结合。
实施例7：ELISA检测与不同种属间受体IFNαR2蛋白的结合能力
分别包被人、小鼠和食蟹猴IFNαR2蛋白，包被浓度为2μg/ml，100ul/孔4℃过夜；用300ul/孔3％脱脂奶粉37℃封闭1h；每孔加入不同浓度融合蛋白及其对照样品各100ul，37℃孵育1h；然后加入Anti-IL-2 Rabbit pAb，37℃孵育1h，再加入Anti-Rabbit IgG HRP 37℃孵育1h；每孔加入100ul TMB显色5min后加入50ul终止液，酶标仪上读取OD450。
结果见图7。实验证实IL-2-Fc-IFNα与人、小鼠、食蟹猴受体IFNαR2蛋白均有结合，展示了种属交叉。
实施例8：检测与FcRn蛋白的结合能力
8.1ELISA检测与FcRn蛋白的结合能力
包被FcRn蛋白(10359-H02H，义翘神州)，包被浓度为2μg/ml，100ul/孔4℃过夜；用300ul/孔3％脱脂奶粉37℃封闭1h；每孔加入不同浓度融合蛋白及其对照样品各100ul，37℃孵育1h；然后加入Anti-IFNα2 Rabbit pAb，37℃孵育1h，再加入Anti-Rabbit IgG  HRP 37℃孵育1h；每孔加入100ul TMB显色5min后加入50ul终止液，，酶标仪上读取OD450。
结果见图8A。实验证实和Herceptin抗体(赫赛汀)相比，IL-2-Fc-IFNα与FcRn蛋白有显著增高的结合力
8.2 Fortebio检测与FcRn蛋白的结合能力
用ProA、HIS探针Load待测蛋白(浓度为4μg/ml)，然后与不同浓度FcRn(4ug/ml、1ug/ml、0.25ug/ml和0)蛋白结合、解离，计算待测分子与其的亲和力。
结果见图8B。实验证实和Herceptin抗体(赫赛汀)相比，IL-2-Fc-IFNα与FcRn蛋白有显著增高的亲和力。
实施例9：对人PBMC细胞刺激后免疫细胞亚群检测
按150000个细胞/孔将人新鲜PBMC铺板至96孔板中，按照试验设计加入500nM的蛋白，继续培养72h；孵育结束后，按流式检测要求染色分别标记Anti-Foxp3抗体(FITC)、Anti-CD4抗体(PE)、Anti-CD56抗体(PE)、Anti-CD8抗体(PE)和Anti-Granzyme B抗体(FITC)，采集并分析流式数据，进行数据汇总并分析。
结果见图9。实验证实IL-2-Fc-IFNα刺激PBMC中NK和CD8阳性T细胞激活、表达Granzyme B的能力与野生型IL-2保持相同活性；刺激Treg细胞(FOXP3+细胞)的增殖能力比野生型IL-2明显降低。
实施例10：对CTLL-2小鼠T淋巴细胞刺激增殖能力检测
培养扩增小鼠CTLL-2细胞至所需细胞量，按100000个细胞/孔将小鼠CTLL-2细胞铺板至96孔板中，按照试验设计加入不同浓度(100nM、4nM、800pM、400pM、200pM、100pM、50pM、25pM、0)蛋白，继续培养72h；采用CCK-8法测定活细胞Formazan表达量，进而评价细胞活力。具体操作按照说明书操作,培养结束后加入10％CCK-8溶液，混匀后，共孵育2-4小时，在酶标仪中采集吸光值数据。使用Excel软件分析处理数据，使用GraphPad Prism 7软件，根据吸光值数据拟合药效剂量曲线，计算IC50。
结果见图10。实验证实IL-2-Fc-IFNα刺激CTLL-2细胞增殖的能力比野生型IL-2高10倍。
实施例11：对肿瘤细胞的体外直接杀伤能力检测
直接杀伤能力是在没有免疫细胞的存在下，测定IFN-a对肿瘤细胞的直接杀伤。
11.1对人胃癌细胞株NCI-N87的直接杀伤能力检测
培养扩增人胃癌细胞NCI-N87至所需细胞量，按4000个细胞/孔将NCI-N87细胞铺板至96孔板中，按照试验设计加入不同浓度(4000nM、1000nM、250nM、100nM、62.5nM、 15.625nM、10nM、4nM、1.5625nM、1nM、244.14pM、156.25pM、100pM、100pM、10pM、1pM、0)蛋白，继续培养5天；采用CCK-8法测定活细胞Formazan表达量，进而评价细胞活力。具体操作按照说明书操作,培养结束后加入10％CCK-8溶液，混匀后，共孵育2-4小时，在酶标仪中采集吸光值数据。使用Excel软件分析处理数据，使用GraphPad Prism 7软件，根据吸光值数据拟合药效剂量曲线，计算IC50。
结果见图11A。野生型IFNα对NCI-N87细胞有显著杀伤功能；而IL-2-Fc-IFNα只有在高浓度下对NCI-N87细胞有杀伤作用。阴性对照TTI-622(SIRPa-Fc)对NCI-87细胞没有杀伤功能。
11.2对人乳腺癌细胞株MDA-MB-231的直接杀伤能力检测
培养扩增人乳腺癌细胞MDA-MB-231至所需细胞量，按2000个细胞/孔将MDA-MB-231细胞铺板至96孔板中，按照试验设计加入不同浓度(4000nM、1000nM、250nM、62.5nM、15.625nM、4nM、1nM、244.14pM、61pM、15.26pM、0)蛋白，继续培养5天；采用CCK-8法测定活细胞Formazan表达量，进而评价细胞活力。具体操作按照说明书操作,培养结束后加入10％CCK-8溶液，混匀后，共孵育2-4小时，在酶标仪中采集吸光值数据。使用Excel软件分析处理数据，使用GraphPad Prism 7软件，根据吸光值数据拟合药效剂量曲线，计算IC50。
结果见图11B。野生型IFNα对MDA-MB-231细胞有显著杀伤功能；而IL-2-Fc-IFNα只有在高浓度下对MDA-MB-231细胞有杀伤作用。阴性对照TTI-622(SIRPa-Fc)对MDA-MB-231细胞没有杀伤功能。
11.3对人黑色素瘤细胞株A375的直接杀伤能力检测
培养扩增人乳腺癌细胞A375至所需细胞量，按1000个细胞/孔将A375细胞铺板至96孔板中，按照试验设计加入不同浓度(16000nM、1600nM、160nM、16nM、1.6nM、160pM、16pM、1.6pM、0.16pM、0)蛋白，继续培养5天；采用CCK-8法测定活细胞Formazan表达量，进而评价细胞活力。具体操作按照说明书操作,培养结束后加入10％CCK-8溶液，混匀后，共孵育2-4小时，在酶标仪中采集吸光值数据。使用Excel软件分析处理数据，使用GraphPad Prism 7软件，根据吸光值数据拟合药效剂量曲线，计算IC50。
结果见图11C。野生型IFNα对A375细胞有显著杀伤功能；而IL-2-Fc-IFNα只有在高浓度下对A375细胞有杀伤作用。阴性对照TTI-622(SIRPa-Fc)对A375细胞没有杀伤功能。
11.4对人Burkitt's淋巴瘤细胞株Daudi B细胞的直接杀伤能力检测
培养扩增人Burkitt's淋巴瘤细胞Daudi B细胞至所需细胞量，按10000个细胞/孔将Daudi B细胞铺板至96孔板中，按照试验设计加入不同浓度(62.5nM、12.5nM、2.5nM、500pM、250pM、125pM、62.5pM、31.25pM、15.625pM、7.813pM、4pM、2pM、0)蛋白，继续培养5天；采用CCK-8法测定活细胞Formazan表达量，进而评价细胞活力。具体操作按 照说明书操作,培养结束后加入10％CCK-8溶液，混匀后，共孵育2-4小时，在酶标仪中采集吸光值数据。使用Excel软件分析处理数据，使用GraphPad Prism 7软件，根据吸光值数据拟合药效剂量曲线，计算IC50。
结果见图11D。野生型IFNα对Daudi B细胞有显著杀伤功能；而IL-2-Fc-IFNα只有在较高浓度下对Daudi B细胞有杀伤作用。阴性对照TTI-622(SIRPa-Fc)对Daudi B细胞没有杀伤功能。
实验证实本发明的融合蛋白的直接杀伤功能相较于野生型IFNα2较弱，但仍保留IFNα臂直接杀伤功能。
实施例12：对肿瘤细胞的体外综合杀伤能力检测
综合杀伤功能是直接杀伤能力和间接杀伤能力的总和。综合杀伤能力在免疫细胞PBMC的存在下获得。
12.1对人胃癌细胞株NCI-N87的综合杀伤能力检测
培养扩增人胃癌细胞NCI-N87至所需细胞量，按4000个细胞/孔将NCI-N87细胞铺板至96孔板中，待6小时细胞贴壁；采购新鲜的人PBMC，按照效靶比10：1进行铺板，按照试验设计加入不同浓度(800nM、80nM、8nM、800pM、80pM、8pM、0.8pM、0)蛋白，继续培养5天；PBS三次洗板，移除PBMC，采用CCK-8法测定活细胞Formazan表达量，进而评价细胞活力。具体操作按照说明书操作,培养结束后加入10％CCK-8溶液，混匀后，共孵育2-4小时，在酶标仪中采集吸光值数据。使用Excel软件分析处理数据，使用GraphPad Prism 7软件，根据吸光值数据拟合药效剂量曲线，计算IC50。
结果见图12A。IL-2-Fc-IFNα对NCI-N87细胞的综合杀伤能力强于野生型IL-2。
12.2对人乳腺癌细胞株MDA-MB-231的综合杀伤能力检测
培养扩增人乳腺癌细胞MDA-MB-231至所需细胞量，按4000个细胞/孔将MDA-MB-231细胞铺板至96孔板中，待6小时细胞贴壁；采购新鲜的人PBMC，按照效靶比10：1进行铺板，按照试验设计加入不同浓度(800nM、80nM、8nM、800pM、80pM、8pM、0.8pM、0)蛋白，继续培养5天；PBS三次洗板，移除PBMC，采用CCK-8法测定活细胞Formazan表达量，进而评价细胞活力。具体操作按照说明书操作,培养结束后加入10％CCK-8溶液，混匀后，共孵育2-4小时，在酶标仪中采集吸光值数据。使用Excel软件分析处理数据，使用GraphPad Prism 7软件，根据吸光值数据拟合药效剂量曲线，计算IC50。
结果见图12B。IL-2-Fc-IFN-a对MDA-MB-231细胞的综合杀伤能力和野生型IL-2相似。
12.3对人黑色素瘤细胞株A375的综合杀伤能力检测
培养扩增人黑色素瘤细胞A375至所需细胞量，按4000个细胞/孔将A375细胞铺板至96孔板中，待6小时细胞贴壁；采购新鲜的人PBMC，按照效靶比10：1进行铺板，按照试 验设计加入不同浓度(800nM、80nM、8nM、800pM、80pM、8pM、0.8pM、0)蛋白，继续培养5天；PBS三次洗板，移除PBMC，采用CCK-8法测定活细胞Formazan表达量，进而评价细胞活力。具体操作按照说明书操作,培养结束后加入10％CCK-8溶液，混匀后，共孵育2-4小时，在酶标仪中采集吸光值数据。使用Excel软件分析处理数据，使用GraphPad Prism 7软件，根据吸光值数据拟合药效剂量曲线，计算IC50。
结果见图12C。IL-2-Fc-IFN-a对A375细胞的综合杀伤能力显著强于野生型IL-2。
12.4对人Burkitt's淋巴瘤细胞株Daudi B细胞的综合杀伤能力检测
培养扩增人Burkitt's淋巴瘤细胞Daudi B细胞至所需细胞量，从供体处采集分离新鲜PBMC，按10000个细胞/孔将Daudi B细胞铺板至96孔板中,设置两个复孔，PBMC(效靶比设置为10：1和5：1)及不同浓度IAMA005(16nM、160nM)按实验排版设计加入对应孔板中,共3块板，按不同检测时间分别取用，Co-Culture培养24h、48h、72h后分布留取培养上清至LDH检测保存液中，并存放于-80度冰箱。每孔细胞分别重悬后按流式检测要求染色并进行检测，采集并分析流式数据，进行数据汇总并分析。结果见图12D。
培养扩增人Burkitt's淋巴瘤细胞Daudi B细胞至所需细胞量，对Daudi B细胞进行CFSE染色，再按20000个细胞/孔将Daudi B细胞铺板至96孔板中；采购新鲜的人PBMC，按照效靶比10：1进行铺板，按照试验设计加入不同浓度(800nM、80nM、8nM、800pM、80pM、8pM、0.8pM、0)蛋白，继续培养5天；PBS三次洗板并进行低速离心，移除细胞碎片，采用荧光酶标仪法测定活细胞荧光强度，进而评价细胞活力。使用Excel软件分析处理数据，使用GraphPad Prism 7软件，根据吸光值数据拟合药效剂量曲线，计算IC50。
结果见图12E。IL-2-Fc-IFN-a对Daudi B细胞的综合杀伤能力维持与野生型IL-2相当的活性。
实施例13：ExVivo类器官系统中的药效研究
收集乳腺癌患者胸水样本(鉴定为HER2 2+阳性)，分离样本中的细胞，包括肿瘤细胞和免疫细胞。候选药物和参比药物在体外与肿瘤细胞免疫细胞复合物共孵育。处理4-5天后，收集培养基和细胞进行分析，包括肿瘤细胞、Granzyme B、IFN-γ等。
结果见图13。在该例样本的ExVivo评价系统中，IL-2-Fc-IFN-a刺激的胸水免疫细胞抗肿瘤活性明显提升，其分泌的抗肿瘤活性细胞因子Granzyme B和IFN-γ亦显著升高。
上文结合附图描述了本公开的示范实施例。然而，本领域技术人员应当理解的是，本公开不局限于所公开的具体结构。在不脱离本公开的精神和范围的情况下，能够对本公开的示范实施例进行多种变化和改变。所有这些变化和改变均包含在由本公开的权利要求所限定的保护范围内。
参考文献
[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 W H,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-2therapy[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 (28)

  1. 一种融合蛋白，其包含IL-2部分和Fc部分，其中所述IL-2部分包含与野生型IL-2蛋白相比具有一个或多个突变的氨基酸序列，所述氨基酸序列为与SEQ ID NO:2具有至少90％同一性的氨基酸序列。
  2. 权利要求1的融合蛋白，其中所述突变包括参照SEQ ID NO:1中氨基酸位置的选自以下的一种或多种取代：R38A、L80F、R81D、L85V、I86V和I91F。
  3. 权利要求1或2的融合蛋白，其中所述Fc部分包含人IgG Fc，如人IgG1 Fc、IgG2 Fc、IgG3 Fc、IgG4 Fc或其变体。
  4. 权利要求3的融合蛋白，其中所述Fc变体包含选自以下的一种或多种突变：L234A和L235A突变、以及M252Y、S254T和T256E突变。
  5. 权利要求1-4中任一项的融合蛋白，其中所述融合蛋白还包含IFNα部分。
  6. 权利要求5的融合蛋白，其中所述IFNα部分包含与SEQ ID NO:6或5具有至少90％同一性的氨基酸序列。
  7. 权利要求6的融合蛋白，其中所述IFNα部分包括参照SEQ ID NO:5中氨基酸位置的选自以下的一种或多种取代：R144A和R149A。
  8. 权利要求1-7中任一项的融合蛋白，其包含：
    (a)IL-2部分可操作地连接于Fc部分；
    (b)IL-2部分可操作地连接于Fc部分且Fc部分可操作地连接于IFNα部分；
    (c)IL-2部分可操作地连接于IFNα部分且IFNα部分可操作地连接于Fc部分；或
    (d)IFNα部分可操作地连接于IL-2部分且IL-2部分可操作地连接于Fc部分。
  9. 权利要求8的融合蛋白，其从N末端到C末端包含：
    (a)IL-2部分可操作地连接于Fc部分；
    (b)IL-2部分可操作地连接于Fc部分且Fc部分可操作地连接于IFNα部分；
    (c)Fc部分可操作地连接于IL-2部分；或
    (d)IFNα部分可操作地连接于Fc部分且Fc部分可操作地连接于IL-2部分。
  10. 权利要求8或9的融合蛋白，其中所述可操作地连接是直接连接或通过肽接头进行连接，任选地所述肽接头为GS系列接头，例如如SEQ ID NO:11所示的肽接头。
  11. 权利要求1-10中任一项的融合蛋白，其包含如SEQ ID NO:3、4、9或10所示的氨基酸序列。
  12. 一种融合蛋白，其包含IFNα部分和Fc部分，其中所述IFNα部分包含与野生型IFNα蛋白相比具有一个或多个突变的氨基酸序列，所述氨基酸序列为与SEQ ID NO:6具有至少90％同一性的氨基酸序列。
  13. 权利要求12的融合蛋白，其中所述IFNα部分包括参照SEQ ID NO:5中氨基酸位置的选自以下的一种或多种取代：R144A和R149A。
  14. 权利要求12或13的融合蛋白，其中所述Fc部分包含人IgG Fc，如人IgG1 Fc、IgG2 Fc、IgG3 Fc、IgG4 Fc或其变体。
  15. 权利要求14的融合蛋白，其中所述Fc变体包含选自以下的一种或多种突变：L234A和L235A突变、以及M252Y、S254T和T256E突变。
  16. 权利要求12-15中任一项的融合蛋白，其从N末端到C末端包含：
    (a)IFNα部分可操作地连接于Fc部分；或
    (b)Fc部分可操作地连接于IFNα部分。
  17. 权利要求16的融合蛋白，其包含如SEQ ID NO:7或8所示的氨基酸序列。
  18. 一种核酸分子，其包括编码权利要求1-16中任一项的融合蛋白的核酸序列。
  19. 包含权利要求18的核酸分子的载体。
  20. 包含权利要求18的核酸分子或权利要求19的载体的宿主细胞。
  21. 一种药物组合物，其包含权利要求1-16中任一项的融合蛋白或编码其的核酸分子，以及药学上可接受的载剂。
  22. 一种用于生产权利要求1-16中任一项的融合蛋白的方法，其包括以下步骤：
    -在包含编码所述融合蛋白的载体的宿主细胞中表达所述融合蛋白；和
    -从宿主细胞培养物中分离出所述融合蛋白。
  23. 一种调节受试者中的免疫应答的方法，其包括向受试者施用权利要求1-16中任一项的融合蛋白或权利要求21的药物组合物。
  24. 一种用于治疗或预防受试者中的癌症或感染性疾病的方法，其包括向受试者施用有效量的权利要求1-16中任一项的融合蛋白或权利要求21的药物组合物。
  25. 权利要求24的方法，其中所述癌症选自乳腺癌、胃癌、黑色素瘤、淋巴瘤、肺癌、结肠癌、卵巢癌、膀胱癌、肾细胞癌、肝癌、前列腺癌、胰腺癌和白血病。
  26. 权利要求1-16中任一项的融合蛋白在制备用于预防、治疗和/或管理癌症或感染性疾病的药物中的用途。
  27. 权利要求1-16中任一项的融合蛋白，其用于治疗或预防癌症或感染性疾病。
  28. 一种试剂盒，包括包含权利要求1-16中任一项的融合蛋白或权利要求21的药物组合物的容器。
PCT/CN2023/094205 2022-05-13 2023-05-15 新融合蛋白及其用途 WO2023217288A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202380012626.1A CN117881704A (zh) 2022-05-13 2023-05-15 新融合蛋白及其用途

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210521837.5A CN114853909A (zh) 2022-05-13 2022-05-13 新型IL-2与INFα和Fc融合蛋白的设计、制备及用途
CN202210521837.5 2022-05-13

Publications (1)

Publication Number Publication Date
WO2023217288A1 true WO2023217288A1 (zh) 2023-11-16

Family

ID=82637234

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/094205 WO2023217288A1 (zh) 2022-05-13 2023-05-15 新融合蛋白及其用途

Country Status (2)

Country Link
CN (2) CN114853909A (zh)
WO (1) WO2023217288A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114853909A (zh) * 2022-05-13 2022-08-05 南京吉盛澳玛生物医药有限公司 新型IL-2与INFα和Fc融合蛋白的设计、制备及用途

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101970492A (zh) * 2007-10-31 2011-02-09 Xencor公司 与FcRn结合改变的Fc变体
CN108727504A (zh) * 2018-04-16 2018-11-02 中国科学院生物物理研究所 一种ifn与抗pd-l1抗体的融合蛋白及其应用
CN112218884A (zh) * 2018-05-07 2021-01-12 免疫分子中心 由白介素-2突变蛋白和i型干扰素构成的融合蛋白
CN112724259A (zh) * 2020-11-16 2021-04-30 天津林达生物科技有限公司 人血清白蛋白与白介素2的融合蛋白及其用途
TW202144391A (zh) * 2020-03-19 2021-12-01 大陸商信達生物製藥（蘇州）有限公司 白介素２突變體及其用途
CN114853909A (zh) * 2022-05-13 2022-08-05 南京吉盛澳玛生物医药有限公司 新型IL-2与INFα和Fc融合蛋白的设计、制备及用途

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101970492A (zh) * 2007-10-31 2011-02-09 Xencor公司 与FcRn结合改变的Fc变体
CN108727504A (zh) * 2018-04-16 2018-11-02 中国科学院生物物理研究所 一种ifn与抗pd-l1抗体的融合蛋白及其应用
CN112218884A (zh) * 2018-05-07 2021-01-12 免疫分子中心 由白介素-2突变蛋白和i型干扰素构成的融合蛋白
TW202144391A (zh) * 2020-03-19 2021-12-01 大陸商信達生物製藥（蘇州）有限公司 白介素２突變體及其用途
CN112724259A (zh) * 2020-11-16 2021-04-30 天津林达生物科技有限公司 人血清白蛋白与白介素2的融合蛋白及其用途
CN114853909A (zh) * 2022-05-13 2022-08-05 南京吉盛澳玛生物医药有限公司 新型IL-2与INFα和Fc融合蛋白的设计、制备及用途

Also Published As

Publication number Publication date
CN114853909A (zh) 2022-08-05
CN117881704A (zh) 2024-04-12

Similar Documents

Publication Publication Date Title
US11535657B2 (en) Molecules that selectively activate regulatory T cells for the treatment of autoimmune diseases
US20220112260A1 (en) Molecules that selectively activate regulatory t cells for the treatment of autoimmune diseases
JP6853317B2 (ja) 高度に低下された受容体結合親和性を有するサイトカインを含むフソカイン
JP6416628B2 (ja) 標的変異体αヘリックスバンドルサイトカイン
CA2955006C (en) Modified il-2 variants that selectively activate regulatory t cells for the treatment of autoimmune diseases
JP6660297B2 (ja) 疾患及び障害を処置するためのインターロイキン−１０の使用方法
JPH0542413B2 (zh)
WO2023217288A1 (zh) 新融合蛋白及其用途
WO2022057909A1 (zh) 双特异性重组蛋白及其用途
US20230027899A1 (en) Cd122 with altered icd stat signaling
EP4019536A1 (en) Immunocytokine, preparation for same, and uses thereof
WO2023070056A2 (en) Heterodimeric fc cytokines and uses thereof
KR20040108786A (ko) 키메라 길항제 ＡｎＴＨ１
JP2023553934A (ja) ヒトＩＬ－１０Ｒａ受容体を活性化するためのイムノサイトカインおよびその使用
AU2022369312A1 (en) Heterodimeric fc cytokines and uses thereof
WO2023070038A2 (en) Human il-12p40 variants and uses thereof
CN116685345A (zh) Il-7融合蛋白及相关方法
KR20080113759A (ko) 신규한 단백질 유입 도메인 및 이의 용도

Legal Events

Date Code Title Description
DPE2 Request for preliminary examination filed before expiration of 19th month from priority date (pct application filed from 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23803056

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 202380012626.1

Country of ref document: CN