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WO2021063313A1 - 改变效应功能的Fc变体及其融合蛋白 - Google Patents

改变效应功能的Fc变体及其融合蛋白 Download PDF

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WO2021063313A1
WO2021063313A1 PCT/CN2020/118409 CN2020118409W WO2021063313A1 WO 2021063313 A1 WO2021063313 A1 WO 2021063313A1 CN 2020118409 W CN2020118409 W CN 2020118409W WO 2021063313 A1 WO2021063313 A1 WO 2021063313A1
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fusion protein
cells
variant
amino acid
seq
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PCT/CN2020/118409
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English (en)
French (fr)
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路力生
霍永庭
刘云鹏
李艳敏
韩喆
欧颖烨
芦迪
涂晶晶
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深圳市菲鹏生物制药股份有限公司
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    • 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]
    • C07K14/5428—IL-10
    • 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]
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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]
    • C07K14/5412—IL-6
    • 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]
    • C07K14/5434—IL-12
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

Definitions

  • the present invention relates to the field of biopharmaceuticals, and in particular to an Fc variant with altered effector function, the Fc region of which contains one or more amino acid changes, which has an altered effector function; further, the present invention relates to an Fc variant with altered effector function.
  • Body fusion protein
  • the Fc region of an antibody interacts with many Fc receptors and ligands to perform a series of important functions, called effector functions.
  • the Fc receptor of IgG antibody is called Fc ⁇ R
  • IgE is Fc ⁇ R
  • IgA is Fc ⁇ R
  • Three subclasses of FcyR have been identified: FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16).
  • FcRn neonatal Fc receptor
  • the effector functions mediated by the Fc region of an antibody can be divided into two types: (1) The effector functions that play a role after the antibody binds to the antigen (these functions involve participation in the complement cascade or Fc receptor (FcR)-load cell); And (2) Effector functions that do not depend on antigen binding (these functions provide continuity in the blood circulation and the ability to cross cell barriers through endocytosis).
  • the Fc region of antibodies is commonly used to prepare various fusion proteins.
  • the Fc portion of the fusion protein can bind to Fc ⁇ Rs receptors expressed on various immune leukocytes, thereby producing Fc-segment-mediated cytotoxicity (ADCC) and complement-dependent cells.
  • ADCC Fc-segment-mediated cytotoxicity
  • CDC Toxicity
  • an Fc variant which aims to attenuate the Fc receptors expressed on Fc and various types of immune leukocytes, namely Fc ⁇ Rs (mainly including FcR gamma I, FcR gamma IIa, FcR gamma IIb and FcR gamma IIIa), thereby reducing or eliminating Fc segment-mediated cytotoxicity (antibody-dependent cell mediated cytotoxicity, ADCC) or complement C1q-mediated complement-dependent cytotoxicity (complement-dependent cytotoxicity, CDC) effect, and keep its affinity with neonatal Fc receptor (FcRn) unchanged or enhanced, without affecting the half-life in vivo.
  • Fc ⁇ Rs mainly including FcR gamma I, FcR gamma IIa, FcR gamma IIb and FcR gamma IIIa
  • the Fc variant contains an amino acid substitution at at least one selected from the 223, 235, 237, 238, 265, 297, 327, and 434 positions.
  • the Fc is IgG Fc, wherein IgG Fc is one of IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, preferably IgG1 Fc, more preferably human IgG1 Fc.
  • amino acid substitution is selected from at least one of E223P, L235A, G237A, P238A, D265A, N297A, A327Q, and N434A.
  • amino acid substitution is selected from at least three of E223P, L235A, G237A, P238A, D265A, N297A, A327Q, and N434A.
  • the amino acid substitution is a three amino acid substitution selected from: E223P, P238A, D265A; N297A, L235A, N434A; and A327Q, G237A, L235A.
  • the present invention provides an Fc variant fusion protein. Specifically, the present invention relates to the following embodiments:
  • Embodiment 1 A fusion protein whose structure is as shown in the following formula:
  • the peptide linker is (GS) n , where n is an integer from 1 to 10, and B is IgG Fc.
  • Embodiment 2 The fusion protein of embodiment 1, wherein the A is IL-10, preferably human IL-10.
  • Embodiment 3 The fusion protein of any one of embodiments 1-2, wherein IL-10 is connected to the N-terminus or C-terminus of IgG Fc through the peptide linker.
  • Embodiment 4 The fusion protein of embodiment 1, wherein IgG Fc is one of IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, preferably IgG1 Fc, more preferably human IgG1 Fc.
  • Embodiment 5 The fusion protein of any one of embodiments 1-4, wherein the amino acid sequence of IL-10 is SEQ ID NO: 17.
  • Embodiment 6 The fusion protein of any one of the preceding embodiments, wherein the IgG Fc is modified, and compared with wild-type IgG Fc, the modified IgG Fc weakens the affinity of the fusion protein to Fc ⁇ R.
  • Embodiment 7 The fusion protein of embodiment 6, wherein according to EU numbering, the IgG Fc contains an amino acid substitution at at least one selected from positions 223, 235, 237, 238, 265, 297, 327, and 434 .
  • Embodiment 8 The fusion protein of embodiment 7, wherein the amino acid substitution is selected from at least one of E223P, L235A, G237A, P238A, D265A, N297A, A327Q, and N434A.
  • Embodiment 9 The fusion protein of embodiment 6 or 7, wherein the amino acid substitution is selected from at least three of E223P, L235A, G237A, P238A, D265A, N297A, A327Q, and N434A.
  • Embodiment 10 The fusion protein of embodiment 9, wherein the amino acid substitution is a three-amino acid substitution selected from: E223P, P238A, D265A; N297A, L235A, N434A; and A327Q, G237A, L235A.
  • the amino acid substitution is a three-amino acid substitution selected from: E223P, P238A, D265A; N297A, L235A, N434A; and A327Q, G237A, L235A.
  • Embodiment 11 The fusion protein of embodiment 10, wherein the amino acid sequence of the fusion protein is SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.
  • the present invention provides a nucleic acid that encodes any one of the aforementioned Fc variants or any one of the Fc variant fusion proteins.
  • the nucleotide sequence of the nucleic acid is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO :twenty two.
  • the present invention provides a pharmaceutical composition comprising any one of the aforementioned Fc variants or any one of the Fc variant fusion proteins and a pharmaceutically acceptable carrier.
  • the present invention provides a use of any one of the above-mentioned pharmaceutical compositions in the preparation of a medicament for treating cancer in an individual in need thereof.
  • the cancer is a solid tumor.
  • the solid tumor is colon cancer, melanoma, colorectal cancer, breast cancer.
  • the individual is a human.
  • Fc or "Fc region” is used herein to define the C-terminal region of an antibody heavy chain that contains at least a part of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the IgG Fc region contains IgG CH2 and IgG CH3 domains.
  • the "CH2 domain" of the human IgG Fc region generally extends from the amino acid residue at about position 231 to the amino acid residue at about position 340.
  • the numbering of amino acid residues in the Fc region or constant region is in accordance with the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service , National Institutes of Health, Bethesda, MD, 1991.
  • the Fc described herein includes wild-type Fc and modified Fc.
  • the modified IgG Fc weakens the affinity of the fusion protein described herein to Fc ⁇ R. Affinity can be determined using methods known in the art or methods disclosed herein.
  • substitution refers to the replacement of one amino acid with another amino acid in a polypeptide.
  • the amino acid is replaced with another amino acid having similar structure and/or chemical properties, such as a conservative amino acid replacement.
  • Constant amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic properties of the residues involved.
  • non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine;
  • sexual amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine;
  • positively charged (basic) amino acids include arginine, lysine, and histidine ;
  • negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Non-conservative substitutions would require swapping members of one of these categories for members of another category.
  • an amino acid substitution may also result in the replacement of one amino acid with another amino acid having different structural and/or chemical properties, such as replacing an amino acid from one group (e.g., polar) with another from a different group (e.g., basic).
  • An amino acid substitution may be used to generate amino acid substitutions. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and so on. For example, E223P indicates that the E at position 233 in Fc is replaced by P, and P238A indicates that P at position 238 in Fc is replaced by A.
  • IgG class antibody refers to an antibody having the structure of a naturally-occurring immunoglobulin G (IgG) molecule.
  • the heavy chain of the IgG antibody has the domain structure VH-CH1-CH2-CH3.
  • the light chain of the IgG antibody has the domain structure VL-CL.
  • the IgG class antibody basically consists of two Fab fragments and an Fc domain connected via an immunoglobulin hinge region.
  • IgG class antibodies include, for example, IgG1, IgG2, IgG3, and IgG4.
  • IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc represent the Fc or Fc regions of IgG1, IgG2, IgG3, and IgG4, respectively.
  • the IgG Fc in the fusion protein herein can be IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, preferably IgG1 Fc, more preferably
  • fusion protein refers to a fusion polypeptide molecule comprising IL-10, IL-6 or IL-12 molecules and the Fc portion of IgG1, wherein the components of the fusion protein are connected to each other by peptide bonds, or directly Ground or via a peptide linker.
  • peptide linker is a peptide comprising one or more amino acids, usually about 2-20 amino acids.
  • Peptide linkers are known in the art or described herein.
  • Suitable, non-immunogenic peptide linkers include, for example, (GS) n linkers, where n is an integer from 1-10. In one embodiment, n is 1-6, preferably 4.
  • Fusion means that the components are connected by peptide bonds directly or via one or more peptide linkers.
  • Binding affinity refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise indicated, as used herein, "binding affinity” refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair.
  • the affinity of a molecule X for its partner Y can usually be expressed in terms of the dissociation constant (K D ), which is the ratio of dissociation and association rate constants (K dissociation and K binding, respectively).
  • K D dissociation constant
  • K dissociation and K binding the ratio of dissociation and association rate constants
  • Polynucleotide or “nucleic acid” are used interchangeably herein and refer to polymers of nucleotides of any length, and include DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or can be incorporated into polymers by DNA or RNA polymerase or through synthetic reactions Of any substrate. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. The nucleotide sequence can be interrupted by non-nucleotide building blocks. Polynucleotides may include modifications made after synthesis, such as conjugation to a label.
  • Modification refers to any manipulation of the peptide backbone (e.g., amino acid sequence) or post-translational modification (e.g., glycosylation) of the polypeptide. Modifications also include the substitution, deletion or insertion of amino acids in the amino acid sequence.
  • Natural IL-10 (also known as “wild-type IL-10") means naturally occurring IL-10, as opposed to “modified IL-10", which has been modified from naturally occurring IL-10, for example, to change One or more of its characteristics, such as stability.
  • the modified IL-10 molecule may, for example, contain modifications in the amino acid sequence, such as amino acid substitutions, deletions or insertions.
  • Natural IL-6 (also known as “wild-type IL-6”) means naturally occurring IL-6, as opposed to “modified IL-6", which is modified from naturally occurring IL-6, for example, to change One or more of its characteristics, such as stability.
  • the modified IL-6 molecule may, for example, contain modifications in the amino acid sequence, such as amino acid substitutions, deletions or insertions.
  • “Natural IL-12” also known as “wild-type IL-12” means naturally occurring IL-12, as opposed to “modified IL-12", which is modified from naturally occurring IL-12, for example, to change One or more of its characteristics, such as stability.
  • the modified IL-12 molecule may, for example, contain modifications in the amino acid sequence, such as amino acid substitutions, deletions or insertions.
  • vector refers to a nucleic acid molecule capable of multiplying another nucleic acid to which it is linked.
  • the term includes a vector that is a self-replicating nucleic acid structure and a vector integrated into the genome of a host cell into which it is introduced.
  • Certain vectors can direct the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells”, which include initially transformed cells and progeny derived therefrom (regardless of the number of passages).
  • the offspring may not be exactly the same as the parent cell in nucleic acid content, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the original transformed cell.
  • the host cell is any type of cell system that can be used to produce the fusion protein of the present invention.
  • Host cells include cultured cells, such as mammalian cultured cells such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells , Yeast cells, bacterial cells, such as Escherichia coli, insect cells and plant cells, etc., but also include cells contained in transgenic animals, transgenic plants, or cultured plants or animal tissues.
  • mammalian cultured cells such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells
  • Yeast cells bacterial cells, such as Escherichia coli, insect cells and plant cells, etc., but also include cells contained in transgenic animals, transgenic plants, or cultured plants or animal tissues.
  • the “effective amount” of an agent refers to the amount necessary to cause physiological changes in the cells or tissues to which it is administered.
  • a “therapeutically effective amount” of an agent such as a pharmaceutical composition refers to an amount effective to achieve the desired therapeutic or preventive result (in the necessary dose and for the necessary time).
  • a therapeutically effective amount of an agent for example, eliminates, reduces, delays, minimizes, or prevents the adverse effects of the disease.
  • mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats) ).
  • domesticated animals e.g., cattle, sheep, cats, dogs, and horses
  • primates e.g., human and non-human primates such as monkeys
  • rabbits e.g., mice and rats
  • rodents e.g., mice and rats
  • composition refers to a formulation in a form that allows the biological activity of the active ingredients contained therein to be effective, and does not contain other ingredients that have unacceptable toxicity to subjects who will receive the formulation.
  • “Pharmaceutically acceptable carrier” refers to ingredients in the pharmaceutical composition that are non-toxic to the subject other than the active ingredients.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • treatment/treatment refers to an attempt to change the natural course of a disease in an individual being treated, and may be for prevention or clinical intervention implemented during the course of clinical pathology.
  • the desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or reducing the disease state, and regressing or improving prognosis.
  • FIG. 1 Plasmid pcDNA3.4A(a), pcDNA3.4A-R0354(b), pcDNA3.4A-R0355(c), pcDNA3.4A-R0356(d), pcDNA3.4A-R0359VCHE(e), pcDNA3.4A -Map of R0359VCLE(f), and pcDNA3.4A-R0330(g).
  • Figure 3 The amino acid sequences of R0354, R0355, R0356, R0359 (consisting of R0359VCHE and R0359VCLE) and R0330 and human IL-10.
  • Figure 4 Purity analysis and chromatogram: a). SE-HPLC purity of affinity eluate; b). SE-HPLC purity after pH treatment of affinity elution; c). SE- elution fraction after fine purification HPLC purity; d) chromatograms of the combined elution fractions.
  • CD8+ T cell IL-10R increases after activation.
  • FIG. 7 Anti-tumor activity in mice: a) Mouse colon cancer (CT26WT) anti-tumor model; b) Mouse colon cancer (CT26WT) tumor survival curve; c) Mouse melanoma (B16-F1) anti-tumor model D) Mouse colon cancer (MC38) anti-tumor model.
  • C26WT Mouse colon cancer
  • C26WT Mouse colon cancer
  • B16-F1 Mouse colon cancer
  • MC38 Mouse colon cancer
  • an Fc variant is disclosed.
  • the Fc variant is IgG Fc, wherein IgG Fc is one of IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, preferably IgG1 Fc, More preferred is human IgG1 Fc.
  • the IgG Fc contains an amino acid substitution in at least one selected from the 223, 235, 237, 238, 265, 297, 327, and 434 positions.
  • amino acid substitution is selected from at least one of E223P, L235A, G237A, P238A, D265A, N297A, A327Q, and N434A.
  • amino acid substitution is selected from at least three of E223P, L235A, G237A, P238A, D265A, N297A, A327Q, and N434A.
  • the amino acid substitution is a three amino acid substitution selected from: E223P, P238A, D265A; N297A, L235A, N434A; and A327Q, G237A, L235A.
  • Fc variants containing the deletion or mutation of position 447 or other Fc variants that do not affect Fc ⁇ The mutation site of receptor affinity is also within the protection scope of the present invention.
  • Fc fusion proteins There are many forms of Fc fusion proteins.
  • the Fc variant of the present invention can be made into any form of fusion protein as required, or can be fused with other types of proteins.
  • the examples of the fusion protein disclosed in the present invention are only one of them. This example does not limit the scope of protection of the present invention.
  • a fusion protein is disclosed, the structure of which is as shown in the following formula:
  • A is IL-10, IL-6 or IL-12
  • the peptide linker is (GS) n , where n is an integer of 1-10, and B is IgG Fc.
  • A can also be an active fragment of IL-10, IL-6 or IL-12.
  • IgG1 Fc comprises three amino acid substitutions selected from the group consisting of: E223P, P238A, D265A; N297A, L235A, N434A; and A327Q, G237A, L235A.
  • IL-10 may be natural IL-10 of an animal, such as a mammal, such as human, or a modified IL-10 obtained by substituting or inserting one or more amino acids of IL-10.
  • IL-10 active fragment refers to the peptide chain obtained by truncating one or more amino acids from the N-terminus or C-terminus or from the N-terminus and C-terminus of IL-10 from natural IL-10 or modified IL-10. , The peptide chain retains the functional activity of IL-10.
  • IL-10 activity can be obtained by conventional methods, such as obtaining IL-10 fragments by chemical synthesis or recombinant expression methods, and measure whether the IL-10 fragments have the original IL by methods known in the art or disclosed in this application. -10 activity.
  • the polynucleotide and nucleic acid coding region of the present invention may be combined with another coding region that encodes a signal peptide that directs the secretion of the polypeptide encoded by the polynucleotide of the present invention.
  • the DNA encoding the signal sequence can be placed upstream of the nucleic acid encoding the fusion protein of the invention or a fragment thereof.
  • the protein secreted by mammalian cells has a signal peptide or secretion leader sequence. Once the growing protein chain is exported across the rough endoplasmic reticulum, the signal peptide or secretion leader sequence is cut away from the mature protein.
  • polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cut from the translated polypeptide to generate a secreted or "mature" form of the polypeptide.
  • the signal peptide may be a universal signal peptide, for example, an IgG universal signal peptide.
  • the coding sequence of the signal peptide may be ATGGGATGGTCATGCATAATACTCTTTCTTGTGGCTACTGCTACCGGGGTTCACTCT (SEQ ID NO: 11).
  • A-peptide linker-B means that the peptide linker connects the carboxyl terminal (C terminal, or 3'end) of A and the amino terminal (N terminal, or 5'end) of B, respectively
  • B-peptide Linker-A means that the peptide linker connects the carboxyl end (C end, or 3'end) of B and the amino end (N end, or 5'end) of A, respectively.
  • n is 1-6, for example, 1,2,3,4,5,6, preferably n is 4.
  • IgG1 Fc is human IgG1 Fc.
  • the Fc of the IgG class of antibodies confers favorable pharmacokinetic properties on the fusion protein, including long serum half-life (attributed to better accumulation in the target tissue and favorable tissue-to-blood distribution ratio), it can also cause unwanted fusion
  • the protein targets cells that express Fc receptors, not cells of interest.
  • activation of the Fc receptor signaling pathway can cause cytokine release that leads to (pro-inflammatory) cytokine receptor activation and severe side effects during systemic administration.
  • the fusion protein of the present invention not only retains the immunological and anti-tumor activity of IL-10, but more importantly, optimizes the combination of site-specific gene mutations in the Fc segment, and weakens the IL-10-Fc fusion protein and the Fc receptor expressed on various immune leukocytes.
  • Fc ⁇ Rs (mainly including FcR gamma I, FcR gamma IIa, FcR gamma IIb and FcR gamma IIIa), thereby reducing or eliminating Fc-segment-mediated cytotoxicity (ADCC) or binding to complement C1q-mediated Complement-dependent cytotoxicity (CDC) does not produce cytotoxicity, prevents effector T cells from being killed, and ultimately effectively improves the anti-tumor effect of IL-10.
  • ADCC Fc-segment-mediated cytotoxicity
  • CDC complement C1q-mediated Complement-dependent cytotoxicity
  • CDC assays can be implemented (see, for example, Gazzano-Santoro et al. J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).
  • IL-10 is human IL-10 or hIL-10.
  • IL-6 is human IL-6 or hIL-6.
  • IL-12 is human IL-12 or hIL-12.
  • the three amino acid substitutions contained in the IgG1 Fc are E223P, P238A, D265A; N297A, L235A, N434A; or A327Q, G237A, L235A.
  • the numbering refers to the numbering of Fc amino acids, and the numbering is carried out according to the EU numbering system.
  • amino acid sequence of the fusion protein is SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.
  • the invention relates to a nucleic acid encoding an Fc variant or fusion protein of the invention.
  • nucleic acid encoding a fusion protein due to the degeneracy of the genetic code, there can be many types of nucleic acid encoding a fusion protein.
  • Those skilled in the art can optimize the nucleic acid sequence according to the host cell, so that the nucleic acid sequence can be expressed in the host cell at an optimized level, for example, with a higher expression level.
  • the nucleic acid sequence encoding the fusion protein can also be effectively linked to regulatory elements such as promoters, introns, enhancers, etc., so that these regulatory elements perform their respective functions in the host cell.
  • the fusion protein may also include a signal peptide so that the encoded fusion protein can be secreted into the culture medium, thereby obtaining a supernatant by centrifugation, and further purifying the fusion protein from the supernatant.
  • Nucleic acids containing fusion protein coding sequences, regulatory elements, and signal peptide coding sequences are also within the scope of the nucleic acid of the present invention.
  • nucleotide sequence of the nucleic acid is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.
  • the invention relates to an expression vector comprising a nucleic acid encoding an Fc variant or fusion protein of the invention.
  • the expression vector can be a part of a plasmid, a virus or can be a nucleic acid fragment.
  • the expression vector contains an expression cassette in which a polynucleotide (i.e. coding region) encoding a fusion protein (fragment) is cloned in an operative connection with a promoter and/or other transcription or translation control elements.
  • a polynucleotide i.e. coding region
  • fragment a fusion protein
  • the induced promoter function results in the transcription of the mRNA encoding the desired gene product and if the nature of the connection between the two DNA fragments does not interfere with the ability of the expression regulatory sequence to direct the expression of the gene product or does not interfere with the ability of the DNA template to be transcribed, then Two DNA fragments (such as the polypeptide coding region and the promoter associated with it) are "operably linked.”
  • the promoter can realize the transcription of the nucleic acid encoding the polypeptide, then the promoter region will be operatively linked to the nucleic acid.
  • the promoter may be a cell-specific promoter, which only directs substantial transcription of DNA in a predetermined cell.
  • transcription control elements such as enhancers, operators, repressors, and transcription termination signals can be operatively linked to polynucleotides to direct cell-specific transcription.
  • transcription control regions include, but are not limited to, transcription control regions that function in vertebrate cells, such as but not limited to promoters and enhancer segments from cytomegalovirus (for example, immediate early promoter, linked to intron-A), ape Virus 40 (e.g. early promoter) and retrovirus (e.g. Rous sarcoma virus).
  • transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit positive beta protein, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers and inducible promoters (e.g., tetracycline inducible promoters). Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to, ribosome binding sites, translation start and stop codons, and elements derived from viral systems (specifically, internal ribosome entry sites or IRES, also known as CITE sequences). The expression cassette may also contain other features, such as an origin of replication and/or chromosomal integration elements, such as retroviral long terminal repeat (LTR) or adeno-associated virus (AAV) inverted terminal repeat (ITR).
  • LTR retroviral long terminal repeat
  • AAV adeno-associated virus
  • the polynucleotide and nucleic acid coding region of the present invention may be linked to another coding region that encodes a secretory peptide or a signal peptide that directs the secretion of the polypeptide encoded by the polynucleotide of the present invention.
  • the DNA encoding the signal sequence can be placed upstream of the nucleic acid encoding the fusion protein of the invention or a fragment thereof.
  • the protein secreted by mammalian cells has a signal peptide or secretion leader sequence.
  • the signal peptide or secretion leader sequence is cut away from the mature protein.
  • polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cut from the translated polypeptide to generate a secreted or "mature" form of the polypeptide.
  • natural signal peptides are used, such as immunoglobulin heavy chain or light chain signal peptides, or functional derivatives that retain the ability of the sequence to direct the secretion of the polypeptide to which it is operatively linked.
  • a heterologous mammalian signal peptide or a functional derivative thereof can be used.
  • the wild-type leader sequence can be replaced with the leader sequence of human tissue plasminogen activator (TPA) or mouse ⁇ -glucuronidase.
  • TPA tissue plasminogen activator
  • DNA encoding a short protein sequence that can be used to facilitate later purification (such as a histidine tag) or to help label the fusion protein can be incorporated into or at the end of the polynucleotide encoding the fusion protein (fragment).
  • a host cell comprising one or more polynucleotides of the invention.
  • a host cell comprising one or more vectors of the invention.
  • the polynucleotide and the vector may be incorporated individually or in combination with any of the features described herein with respect to the polynucleotide and the vector, respectively.
  • the host cell comprises a vector (e.g., has been transformed or transfected with the vector), which comprises a polynucleotide encoding (part of) the fusion protein of the invention.
  • the term "host cell” refers to any type of cell system that can be engineered to produce the fusion protein or fragments thereof of the present invention.
  • Host cells suitable for replication and supporting the expression of fusion proteins are well known in the art.
  • specific expression vectors can be used to transfect or transduce such cells, and a large number of vector-containing cells can be cultured for inoculation of large-scale fermenters, so as to obtain a sufficient amount of fusion protein for clinical applications.
  • Suitable host cells include prokaryotic microorganisms such as Escherichia coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells and the like.
  • polypeptides can be produced in bacteria, especially when glycosylation is not required. After expression, the polypeptide can be separated from the bacterial cell paste in the soluble fraction and can be further purified.
  • eukaryotic microorganisms such as filamentous fungi or yeast are also suitable for cloning or expression hosts for vectors encoding polypeptides, including their glycosylation pathways that have been "humanized", resulting in the production of partially or fully human glycosyl
  • Suitable host cells for expressing (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include insect cells.
  • a large number of baculovirus strains have been identified that can be used with insect cells, especially for the transfection of Spodoptera frugiperda cells.
  • Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (description of PLANTIBODIES TM technology for generating antibodies in transgenic plants).
  • Vertebrate cells can also be used as hosts.
  • mammalian cell lines adapted to grow in suspension can be useful.
  • monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293 or 293T cells, as described in, for example, Graham et al., J Gen Virol 36,59 (1977)), baby hamster kidney cells (BHK), mouse sertoli (sertoli) cells (TM4 cells, as described in, for example, Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), macaque kidney cells (MDCK), bovine mouse liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2), mice Breast tumor cells (MMT 060562), TRI cells (as described, for example, in Mather et al., Annals NYAcad Sci 383, 44-68 (1982)), MRC5 cells and FS4 cells.
  • mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr - CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63 and Sp2/0.
  • CHO Chinese hamster ovary
  • myeloma cell lines such as YO, NS0, P3X63 and Sp2/0.
  • Host cells include cultured cells, such as mammalian cultured cells, yeast cells, insect cells, bacterial cells, and plant cells, but also include cells contained in transgenic animals, transgenic plants, or cultured plants or animal tissues.
  • the host cell is a bacterial cell, preferably an Escherichia cell, particularly preferably an Escherichia coli cell.
  • the Fc variant or fusion protein prepared as described herein can be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion Resistance to chromatography and so on.
  • the actual conditions used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be obvious to those skilled in the art.
  • affinity chromatography purification antibodies, ligands, receptors, or antigens to which the fusion protein binds can be used.
  • the purity of the fusion protein can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and the like.
  • compositions Composition, formulation and route of administration
  • the invention provides a pharmaceutical composition comprising any of the Fc variants or fusion proteins provided herein.
  • the pharmaceutical composition comprises any Fc variant or fusion protein provided herein and a pharmaceutically acceptable carrier.
  • Any of the fusion proteins provided herein can be used in the treatment method.
  • the fusion protein of the present invention will be formulated, administered, and administered in a manner consistent with good medical practice.
  • Factors considered in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the location of drug delivery, the method of administration, the schedule of administration, and other factors known to medical practitioners.
  • the fusion protein of the present invention for use as a medicine is provided. In another aspect, the fusion protein of the present invention for use in the treatment of diseases is provided. In certain embodiments, a fusion protein of the invention for use in a method of treatment is provided. In one embodiment, the present invention provides a fusion protein as described herein for use in the treatment of diseases in individuals in need thereof. In certain embodiments, the present invention provides a fusion protein for use in a method of treating an individual suffering from a disease, the method comprising administering to the individual a therapeutically effective amount of the fusion protein. In certain embodiments, the disease to be treated is cancer. Exemplary cancers include colon cancer, melanoma, colorectal cancer, and breast cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, such as an anti-cancer agent.
  • the "individual" according to any of the above embodiments is a mammal, preferably a human.
  • the present invention provides the use of the fusion protein of the present invention in the manufacture or preparation of medicines for the treatment of diseases in individuals in need thereof.
  • the medicament is used in a method of treating a disease, the method comprising administering a therapeutically effective amount of the medicament to an individual suffering from the disease.
  • the disease to be treated is cancer.
  • the disease is colon cancer.
  • the disease is melanoma.
  • the disease is colorectal cancer.
  • the disease is breast cancer.
  • the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, such as an anti-cancer agent.
  • the "individual" according to any of the above embodiments is a mammal, preferably a human.
  • the invention provides a method for treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of the fusion protein of the invention.
  • the individual is administered a composition comprising the fusion protein of the invention in a pharmaceutically acceptable form.
  • the disease to be treated is cancer.
  • the disease is colon cancer.
  • the disease is melanoma.
  • the disease is colorectal cancer.
  • the disease is breast cancer.
  • the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, such as an anti-cancer agent.
  • the "individual" according to any of the above embodiments is a mammal, preferably a human.
  • an effective amount of the fusion protein of the invention is administered to the cell. In other embodiments, a therapeutically effective amount of the fusion protein of the invention is administered to the individual to treat the disease.
  • the appropriate dosage of the fusion protein of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the weight of the patient, the fusion The type of protein, the severity and progress of the disease, whether the fusion protein is administered for preventive or therapeutic purposes, previous or simultaneous therapeutic intervention, the patient's clinical history and response to the fusion protein, and the judgment of the attending physician.
  • the practitioner responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage for the individual subject in any event.
  • Various dosing regimens are encompassed herein, including but not limited to single or multiple administrations at various time points, bolus administrations, and pulse infusions.
  • the percentage concentration in the examples is the mass volume percentage concentration.
  • a 2% agarose gel means that 100ml of gel contains 2g of agarose.
  • 10% fetal bovine serum means that a volume of 100ml contains 10g fetal bovine serum.
  • the Fc segment of human IgG1 antibody was subjected to site-directed mutation according to the EU numbering method to construct three plasmids: PCDNA3.4A-R0354, PCDNA3.4A-R0355 and PCDNA3.4A-R0356.
  • the molecular structures of the three target fragments are as follows: R0354: IgG general signal peptide-IL10-GSGSGS-hIgG1 Fc (hinge region+CH2+CH3, containing E223P, P238A, D265A mutations); R0355: IgG general signal peptide-IL10-GSGSGS-hIgG1 Fc (hinge region+CH2+CH3, containing N297A, L235A, N434A mutations); R0356: IgG universal signal peptide-IL10-GSGSGS-hIgG1 Fc (hinge region + CH2 + CH3, containing A327Q, G237A, L235A mutations).
  • PCDNA3.4A-R0359 and PCDNA3.4A-R0330 were constructed.
  • PCDNA3.4A-R0359 includes PCDNA3.4A-R0359VCHE and PCDNA3.4A-R0359VCLE, the molecular structure is as follows: PCDNA3.4A-R0359VCHE: IgG general signal peptide-anti-HIV VH-hIgG1.3CH (CH1+hinge area+CH2+CH3), PCDNA3.4A-R0359VCLE: IgG universal signal peptide-anti-HIV VL-hIgKC, the polypeptide expressed by the isotype control PCDNA3.4A-R0359 is R0359.
  • R0330 The molecular structure of R0330 is as follows: IgG general signal peptide-IL10-GSGSGSGS-hIgG1 Fc (hinge region + CH2 + CH3, including L234A, L235E, G237A, and deletion of K at position 447, which is a Bristol-Myers Squibb Fc variant, used as the original Invented comparison);
  • PCR instrument Tprofessional TR20
  • electrophoresis instrument DYY-TC
  • gel imager Smart Gel N
  • centrifuge H1650-W
  • micro thermostat HW-8C
  • ultra-clean Workbench SDJ series
  • constant temperature oscillator H2-9211K
  • constant temperature water bath HH-4A
  • the main reagents used in the experiment pcDNA TM 3.4 TOPO TM TA Cloning Kit (Invitrogen A14697); 5-alpha Competent E. coli (NEB C2987I); DL2000 DNA Marker (TAKARA 3427A); Q5 high-fidelity DHA polymerization Enzyme (High-Fidelity DNA Polymerase) (NEB M0491L); AxyPrep DNA Gel Extraction Kit (AxyGEN AP-GX-50); Hind III (NEB); EcoRI (NEB); T4DNA ligase (TAKARA) 2011A); Endotoxin-free plasmid large-scale extraction kit (TIANGEN DP117);
  • the amino acid sequences of R0354, R0355, R0356, R0359 and R0330 were optimized by humanized bases, and their coding nucleotide sequences were artificially synthesized.
  • the coding nucleic acid sequences of R0354, R0355, R0356, R0359, and R0330 are shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and 9, and SEQ ID NO: 19, respectively.
  • the PCDNA3.4A vector is selected according to the purpose of the plasmid to be constructed, the output of the foreign gene expression product, the difficulty of vector DNA preparation, and the analysis results of the restriction site of the vector and the target fragment.
  • the vector was purchased from Invitrogen, and the constructed plasmid map is shown in Figure 1.
  • the primers were designed by SnapGene software (SnapGene Version 1.1.3), and the sequence of a pair of specific oligonucleotide primers for amplification of R0354, R0355, and R0356 was finally determined to be 1F 5'-CCGCAAGCTTGCCACCATGGGATGG TCATGCATAATACTC-3' (SEQ ID NO: 12) 1R 5'-CCGGAATTCTCATTACTTGCCGGGGCTC AGGCTCAG-3' (SEQ ID NO: 13), the length of the amplified product fragment is 1288bp; the sequence of a pair of specific oligonucleotide primers for amplification of R0359VCHE is 2F 5'-CCGCAAGCTTGCCACCATGGGATGGTCATGCATACATA-3' (SEQ ID NO: 14); 2R 5'-CCGGAATTCTCATTACTTGCCAGGGGACAGAGACAGGGACTT C-3' (SEQ ID NO: 15), the length of the amplified product fragment is
  • Total reaction volume 50 ⁇ L, including PCR 5 ⁇ buffer 10 ⁇ L, High GC buffer 10 ⁇ L, DNA template 0.5 ⁇ L, Q5 High-Fidelity DNA Polymerase 0.5 ⁇ L, dNTP (25mM) 4 ⁇ L, and 1.5 ⁇ L each of the specific upper and lower primers (final concentration 200nmol/L), and add sterile double-distilled water to a total volume of 50 ⁇ L;
  • Reaction conditions pre-denaturation at 94°C for 5 minutes, followed by successive denaturation at 94°C for 30 seconds, followed by annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute and 30 seconds, a total of 30 cycles.
  • the target DNA molecule and carrier molecule were digested with Hind III (NEB) and EcoRI (NEB) to obtain the corresponding sticky ends.
  • the digestion reaction was carried out at 37°C in a water bath for 1 h (reaction system: Hind III 1 ⁇ L; EcoR I 1 ⁇ L; 10 ⁇ buffer 5 ⁇ L; plasmid/DNA 10 ⁇ L ⁇ 12 ⁇ L ⁇ 1 ⁇ g; total volume 50 ⁇ L (fill with sterile water to the total volume); reaction conditions: temperature 37°C, enzyme digestion for 1 hour); 2% agarose gel electrophoresis to recover the carrier enzyme Cut large fragments, and use Axygen agarose gel recovery kit to directly recover the digested fragments of the target gene.
  • the large fragment recovered by digestion of the vector is ligated with the digested fragment of the target gene, using T4 ligase (TAKARA 2011A), and reacted at 16°C for 4 hours (reaction system: vector digestion fragment 0.5 ⁇ L; target gene digestion fragment 3.8 ⁇ L; T4 DNA Ligase 0.5 ⁇ L; 10 ⁇ T4 DNA buffer 0.5 ⁇ L).
  • All the ligation products are mixed with 120ul DH5 ⁇ competent bacteria (NEB C2987I), then ice bath for 30 minutes, heat shock at 42°C for 90 seconds, immediately place on ice for 2 minutes, add 450ul LB medium preheated to room temperature, 37°C constant temperature shaker ( H2-9211K) incubate for 60 minutes, centrifuge the bacterial solution with a centrifuge (H1650-W), mix it with a pipette, and evenly spread it on an LB plate containing 100ug of ampicillin per ml, 37°C constant temperature incubator ( HW-8C) Inverted culture overnight.
  • H2-9211K 37°C constant temperature shaker
  • transient transfection to express the proteins and constructed the corresponding four expression vector plasmids, namely PCDNA3.4A-R0354, PCDNA3.4A-R0355, PCDNA3.4A-R0356 and PCDNA3.4A- R0359; PEI (polysciences; MW40000) was used to transfect Expi293 (ThermoFisher Scientific; A14635) for a large amount of transient expression; the samples were collected by centrifugation after continuous cultivation for 7 days.
  • the Expi293 (ThermoFisher Scientific; A14635) cells were passaged one day before transient transfection.
  • Dynamis medium (gibco; A2617502) was used to inoculate a 1L shaker flask (conning; 431147) at a density of 2E6 and placed in a cell culture shaker (Adolf Kuhner; ISF4 -XC) 37°C; 8% CO 2 ; culture at 120 rpm;
  • Buffer Buffer A: 1*PBS (10mM PB, 150mM NaCl, pH7.2, Guangzhou Chemical Reagent Factory, unless otherwise specified, the following reagents are from Guangzhou Chemical Reagent Factory); Buffer B (20mM NaAc, pH3.4); CIP buffer (0.1M NaOH); Neutralization buffer (1M Tris, pH8.0 (aMResCO, 0497-500G)); Buffer: 0.5M HCitrate (Chinese National Medicine, Shanghai Test, 10007108); Ultrafiltration Concentration Tube (Millipore, Ultra 15mL Centrifugal Filters, 30KDa).
  • chromatography packing 1 GE Healthcare, MabSelect SuRe LX
  • chromatography column 1 GE Healthcare, XK16/20, column volume (CV), 26ml
  • chromatography system GE Healthcare, AKTA pure150; portable pH meter (Horiba); Microplate reader (Epoch, BioTek); Centrifuge (Xiangyi, H2050R); Chromatography filler 2: Boglong, Chromdex PG200, Chromatography column 2: GE Healthcare, XK26/40, column volume (Colunm volume, CV) 188.91ml.
  • the purification process taking the fusion protein R0354 as an example is as follows:
  • fusion proteins R0330, R0354, R0355 and R0356 were derived from the fusion protein prepared in Example 2. Its molecular weight is 90.24kDa, 90.38kDa, 90.34kDa and 90.62kDa.
  • Fc receptor FcR gamma I, FcR gamma IIa, FcR gamma IIb, FcR gamma IIIa, and FcRn recombinant proteins were purchased from Acro Biosystems; control substance hIgG1 (403502, Biolegend) points and hIgG4 (403702, Biolegend)
  • Buffer system 1*phosphate buffered saline solution (SBJ-0032, Sembega), which contains 0.02% Tween 20 (44112-100G-F, Merck) (PBST)
  • Regeneration fluid system 10mM glycine (G8898-500G, Merck)
  • FcR gamma I was diluted with 1*PBST buffer to 2ug/ml, and solidified at this concentration to the Anti-Penta-HIS (HIS1K) (18-5120, Fortebio) probe of the molecular interaction analyzer ForteBIO (Octet OK e)
  • HIS1K Anti-Penta-HIS
  • Fortebio molecular interaction analyzer ForteBIO
  • the analytes R0330, R0354, R0355, R0356, hIgG1 and hIgG4 corresponding to this receptor were diluted to 66.67nM with 1*PBST buffer, respectively.
  • FcRn, FcR gamma IIa, FcR gamma IIb, and FcR gamma IIIa were diluted with 1*PBST to 3ug/ml, and solidified on the HIS1K probe at this concentration.
  • Each receptor corresponds to the analytes R0330, R0354, R0355, R0356 , HIgG1 and hIgG4 were diluted to 2 ⁇ M with 1*PBST buffer respectively.
  • Table 1 Fortebio method to evaluate R0330, R0354, R0355, R0356, hIgG1, hIgG4 and FcR gamma I (Table 1a), FcR gamma IIa (Table 1b), FcR gamma IIb (Table 1c), FcR gamma IIIa (Table 1d) And FcRn (Table 1e) receptor affinity
  • PBMC from normal people is voluntary donated blood by company employees, number 80
  • CD8+T separation kit Movable Batch
  • DPBS Hyclone, cat: SH3002802
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • X-VIVO LONZA, cat: 04-418Q
  • anti-CD8 fluorescent antibody Biolegend, cat: 344732
  • anti-CD45RO fluorescent antibody Biolegend, cat: 304220
  • anti-hIL-10R fluorescent antibody Biolegend, cat: 501404
  • PE Rat IgG1, ⁇ isotype control antibody Isotype Control Antibody
  • Anti-CD3 mouse Monoclonal antibody self-produced
  • anti-CD28 mouse monoclonal antibody self-produced
  • 24-well plates pipettes and other conventional consumables for cell culture.
  • CD8+T cell isolation and culture Take about 1 ⁇ 10 8 freshly isolated PBMCs from normal humans, and isolate CD8+T cells according to the instructions of the CD8+T isolation kit. Coat anti-CD3 mouse monoclonal antibody at a concentration of 1 ⁇ g/mL in a 24-well plate in advance, adjust the cells to 3 ⁇ 10 6 cells/mL with X-VIVO medium containing 10% FBS, and add anti-CD28 mouse monoclonal antibody Its concentration is 1 ⁇ g/mL. Finally, the cells were cultured in a 24-well plate at 37°C in a carbon dioxide incubator with a volume of 1 mL/well.
  • CD8+T cells cultured in 24-well plates were collected on day 0, day 3 and day 6. After washing with PBS, add anti-CD8 fluorescent antibody, CD45RO fluorescent antibody, and anti-hIL-10R Fluorescent antibody, the control group was added with anti-CD8 fluorescent antibody, CD45RO fluorescent antibody, PE Rat IgG1, and ⁇ isotype control antibody. After incubating at 4°C for 30 minutes, the cells were washed again with PBS, and the expression of IL-10R was detected by flow cytometry.
  • the figure shows the expression of IL-10R on CD8+ T cells stimulated by the above method on day 0, day 3, and day 6: CD8+ T cells have less IL-10R on day 1, and CD8+ on day 3.
  • the IL-10R of T cells increased significantly, and the IL-10R of CD8+ T cells continued to increase significantly on the 6th day.
  • IL-10 fusion protein promotes the secretion of INF- ⁇ from CD8+ T cells after activation
  • CD8+T cell isolation and culture Take about 1 ⁇ 10 8 freshly isolated PBMCs from normal humans, and isolate CD8+T cells according to the instructions of the CD8+T isolation kit. Coat anti-CD3 mouse monoclonal antibody at a concentration of 1 ⁇ g/mL in a 24-well plate in advance, adjust the cells to 3 ⁇ 10 6 cells/mL with X-VIVO medium containing 10% FBS, and add anti-CD28 mouse monoclonal antibody Its concentration is 1 ⁇ g/mL. Finally, the cells were cultured in a 24-well plate at a volume of 1 mL/well in a carbon dioxide incubator at 37°C for three days.
  • Cell detection After isolating CD8+ T cells, take a small amount of cells, perform CD8 phenotype identification, and detect the purity of the separated cells.
  • IL-10 fusion protein stimulated CD8+ T cells after activation collect CD8+ T cells after three days of activation, and resuspend the cells with X-VIVO containing 10% FBS to a density of 2 ⁇ 10 6 cells/mL, with 250 ⁇ L/well
  • a certain dose of IL-10 fusion protein R0354, R0355, R0356 and isotype control protein R0359 were added at the same time, and the culture was continued for three days at 37°C in a carbon dioxide incubator.
  • the anti-CD3 mouse monoclonal antibody was added again to make the concentration 1 ⁇ g/mL, and the culture was continued for 4h.
  • INF ⁇ detection Collect the cell culture supernatant after reaching the end of the culture, and use the INF ⁇ detection kit to detect the INF ⁇ content according to the instructions of the INF ⁇ detection kit.
  • the picture on the right shows the donor’s CD8+ T cells in this experiment after being activated for 3 days with anti-CD3 antibody (10 ⁇ g/mL, coated in 96-well plate in advance) and anti-CD28 (1 ⁇ g/mL, free), and then used IL-10 fusion protein: INF ⁇ secretion of cells after R0354, R0355, and R0356 continue to be cultured for 3 days (R0359 is the isotype control).
  • the experimental results show that the three concentration gradients of IL-10 fusion protein (10/1/0.1 ⁇ g/mL) can stimulate the activated CD8+ T cells to secrete INF ⁇ , and the activity is R0355>R0354>R0356.
  • mice Female, Balb/c mice; RPMI 1640 medium (Gibco, 11875085), FBS (Gibco, 10091-148), 0.25% trypsin-EDTA (Gibco, 25200056), penicillin-streptomycin (Gibco, 15140122), DMSO (Sigma, D2650), DPBS (Hyclone, SH30028.02), CT26WT cells (Shanghai Cell Bank, TCM37)
  • CT26WT mouse colon cancer in vivo tumor model establishment complete medium: RPMI 1640 medium + 10% fetal bovine serum (Gibco, cat: 10091-148) + 1% double antibiotics (1:1 penicillin- Streptomycin) culture mouse colon cancer cell CT26WT, take the cells in the logarithmic growth phase, and stain with trypan blue to determine the number of viable cells. After collecting the cells, they were washed twice with serum-free RPMI 1640 medium to remove the complete medium. The forelimb axillary skin of female Balb/c mice was disinfected with 75% alcohol, and CT26WT mouse colon cancer cells with adjusted density were inoculated under the forelimb axillary skin.
  • the inoculation volume of each mouse was 0.2 mL.
  • Cell density setting According to the number of cells inoculated, Balb/c mice are divided into 3 groups, 5 in each group, namely: density 1 (1 ⁇ 10 5 cells/0.2 mL/mouse), density 2 (5 ⁇ 10 5 cells/0.2 mL/piece), density 3 (1 ⁇ 10 6 cells/0.2 mL/piece).
  • Example 7 In vivo anti-tumor evaluation of IL-10 fusion protein in tumor model
  • Example 7.1 In vivo anti-tumor evaluation of R0330, R0354, R0355, R0356 on CT26 mouse colon cancer
  • mice Female, Balb/c mice; CT26WT cells; RPMI 1640 medium (Gibco, 11875085), FBS (Gibco, 10091-148), 0.25% trypsin-EDTA (Gibco, 25200056), penicillin-streptomycin (Gibco, 15140122), DMSO (Sigma, D2650), DPBS (Hyclone, SH30028.02)
  • Cell culture culture mouse colon cancer cells (CT26WT) in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (Gibco), 1% glutamine and 1% penicillin-streptomycin (1:1) )in.
  • Inoculation Collect CT26WT cells in logarithmic growth phase and adjust the cell concentration to 1X10 5 /mL. Take 70 female BALB/C mice, subcutaneously inoculate CT26WT cells, the inoculation volume is 0.1mL/mouse, that is, 1X10 5 /mouse.
  • mice were randomly divided into 6 groups according to tumor volume, with 10 mice in each group, and the administration was started (see Table 2 below for the administration schedule).
  • Example 7.2 In vivo anti-tumor evaluation of R0356 on B16-F1 mouse melanoma
  • mice Female, C57BL/6 mice; B16-F1 cells (national experimental cell resource sharing platform); DMEM (Gibco, 11965084), FBS (Gibco, 10091-148), 0.25% trypsin-EDTA (Gibco, 25200056 ), penicillin-streptomycin (Gibco, 15140122), DMSO (Sigma, D2650), DPBS (Hyclone, SH30028.02)
  • Mouse melanoma cells (B16-F1) were cultured in DMEM medium (Gibco) containing 10% FBS (Gibco), 1% glutamine, and 1% penicillin-streptomycin.
  • Inoculation Collect B16-F1 cells in the logarithmic growth phase and adjust the cell concentration to 2X10 6 /mL.
  • (1) Take 20 female C57BL/6 mice and inoculate B16-F1 cells subcutaneously with a volume of 0.1 mL/mouse, that is, 2 ⁇ 10 5 /mouse.
  • the tumor volume was measured and recorded, and then the long and short diameters of the tumor were measured with vernier calipers twice a week. Use the formula: (1/2) X long diameter X (short diameter) 2 to calculate the tumor volume (see Table 5).
  • Example 7.3 Evaluation of anti-tumor activity of R0354, R0355, R0356 on MC38 mouse colon cancer
  • mice Female, C57BL/6 mice; MC38 cells (National Experimental Cell Resource Sharing Platform); RPMI Medium 1640 (Gibco, 11875085), FBS (Gibco, 10091-148), 0.25% trypsin-EDTA (Gibco, 25200056) , Penicillin Streptomycin (Gibco, 15140122), DMSO (Sigma, D2650), DPBS (Hyclone, SH30028.02)
  • MC38 Mouse colon cancer cells (MC38) were cultured in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (Gibco), 1% glutamine, and 1% penicillin-streptomycin.
  • MC38 cells of logarithmic growth phase were collected, and adjusted the cell concentration of 1X10 7 / mL.
  • (1) Take 30 female BALB/C mice and inoculate MC38 cells subcutaneously with a volume of 0.1 mL/mouse, that is, 1 ⁇ 10 6 /mouse.
  • mice with a uniform tumor size were selected into the group.
  • the tumor volume was measured and recorded, and then the long and short diameters of the tumor were measured with vernier calipers twice a week. Use the formula: (1/2) X long diameter X (short diameter) 2 to calculate the tumor volume (see Table 7).

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Abstract

本发明公开了一种改变效应功能的Fc变体及其融合蛋白，其Fc区包含一个或多个氨基酸改变，具有改变的效应功能；进一步的，公开了包含改变效应功能的Fc变体的融合蛋白，以及此类Fc变体或融合蛋白的制备方法，编码此类Fc变体或融合蛋白的核酸，包含所述核酸的表达载体，包含所述Fc变体或融合蛋白、核酸或表达载体的宿主细胞，包含此类Fc变体或融合蛋白的药物组合物，以及此类Fc变体或融合蛋白的用途。

Description

改变效应功能的Fc变体及其融合蛋白 技术领域
本发明涉及生物制药领域，尤其涉及一种改变效应功能的Fc变体，其Fc区包含一个或多个氨基酸改变，其具有改变的效应功能；进一步的，本发明涉及包含改变效应功能的Fc变体的融合蛋白。
背景技术
抗体的Fc区与许多Fc受体及配体相互作用，行使一系列重要的功能，称为效应器功能(effector function)。IgG抗体的Fc受体被称为FcγR，IgE的为FcεR，IgA的为FcαR等等。已经鉴定了FcγR的三个亚类：FcγRI(CD64)、FcγRII(CD32)和FcγRIII(CD16)。Fc受体的另一种类型是新生Fc受体(FcRn)。
由抗体Fc区介导的效应功能可以分为两种：(1)在抗体与抗原结合之后发挥作用的效应功能(这些功能涉及参与补体级联反应或Fc受体(FcR)-负荷细胞)；和(2)不依赖于抗原结合而发挥作用的效应功能(这些功能提供在血循环中的持续性和通过胞吞作用穿过细胞屏障的能力)。
近年来抗体Fc区常用来制备各种融合蛋白，融合蛋白的Fc段能够与各类免疫白细胞上表达的FcγRs受体结合，从而产生Fc段介导的细胞毒作用(ADCC)以及补体依赖的细胞毒性(CDC)效应，靶向杀伤功能蛋白受体阳性细胞，会影响融合蛋白发挥活性而为减弱治疗类融合蛋白Fc段与FcγRs受体的亲和力而设计的基因突变，通常会导致融合蛋白与FcRn受体亲和力的下降，降低其在体内的半衰期。
发明内容
本发明旨在至少一定程度上解决相关技术中的技术问题之一。为此，在本发明的第一方面，提供了一种Fc变体，旨在减弱Fc与各类免疫白细胞上表达的Fc受体，即FcγRs(主要包括FcR gamma I,FcR gamma IIa,FcR gamma IIb以及FcR gamma IIIa)之间的亲和力，从而降低或者消除Fc段介导的细胞毒作用(antibody-dependent cellmediated cytotoxicity，ADCC)或结合补体C1q介导的补体依赖的细胞毒性(complement-dependent cytotoxicity，CDC)效应，并且保 持其与新生儿Fc受体(neonatal Fc receptor，FcRn)的亲和力不变或增强，不影响体内半衰期。
根据本发明的实施例，其中依照EU编号方式，所述Fc变体在选自第223，235，237，238，265，297，327，434位的至少一处包含氨基酸取代。
根据本发明的实施例，所述Fc为IgG Fc，其中IgG Fc为IgG1 Fc，IgG2 Fc，IgG3Fc，IgG4 Fc中的一种，优选是IgG1 Fc，更优选人IgG1 Fc。
根据本发明的实施例，其中所述氨基酸取代选自E223P，L235A，G237A，P238A，D265A，N297A，A327Q，和N434A中的至少一种。
根据本发明实施例，其中所述氨基酸取代选自E223P，L235A，G237A，P238A，D265A，N297A，A327Q，和N434A中的至少三种。
根据本发明实施例，所述氨基酸取代为选自：E223P，P238A，D265A；N297A，L235A，N434A；和A327Q，G237A，L235A的三氨基酸取代。
在本发明的第二方面，本发明提供了一种Fc变体融合蛋白，具体地，本发明涉及如下实施方式：
实施方案1.一种融合蛋白，其结构为如下式所示：
A-肽接头-B或B-肽接头-A(I)
其中A为IL-10，IL-6，或IL-12，肽接头为(GS) n，其中n为1-10的整数，B为IgG Fc。
实施方案2.实施方案1所述的融合蛋白，其中所述A是IL-10，优选人IL-10。
实施方案3.实施方案1-2任意一项所述的融合蛋白，其中IL-10通过所述肽接头连接在IgG Fc的N末端或者C末端。
实施方案4.实施方案1所述的融合蛋白，其中IgG Fc为IgG1 Fc，IgG2 Fc，IgG3Fc，IgG4 Fc中的一种，优选是IgG1 Fc，更优选人IgG1 Fc。
实施方案5.实施方案1-4任意一项所述的融合蛋白，其中所述IL-10的氨基酸序列是SEQ ID NO：17。
实施方案6.前述实施方案任一项的融合蛋白，其中所述IgG Fc经过修饰，与野生型IgG Fc相比，修饰后IgG Fc削弱了所述融合蛋白与FcγR的亲和力。
实施方案7.实施方案6所述的融合蛋白，其中依照EU编号方式，所述IgG Fc在选自第223，235，237，238，265，297，327，434位的至少一处包含氨基酸取代。
实施方案8.实施方案7所述的融合蛋白，其中所述氨基酸取代选自E223P，L235A，G237A，P238A，D265A，N297A，A327Q，和N434A中的至少一种。
实施方案9.实施方案6或7所述的融合蛋白，其中所述氨基酸取代选自E223P，L235A，G237A，P238A，D265A，N297A，A327Q，和N434A中的至少三种。
实施方案10.实施方案9所述的融合蛋白，所述氨基酸取代为选自：E223P，P238A，D265A；N297A，L235A，N434A；和A327Q，G237A，L235A的三氨基酸取代。
实施方案11.实施方案10所述的融合蛋白，其中所述融合蛋白的氨基酸序列为SEQ ID NO:2、SEQ ID NO:4、或SEQ ID NO:6。
在本发明的第三方面，本发明提供了一种核酸，其编码上述任意一种Fc变体或任意一种Fc变体融合蛋白。
根据本发明实施例，其中所述核酸的核苷酸序列为SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:20、SEQ ID NO:21、或SEQ ID NO:22。
在本发明的第四方面，本发明提供了一种药物组合物，其包含上述任意一种Fc变体或任意一种Fc变体融合蛋白和药学上可接受的载体。
根据本发明的第五方面，本发明提供了一种上述任意一种药物组合物在制备用于治疗有此需要的个体中癌症的药物中的用途。
根据本发明实施例，其中所述癌症为实体瘤。
根据本发明实施例，其中所述实体瘤为结肠癌，黑色素瘤，结肠直癌，乳腺癌。
根据本发明实施例，其中所述个体是人。
定义
除非在下文另外定义，术语在本文中如本领域中一般使用的那样使用。
本文中术语“Fc”或“Fc区”用于定义抗体重链中至少含有恒定区的一部分的C端区域。该术语包括天然序列Fc区和变体Fc区。IgG Fc区包含IgG CH2和 IgG CH3域。人IgG Fc区的“CH2域”通常自大约位置231处的氨基酸残基延伸至大约位置340处的氨基酸残基。除非本文中另外指定，Fc区或恒定区中氨基酸残基的编号方式依照EU编号系统，也称为EU索引，如记载于Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD,1991。
本文所述的Fc包括野生型Fc以及经过修饰的Fc，例如，与野生型IgG Fc相比，修饰后的IgG Fc削弱了本文所述的融合蛋白与FcγR的亲和力。亲和力可以使用本领域公知的方法或者本文所公开的方法测定。
氨基酸“取代”指多肽中一个氨基酸用另一种氨基酸替换。在一个实施方案中，氨基酸用具有相似结构和/或化学特性的另一种氨基酸替换，例如保守氨基酸替换。“保守”氨基酸取代可以在所涉及残基的极性、电荷、溶解性、疏水性、亲水性、和/或两亲性性质的相似性基础上进行。例如，非极性(疏水性)氨基酸包括丙氨酸、亮氨酸、异亮氨酸、缬氨酸、脯氨酸、苯丙氨酸、色氨酸、和甲硫氨酸；极性中性氨基酸包括甘氨酸、丝氨酸、苏氨酸、半胱氨酸、酪氨酸、天冬酰胺、和谷氨酰胺；带正电荷(碱性)氨基酸包括精氨酸、赖氨酸、和组氨酸；而带负电荷(酸性)氨基酸包括天冬氨酸和谷氨酸。非保守取代会需要用这些类别之一的成员交换另一个类别的成员。例如，氨基酸取代还可导致将一个氨基酸用具有不同结构和/或化学特性的另一种氨基酸替换，例如将来自一个组(例如极性)的氨基酸用来自一个不同组(例如碱性)的另一种氨基酸替换。可以使用本领域公知的遗传或化学方法来生成氨基酸取代。遗传方法可包括定点诱变、PCR、基因合成等等。例如E223P表示Fc中第233位的E被P替换，P238A表示Fc中第238位的P被A。
“IgG类抗体”指具有天然存在免疫球蛋白G(IgG)分子的结构的抗体。IgG类抗体的抗体重链具有域结构VH-CH1-CH2-CH3。IgG类抗体的抗体轻链具有域结构VL-CL。IgG类抗体基本上由经由免疫球蛋白铰链区连接的两个Fab片段和一个Fc域组成。IgG类抗体包括例如IgG1、IgG2、IgG3和IgG4。IgG1 Fc，IgG2 Fc，IgG3Fc，IgG4 Fc分别表示IgG1、IgG2、IgG3和IgG4的Fc或Fc区。本文中融合蛋白中的IgG Fc可以为IgG1 Fc，IgG2 Fc，IgG3Fc，IgG4 Fc，优选是IgG1 Fc，更优选人IgG1 Fc。
如本文中使用的，术语“融合蛋白”指包含IL-10、IL-6或IL-12分子和IgG1的Fc部分形成的融合多肽分子，其中融合蛋白的组件通过肽键彼此连接，或是直接地或是经由肽接头。
术语“肽接头”为包含一个或多个氨基酸，通常约2-20个氨基酸的肽。肽接头是本领域中已知的或本文中记载的。合适的、非免疫原性的肽接头包括例如(GS) n接头，其中n为1-10的整数。在一个实施方案中，n为1-6，优选4。
“融合”意指各组分直接地或经由一种或多种肽接头通过肽键连接。
“亲和力”或“结合亲和力”指分子的单一结合位点与其结合配偶体之间非共价相互作用总和的强度。除非另外指示，如本文中使用的，“结合亲和力”指反映结合对的成员之间1:1相互作用的内在结合亲和力。分子X对其配偶体Y的亲和力通常可以以解离常数(K D)来表述，其为解离与结合速率常数(分别为K 解离和K 结合)的比率。如此，相等的亲和力可能包含不同的速率常数，只要速率常数的比率保持相同。亲和力可以通过本领域知道的常用方法来测量，包括本文中描述的那些方法。用于测量亲和力的一种具体方法是表面等离振子共振(SPR)。
“多核苷酸”或“核酸”在本文中可互换使用，指任何长度的核苷酸聚合物，而且包括DNA和RNA。核苷酸可以是脱氧核糖核苷酸、核糖核苷酸、经过修饰的核苷酸或碱基、和/或它们的类似物，或能由DNA或RNA聚合酶或通过合成反应掺入聚合物的任何底物。多核苷酸可包含经过修饰的核苷酸，诸如甲基化核苷酸及其类似物。核苷酸序列可以被非核苷酸构件中断。多核苷酸可包含合成后进行的修饰，诸如缀合至标记物。
术语“修饰”指对肽主链(例如氨基酸序列)的任何操作或对多肽的翻译后修饰(例如糖基化)。修饰也包括氨基酸序列中氨基酸的取代、缺失或插入。
“天然IL-10”(也称作“野生型IL-10”)意指天然存在IL-10,与“经修饰IL-10”相反，后者自天然存在IL-10经过修饰，例如为了改变它的一项或多项特性，诸如稳定性。经修饰IL-10分子可以例如包含氨基酸序列中的修饰，例如氨基酸取代、缺失或插入。“天然IL-6”(也称作“野生型IL-6”)意指天然存在IL-6,与“经修饰IL-6”相反，后者自天然存在IL-6经过修饰，例如为了改变它的一项或多项特性，诸如稳定性。经修饰IL-6分子可以例如包含氨基酸序列中的修饰，例如氨基酸取代、缺失或插入。“天然IL-12”(也称作“野生型IL-12”)意指天然存在IL-12,与“经修饰IL-12”相反，后者自天然存在IL-12经过修饰，例如为了改变它的一项或多项特性， 诸如稳定性。经修饰IL-12分子可以例如包含氨基酸序列中的修饰，例如氨基酸取代、缺失或插入。
如本文中所使用的，术语“载体”指能够增殖与其连接的另一种核酸的核酸分子。该术语包括作为自身复制型核酸结构的载体及整合入接受其导入的宿主细胞的基因组中的载体。某些载体能够指导与其可操作连接的核酸的表达。此类载体在本文中称为“表达载体”。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可交换使用并指已引入外源核酸的细胞，包括这类细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括初始转化的细胞和自其衍生的后代(不考虑传代数)。后代在核酸内含物上可能与亲本细胞不完全相同，但可以含有突变。本文中包括具有如原始转化细胞中筛选或选择的相同的功能或生物学活性的突变体后代。宿主细胞是能用于生成本发明的融合蛋白的任意类型的细胞系统。宿主细胞包括培养的细胞，例如哺乳动物培养细胞如CHO细胞、BHK细胞、NS0细胞、SP2/0细胞、YO骨髓瘤细胞、P3X63小鼠骨髓瘤细胞、PER细胞、PER.C6细胞或杂交瘤细胞、酵母细胞、细菌细胞，如大肠杆菌，昆虫细胞和植物细胞等，而且还包括在转基因动物、转基因植物或培养的植物或动物组织中包含的细胞。
药剂的“有效量”指引起接受其施用的细胞或组织中的生理学变化必需的量。
药剂例如药物组合物的“治疗有效量”指有效实现期望的治疗或预防结果的量(以必要的剂量且持续必要的时间)。治疗有效量的药剂例如消除、降低、延迟、最小化或预防疾病的不良作用。
“个体”或“受试者”是哺乳动物。哺乳动物包括但不限于驯养的动物(例如牛、绵羊、猫、犬和马)、灵长类(例如人和非人灵长类如猴)、家兔和啮齿动物(例如小鼠和大鼠)。特别地，所述个体或受试者是人。
术语“药物组合物”指其形式使得容许其中含有的活性成分的生物学活性有效，且不含对会接受配制剂施用的受试者有不可接受的毒性的别的成分的制剂。
“药学上可接受的载体”指药物组合物中活性成分以外的对受试者无毒的成分。药学上可接受的载体包括但不限于缓冲剂、赋形剂、稳定剂或防腐剂。
如本文中使用的，“治疗/处理”指试图改变治疗个体中疾病的自然进程，并且可以是为了预防或在临床病理学的过程期间实施的临床干预。治疗的期望效果包括但不限于预防疾病的发生或复发、缓解症状、降低疾病的任何直接或间接病理 学后果、预防转移、减缓疾病进展速率、改善或减轻疾病状态、及消退或改善的预后。
附图说明
图1.质粒pcDNA3.4A(a),pcDNA3.4A-R0354(b),pcDNA3.4A-R0355(c),pcDNA3.4A-R0356(d),pcDNA3.4A-R0359VCHE(e)，pcDNA3.4A-R0359VCLE(f)的图谱，以及pcDNA3.4A-R0330(g)。
图2.信号肽、R0354、R0355、R0356、R0359以及R0330分子的基因序列。
图3.R0354,R0355,R0356，R0359(由R0359VCHE和R0359VCLE组成)以及R0330和人IL-10的氨基酸序列。
图4.纯度分析及层析图谱：a).亲和洗脱物SE-HPLC纯度；b).亲和洗脱pH处理后SE-HPLC纯度；c).精细纯化后洗脱组分SE-HPLC纯度；d)合并的洗脱组分的层析图谱。
图5.活化后CD8+T细胞IL-10R增加。
图6.自产IL-10融合蛋白分子促进CD8+T细胞分泌INFγ。
图7.小鼠体内抗肿瘤活性：a)小鼠结肠癌(CT26WT)抗肿瘤模型；b)小鼠结肠癌(CT26WT)肿瘤生存曲线；c)小鼠黑色素瘤(B16-F1)抗肿瘤模型；d)小鼠结肠癌(MC38)抗肿瘤模型。
具体实施方式
为使本发明的上述目的、特征和优点能够更加浅显易懂，下面结合附图对本发明的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本发明。但是本发明能够以很多不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似改进，因此本发明的保护范围以权利要求书为准，不受下面公开的具体实施的限制。
Fc变体
在本发明的一个实施方案中，公开了一种Fc变体，所述Fc变体为IgG Fc，其中IgG Fc为IgG1 Fc，IgG2 Fc，IgG3Fc，IgG4 Fc中的一种，优选是IgG1 Fc，更优选人IgG1 Fc。
在本发明的一个实施方案中，其中依照EU编号方式，所述IgG Fc在选自第223，235，237，238，265，297，327，434位的至少一处包含氨基酸取代。
在本发明的一个实施方案中，其中所述氨基酸取代选自E223P，L235A，G237A，P238A，D265A，N297A，A327Q，和N434A中的至少一种。
在本发明的一个实施方案中，其中所述氨基酸取代选自E223P，L235A，G237A，P238A，D265A，N297A，A327Q，和N434A中的至少三种。
在本发明的一个实施方案中，所述氨基酸取代为选自：E223P，P238A，D265A；N297A，L235A，N434A；和A327Q，G237A，L235A的三氨基酸取代。
本领域技术人员还可以理解的，一些Fc区位点的突变不会影响Fc受体的亲和力，例如，常见的Fc变体还包含K447位的缺失，或为了在Fc的C末端连接其他蛋白而致K447A突变，Fc区447位缺失或突变不会影响Fc变体与Fcγ受体的亲和力和/或FcRn的亲和力，除以上实施方式外，包含447位缺失或突变的Fc变体或其他不影响Fcγ受体亲和力的突变位点，也在本发明保护范围内。
融合蛋白
Fc融合蛋白的形式有多种，本发明Fc变体可根据需要制成任何形式的融合蛋白，或与其他多种类型的蛋白质进行融合，本发明公开的融合蛋白的实施例仅是其中的一种范例，不对本发明的保护范围造成限制。
在本发明的一个实施方案中，公开了一种融合蛋白，其结构为如下式所示：
A-肽接头-B或B-肽接头-A(I)
其中A为IL-10，IL-6或IL-12，肽接头为(GS) n，其中n为1-10的整数，B为IgG Fc。
在本发明的一个实施方案中，A也可以为IL-10，IL-6或IL-12的活性片段。
在本发明的进一步实施方案中，IgG1 Fc包含选自如下组成的组的三氨基酸取代：E223P，P238A，D265A；N297A，L235A，N434A；和A327Q，G237A，L235A。
在本发明的实施方案中，IL-10可以为动物，例如哺乳动物，例如人的天然IL-10，或者IL-10经取代、插入一个或多个氨基酸所得到的经修饰的IL-10。IL-10活性片段是IL-10指天然IL-10或经修饰的IL-10从N-端或C端或者从N-端和C-端截掉一个或多个氨基酸后所得到的肽链，该肽链保留IL-10的功能活性。本 领域技术人员可以通过常规方法得到所述活性片段，如通过化学合成或重组表达方法得到IL-10的片段，并通过本领域公知的或本申请公开的方法测量IL-10片段是否具有原IL-10的活性。
本发明的多核苷酸和核酸编码区可以与编码信号肽的另外的编码区联合，所述信号肽指导由本发明的多核苷酸编码的多肽的分泌。例如，如果期望分泌所述融合物，那么可以将编码信号序列的DNA置于编码本发明融合蛋白或其片段的核酸上游。依照信号假说，由哺乳动物细胞分泌的蛋白质具有信号肽或分泌前导序列，一旦启动将生长的蛋白质链跨越粗面内质网输出，就将该信号肽或分泌前导序列从成熟的蛋白质切去。本领域中普通技术人员知晓由脊椎动物细胞分泌的多肽一般具有融合至多肽N端的信号肽，其从所翻译的多肽切去以生成分泌性或“成熟”形式的多肽。信号肽可以是通用信号肽，例如，IgG通用信号肽。例如，所述信号肽的编码序列可以是ATGGGATGGTCATGCATAATACTCTTTCTTGTGGCTACTGCTACCGGGGTTC ACTCT(SEQ ID NO:11)。
在本发明的实施方案中，A-肽接头-B表示肽接头分别连接A的羧基端(C端，或3’端)和B的氨基端(N端，或5’端)，B-肽接头-A表示肽接头分别连接B的羧基端(C端，或3’端)和A的氨基端(N端，或5’端)。
在另一个实施方案中，n为1-6，例如，1，2，3，4，5，6，优选n为4。
在另一个实施方案中，IgG1 Fc为人IgG1 Fc。虽然IgG类抗体的Fc赋予融合蛋白以有利的药动学特性，包括长血清半衰期(归于靶组织中较好的积累和有利的组织-血液分布比)，但是它可同时引起不想要的将融合蛋白靶向表达Fc受体的细胞，而非目的细胞。此外，Fc受体信号传导途径的激活可引起导致(促炎症)细胞因子受体激活的细胞因子释放和系统性施用时的严重副作用。本发明的融合蛋白不仅保留了IL-10的免疫抗肿瘤活性，更重要的是对Fc段定点基因突变进行优化组合，减弱了IL-10-Fc融合蛋白与各类免疫白细胞上表达的Fc受体，即FcγRs(主要包括FcR gamma I,FcR gamma IIa,FcR gamma IIb以及FcR gamma IIIa)之间的亲和力，从而降低或者消除Fc段介导的细胞毒作用(ADCC)或结合补体C1q介导的补体依赖的细胞毒性效应(CDC)，从而不产生细胞毒性，避免效应T细胞被杀伤，最终有效的提高了IL-10抗肿瘤效果。评估感兴趣分子的ADCC活性的体外测定法的例子记载于美国专利No.5,500,362；Hellstrom et  al.Proc Natl Acad Sci USA 83,7059-7063(1986)和Hellstrom et al.,Proc Natl Acad Sci USA 82,1499-1502(1985)；美国专利No.5,821，337；Bruggemann et al.J Exp Med 166,1351-1361(1987)。为了评估补体激活，可实施CDC测定法(参见例如Gazzano-Santoro et al.J Immunol Methods 202,163(1996)；Cragg et al.,Blood 101,1045-1052(2003)；和Cragg and Glennie，Blood 103,2738-2743(2004))。
在一个优选的实施方案中，IL-10为人IL-10或h IL-10。
在一个优选的实施方案中，IL-6为人IL-6或h IL-6。
在一个优选的实施方案中，IL-12为人IL-12或h IL-12。
在一个优选的实施方案中，所述IgG1 Fc所包含的三氨基酸取代为E223P，P238A，D265A；N297A，L235A，N434A；或A327Q，G237A，L235A。其中编号为对Fc的氨基酸编号，所述编号根据EU编号系统进行。
在另一个优选的实施方案中，所述融合蛋白的氨基酸序列为SEQ ID NO:2、SEQ ID NO:4、或SEQ ID NO:6。
核酸
在一个实施方案中，本发明涉及编码本发明Fc变体或融合蛋白的核酸。本领域技术人员公知，由于遗传密码的简并性，编码一种融合蛋白的核酸可以有很多种。本领域技术人员可以根据宿主细胞对核酸序列进行优化，使得核酸序列在宿主细胞中能够以优化的水平表达，例如，具有较高的表达水平。为了实现例如融合蛋白的表达，编码融合蛋白的核酸序列可以还可以与启动子，内含子，增强子等调节元件有效连接，使得这些调节元件在宿主细胞中发挥各自的功能。融合蛋白还可以包含信号肽使得所编码的融合蛋白能够分泌到培养液中，从而通过离心获得上清液，并从上清液中进一步纯化所述融合蛋白。包含融合蛋白编码序列、调节元件和信号肽编码序列的核酸也在本发明的核酸的范围内。
在一个优选的实施方案中，所述核酸的核苷酸序列为SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:20、SEQ ID NO:21、或SEQ ID NO:22。
在一个实施方案中，本发明涉及包含编码本发明Fc变体或融合蛋白的核酸的表达载体。
表达载体可以是质粒、病毒的一部分或可以是核酸片段。表达载体包含表达盒，其中在与启动子和/或其它转录或翻译控制元件的有效连接中克隆编码融合蛋 白(片段)的多核苷酸(即编码区)。当基因产物例如多肽的编码区与一种或多种调节序列以某种方式联合从而使得该基因产物的表达置于该调节序列的影响或控制下时，即为有效连接。若诱导启动子功能导致编码期望的基因产物的mRNA的转录并且如果两个DNA片段之间的连接的性质不干扰表达调节序列指导该基因产物表达的能力或不干扰DNA模板被转录的能力，则两个DNA片段(如多肽编码区和与其联合的启动子)为“有效连接的”。如此，如果启动子能够实现编码多肽的核酸的转录，那么该启动子区将是与该核酸有效连接。所述启动子可以是细胞特异性启动子，其仅在预先确定的细胞中指导DNA的实质性转录。除启动子以外，其它转录控制元件例如增强子、操纵基因、阻遏物和转录终止信号能与多核苷酸有效连接以指导细胞特异性转录。多种转录控制区是本领域技术人员已知的。这些包括但不限于在脊椎动物细胞中发挥功能的转录控制区，如但不限于来自巨细胞病毒的启动子和增强子区段(例如立即早期启动子，与内含子-A连接)、猿病毒40(例如早期启动子)和逆转录病毒(如例如劳斯(Rous)肉瘤病毒)。其它转录控制区包括那些自脊椎动物基因如肌动蛋白、热休克蛋白、牛生长激素和家兔阳β蛋白衍生的，以及能够控制真核细胞中基因表达的其它序列。另外的合适的转录控制区包括组织特异性启动子和增强子以及诱导型启动子(例如四环素诱导型启动子)。类似地，多种翻译控制元件是本领域普通技术人员已知的。这些包括但不限于核糖体结合位点、翻译起始和终止密码子以及自病毒系统衍生的元件(具体地，内部核糖体进入位点或IRES，亦称为CITE序列)。表达盒还可以包含其它特征，如复制起点和/或染色体整合元件，如逆转录病毒长末端重复(LTR)或腺伴随病毒(AAV)反向末端重复(ITR)。
本发明的多核苷酸和核酸编码区可以与编码分泌肽或信号肽的另外的编码区连接，所述分泌或信号肽指导由本发明的多核苷酸编码的多肽的分泌。例如，如果期望分泌所述融合蛋白，那么可以将编码信号序列的DNA置于编码本发明融合蛋白或其片段的核酸上游。依照信号假说，由哺乳动物细胞分泌的蛋白质具有信号肽或分泌前导序列，一旦启动将生长的蛋白质链跨越粗面内质网输出，就将该信号肽或分泌前导序列从成熟的蛋白质切去。本领域中普通技术人员知晓由脊椎动物细胞分泌的多肽一般具有融合至多肽N端的信号肽，其从所翻译的多肽切去以生成分泌性或“成熟”形式的多肽。在某些实施方案中，使用天然的信号肽，例如免疫球蛋白重链或轻链信号肽，或该序列的保留指导与其有效连接的多肽分 泌的能力的功能性衍生物。或者，可以使用异源哺乳动物信号肽或其功能性衍生物。例如，可以将野生型前导序列用人组织血纤维蛋白溶酶原激活剂(TPA)或小鼠β-葡糖醛酸糖苷酶的前导序列取代。
可以将编码能用于促进后期纯化(例如组氨酸标签)或辅助标记融合蛋白的短蛋白序列的DNA纳入融合蛋白(片段)编码多核苷酸内或其末端。
在一个实施方案中，提供包含本发明的一种或多种多核苷酸的宿主细胞。在某些实施方案中，提供包含本发明的一种或多种载体的宿主细胞。多核苷酸和载体可以单独地或组合地掺入本文中分别关于多核苷酸和载体所描述的任何特征。在一个这类实施方案中，宿主细胞包含载体(例如已用该载体转化或转染)，所述载体包含编码本发明融合蛋白(的部分)的多核苷酸。如本文中使用的，术语“宿主细胞”指任何能工程化以产生本发明的融合蛋白或其片段的细胞系统种类。适用于复制并支持融合蛋白表达的宿主细胞是本领域中公知的。在适当时，可用特定的表达载体转染或转导这类细胞，并且可以培养大量的含载体细胞以用于接种大规模发酵罐，从而获得充足量的融合蛋白用于临床应用。合适的宿主细胞包括原核微生物如大肠杆菌，或各种真核细胞，如中国仓鼠卵巢细胞(CHO)、昆虫细胞等。例如，可以在细菌中生成多肽，尤其在不需要糖基化时。在表达后，可以将多肽在可溶性级分中从细菌细胞糊分离并可以进一步纯化。除了原核生物外，真核微生物如丝状真菌或酵母也是适合编码多肽的载体的克隆或表达宿主，包括其糖基化途径已被“人源化”，导致生成具有部分或完全人的糖基化模式的多肽的真菌和酵母菌株。参见Gerngross,Nat Biotech22,1409-1414(2004)，和Li etal.,Nat Biotech 24,210-215(2006)。适用于表达(糖基化)多肽的宿主细胞还自多细胞生物体(无脊椎动物和脊椎动物)衍生。无脊椎动物细胞的例子包括昆虫细胞。已鉴定出可与昆虫细胞一起使用的大量杆状病毒株，特别是用于转染草地贪夜蛾(Spodoptera frugiperda)细胞。也可以将植物细胞培养物用作宿主。参见例如美国专利号5,959,177,6,040,498,6,420,548,7,125,978和6,417,429(描述用于在转基因植物中生成抗体的PLANTIB0DIES TM技术)。脊椎动物细胞也可以用作宿主。例如，适应于在悬液中生长的哺乳动物细胞系可以是有用的。可用的哺乳动物宿主细胞系的其它例子是由SV40转化的猴肾CV1系(COS-7)；人胚肾系(293或293T细胞，如例如记载于Graham et al.,J Gen Virol 36,59(1977))、幼仓鼠肾细胞(BHK)、小鼠塞托利(sertoli)细胞(TM4细胞，如例如记载于Mather,Biol  Reprod 23,243-251(1980)的)、猴肾细胞(CV1)、非洲绿猴肾细胞(VERO-76)、人宫颈癌细胞(HELA)、猕猴肾细胞(MDCK)，牛鼠肝细胞(BRL3A)、人肺细胞(W138)、人肝细胞(HepG2)、小鼠乳房肿瘤细胞(MMT 060562)、TRI细胞(如例如记载于Mather et al.,Annals N.Y.Acad Sci 383,44-68(1982)的)、MRC5细胞和FS4细胞。其它可用的哺乳动物宿主细胞系包括中国仓鼠卵巢(CH0)细胞，包括dhfr -CHO细胞(Urlaub et al.，Proc Natl Acad Sci USA77,4216(1980))；和骨髓瘤细胞系如YO、NS0、P3X63和Sp2/0。对于某些适用于蛋白质生产的哺乳动物宿主细胞系的综述，参见例如Yazaki and Wu,Methods in Molecular Biology,Vol.248(B.K.C.Lo,ed.,Humana Press,Totowa，NJ)，pp.255-268(2003)。宿主细胞包括培养的细胞，例如哺乳动物培养细胞、酵母细胞、昆虫细胞、细菌细胞和植物细胞等，但还包括在转基因动物、转基因植物或培养的植物或动物组织中包含的细胞。在一个实施方案中，宿主细胞是细菌细胞，优选为细胞埃希氏菌属细胞，特别优选大肠杆菌细胞。
可以通过本领域已知的技术来纯化如本文中描述的那样制备Fc变体或融合蛋白，所述技术如高效液相层析、离子交换层析、凝胶电泳、亲和层析、大小排阻层析等。用于纯化具体蛋白质的实际条件将部分取决于因素，如净电荷、疏水性、亲水性等，而且对于本领域中的技术人员将是明显的。对于亲和层析纯化，能使用融合蛋白结合的抗体、配体、受体或抗原。可以通过多种公知的分析方法中的任一种来测定融合蛋白的纯度，包括凝胶电泳、高压液相层析等。
组合物、配制剂和施用路径
在一个另外的方面，本发明提供包含本文中提供的任何Fc变体或融合蛋白的药物组合物。在一个实施方案中，药物组合物包含本文中提供的任何Fc变体或融合蛋白以及药学上可接受载体。
治疗方法和组合物
可以将本文中提供的任一种融合蛋白用在治疗方法中。
对于在治疗方法中的使用，将以与优良医学实践一致的方式配制、给药和施用本发明的融合蛋白。在此背景中考虑的因素包括治疗的特定病症、治疗的特定哺乳动物、个体患者的临床状况、病症的起因、药剂的投递部位、施用方法、施用时间安排以及医学从业人员已知的其它因素。
在一个方面，提供用作药物的本发明的融合蛋白。在别的方面，提供用于治疗疾病的本发明的融合蛋白。在某些实施方案中，提供用于治疗方法的本发明的融合蛋白。在一个实施方案中，本发明提供如本文中描述的融合蛋白，用于治疗有此需要的个体中的疾病。在某些实施方案中，本发明提供融合蛋白，用于治疗患有疾病的个体的方法，所述方法包括对所述个体施用治疗有效量的融合蛋白。在某些实施方案中，待治疗的疾病是癌症。例示性癌症包括结肠癌、黑色素瘤、结肠直癌和乳腺癌。在某些实施方案中，所述方法进一步包括对个体施用治疗有效量的至少一种另外的治疗剂，例如抗癌剂。依照上文任意实施方案的“个体”是哺乳动物，优选是人。
在一个别的方面，本发明提供本发明的融合蛋白在制造或制备药物中的用途，所述药物用于治疗有此需要的个体中的疾病。在一个实施方案中，所述药物用于治疗疾病的方法，该方法包括对患疾病的个体施用治疗有效量的药物。在某些实施方案中，待治疗的疾病是癌症。在一个特定实施方案中，该疾病为结肠癌。在一个特定实施方案中，该疾病为黑色素瘤。在一个特定实施方案中，该疾病为结肠直癌。在一个特定实施方案中，该疾病为乳腺癌。在一个实施方案中，所述方法进一步包括对个体施用治疗有效量的至少一种另外的治疗剂，例如抗癌剂。依照上文任意实施方案的“个体”是哺乳动物，优选是人。
在一个别的方面，本发明提供用于在个体中治疗疾病的方法，包括对所述个体施用治疗有效量的本发明的融合蛋白。在一个实施方案中，对所述个体施用组合物，其包含药学可接受的形式的本发明的融合蛋白。在某些实施方案中，待治疗的疾病是癌症。在一个特定实施方案中，该疾病为结肠癌。在一个特定实施方案中，该疾病为黑色素瘤。在一个特定实施方案中，该疾病为结肠直癌。在一个特定实施方案中，该疾病为乳腺癌。在某些实施方案中，所述方法进一步包括对个体施用治疗有效量的至少一种另外的治疗剂，例如抗癌剂。依照上文任意实施方案的“个体”是哺乳动物，优选是人。
在一些实施方案中，对细胞施用有效量的本发明的融合蛋白。在其它实施方案中，对个体施用治疗有效量的本发明的融合蛋白以治疗疾病。
为了预防或治疗疾病，本发明的融合蛋白的合适剂量(当单独或与一种或多种其它另外的治疗剂组合使用时)将取决于待治疗疾病的类型、施用路径、患者的体重、融合蛋白的类型、疾病的严重程度和进程、施用融合蛋白是为了预防还是治 疗目的、先前或同时的治疗干预、患者的临床史和对融合蛋白的响应、以及主治医师的判断。负责施用的从业人员将在任何事件中确定组合物中活性成分的浓度和用于个体受试者的合适剂量。本文中涵盖各种给药方案，包括但不限于在各个时间点的单次或多次施用、推注施用、和脉冲输注。
以下实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
实施例：
除非另有说明，实施例中的百分比浓度为质量体积百分比浓度。例如，2％琼脂糖凝胶表示100ml凝胶中含有2g琼脂糖。10％胎牛血清表示100ml体积中含有10g胎牛血清。
为此,我们开发了能显著提高人的白细胞介素-10融合蛋白类药物的免疫抗肿瘤活性的分子构建及其评价方法，具体如下：
实施例1：IL-10-hFc融合蛋白分子构建
对人源IgG1抗体的Fc段依据EU编号方式进行定点突变，构建PCDNA3.4A-R0354，PCDNA3.4A-R0355以及PCDNA3.4A-R0356三个质粒，其中三个目的片段的分子结构如下：R0354：IgG通用信号肽-IL10-GSGSGSGS-hIgG1 Fc(铰链区+CH2+CH3,含E223P，P238A，D265A突变)；R0355：IgG通用信号肽-IL10-GSGSGSGS-hIgG1 Fc(铰链区+CH2+CH3,含N297A，L235A，N434A突变)；R0356：IgG通用信号肽-IL10-GSGSGSGS-hIgG1 Fc(铰链区+CH2+CH3,含A327Q，G237A，L235A突变)。同时构建其同型对照PCDNA3.4A-R0359和PCDNA3.4A-R0330。PCDNA3.4A-R0359包括PCDNA3.4A-R0359VCHE和PCDNA3.4A-R0359VCLE,分子结构如下PCDNA3.4A-R0359VCHE:IgG通用信号肽-anti-HIV VH-hIgG1.3CH(CH1+铰链区+CH2+CH3),PCDNA3.4A-R0359VCLE:IgG通用信号肽-anti-HIV VL-hIgKC，同型对照PCDNA3.4A-R0359所表达的多肽为 R0359。R0330分子结构如下:IgG通用信号肽-IL10-GSGSGSGS-hIgG1 Fc(铰链区+CH2+CH3,含L234A，L235E,G237A,并在447位缺失K,为百时美施贵宝公司Fc变体，作为本发明的对照)；
以上构建质粒图谱见图1，各融合蛋白核酸序列见图2，氨基酸序列见图3。
实验中所用主要仪器：PCR仪(Tprofessional TR20)；电泳仪(DYY-TC)；凝胶成像仪(Smart Gel N)；离心机(H1650-W)；微量恒温器(HW-8C)；超净工作台(SDJ系列)；恒温振荡器(H2-9211K)；恒温水浴锅(HH-4A)；
实验中所用主要试剂:pcDNA TM3.4 TOPO TM TA Cloning Kit(Invitrogen A14697)；5-alpha感受态大肠杆菌(Competent E.coli)(NEB C2987I)；DL2000 DNA Marker(TAKARA 3427A)；Q5高保真DHA聚合酶(High-Fidelity DNA Polymerase)(NEB M0491L)；AxyPrep DNA凝胶提取试剂盒(Gel Extraction Kit)(AxyGEN AP-GX-50)；Hind III(NEB)；EcoRI(NEB)；T4DNA连接酶(TAKARA 2011A)；无内毒素质粒大提试剂盒(TIANGEN DP117)；
实验步骤
(1)基因合成
将R0354、R0355、R0356、R0359和R0330的氨基酸序列分别进行人源化碱基优化，并人工合成它们的编码核苷酸序列。R0354、R0355、R0356、R0359和R0330的编码核酸序列分别如SEQ ID NO:1,SEQ ID NO:3，SEQ ID NO:5、SEQ ID NO:7和9，和SEQ ID NO:19所示。
(2)载体选择
根据所要构建质粒的用途、外源基因表达产物产量、载体DNA制备的难易，以及载体和目标片段的酶切位点分析结果选择PCDNA3.4A载体。载体购买自Invitrogen公司，所构建的质粒图谱见图1。
(3)引物设计
通过SnapGene软件(SnapGene Version 1.1.3)设计引物，最终确定扩增R0354、R0355、R0356的一对特异性寡核苷酸引物序列为1F 5’–CCGCAAGCTTGCCACCATGGGATGG TCATGCATAATACTC-3’(SEQ ID NO:12)；1R 5’-CCGGAATTCTCATTACTTGCCGGGGCTC AGGCTCAG-3’(SEQ ID NO:13),扩增产物片段长度为1288bp；扩增R0359VCHE一对特异性寡核苷酸引物序列为2F 5’- CCGCAAGCTTGCCACCATGGGATGGTCATGCATAATAC-3’(SEQ ID NO:14)；2R 5’-CCGGAATTCTCATTACTTGCCAGGGGACAGAGACAGGGACTT C-3’(SEQ ID NO:15)，扩增产物片段长度为1432bp；扩增R0359VCLE一对特异性寡核苷酸引物序列为2F 5’-CCGCAAGCTTGCCACCATGGGATGGTCATGCATAATAC-3’；3R 5’-CCGGAATTCTCATTAACATTCACCGCGATTAAAGGACTTTGTC-3’(SEQ ID NO:16)，扩增产物片段长度为748bp；扩增R0330的一对特异性寡核苷酸引物序列为1F 5’–CCGCAAGCTTGCCACCATGGGATGGTCATGCATAATACTC-3’(SEQ ID NO:12)；4R 5’-CCGGAATTCTCATTAGCCAGGGGACAGAGACAGGGACTTC-3’(SEQ ID NO:23),扩增产物片段长度为1270bp。
(4)PCR扩增
使用PCR仪(Tprofessional TR20)，通过Q5高保真DHA聚合酶(High-Fidelity DNA Polymerase)(NEB M0491L)，以合成序列为模板，分别以引物1F/1R；2F/2R；2F/3R进行PCR扩增。
反应总体积：50μL，含PCR 5×缓冲液10μL，高GC缓冲液(High GC buffer)10μL，DNA模板0.5μL，Q5高保真DHA聚合酶(High-Fidelity DNA Polymerase)0.5μL，dNTP(25mM)4μL，以及特异性上、下引物(终浓度200nmol/L)各1.5μL，并加消毒双蒸水至总体积50μL；
反应条件：94℃预变性5分钟，然后依次94℃变性30秒，接着55℃退火30秒，72℃延伸1分30秒分钟，共30个循环。
(5)扩增产物纯化
通过电泳仪(DYY-TC)，用每100ml凝胶中含有2g琼脂糖的琼脂糖凝胶对步骤(4)所得的扩增产物进行电泳，以检测其是否有目的片段，使用DL2000DNA Marker(TAKARA 3427A)以产生序列梯。通过凝胶成像仪(Smart Gel N)观察结果，PCR产物经电泳后显示为单一条带，大小约1.3Kb,1.4Kb，800bp和1.3Kb无杂带，提示PCR产物单一，无非特异扩增。利用Axygen琼脂糖凝胶回收试剂盒(AxyPrep DNA Gel Extraction Kit,AxyGEN AP-GX-50)回收纯化的产物,得到目的DNA分子。
(6)载体和目的片段的限制性酶切
对目的DNA分子和载体分子进行Hind III(NEB)和EcoRI(NEB)双酶切,获得相应的粘末端，酶切反应在37℃水浴反应1h(反应体系：Hind III 1μL；EcoR Ⅰ 1μL；10×缓冲液5μL；质粒/DNA 10μL～12μL≤1μg；总体积50μL(无菌水补至总体积)；反应条件：温度37℃，酶切1小时)；2％琼脂糖凝胶电泳回收载体酶切大片段，利用Axygen琼脂糖凝胶回收试剂盒直接回收目的基因酶切片段。
(7)连接转化
载体酶切回收大片段与目的基因酶切片段连接，采用T4连接酶(TAKARA 2011A)，在16℃反应4小时(反应体系：载体酶切片段0.5μL；目的基因酶切片段3.8μL；T4 DNA连接酶0.5μL；10×T4 DNA缓冲液0.5μL)。全部连接产物与120ul DH5α感受态细菌(NEB C2987I)混匀后冰浴30min，42℃热激90s，立即置冰上放置2min,加入预热至室温的450ul LB培养基，37℃恒温摇床(H2-9211K)培养60min，用离心机(H1650-W)将菌液离心后，用移液器混匀后均匀涂布于每毫升含100ug的氨苄青霉素的LB平板上，37℃恒温培养箱(HW-8C)倒置培养过夜。
(8)挑取单克隆菌PCR验证
挑取9个单菌落接种于每毫升含100ug的氨苄青霉素的LB培养液中，250rpm，37℃恒温摇床培养5h，对菌液进行扩增，选择阳性菌液，再进行测序验证。
(9)测序与结果判断
将测序结果与设计序列进行比对，获得三个目的基因的序列及两个同型对照的序列，并用无内毒素质粒大提试剂盒(TIANGEN DP117)提取质粒。五个分子的基因序列见图2。
实施例2.融合蛋白R0330、R0354、R0355、R0356以及R0359的制备以及纯化方法
1)IL-10融合蛋白R0330/R0354/R0355/R0356/R0359制备方法
为了得到更多的目的蛋白，我们采用瞬时转染来表达蛋白，构建相应的4个表达载体质粒,分别是PCDNA3.4A-R0354，PCDNA3.4A-R0355，PCDNA3.4A- R0356以及PCDNA3.4A-R0359；采用PEI(polysciences；MW40000)转染Expi293(ThermoFisher Scientific；A14635)进行大量的瞬时表达；通过连续培养7天，离心收样。
实验步骤：
瞬时转染前一天的Expi293(ThermoFisher Scientific；A14635)细胞传代，用Dynamis培养基(gibco；A2617502)按2E6的密度接种1L摇瓶(conning；431147)，放入细胞培养摇床(Adolf Kuhner；ISF4-XC)中37℃；8％CO 2；120rpm培养；
转染当天，Expi293细胞用细胞计数仪(Countstar；IC1000)计数，用新鲜DY培养稀释调整细胞密度为2.9E6；准备转染；PEI：DNA＝3:1；将DNA-PEI混合物加入Expi293细胞中，混匀，放入细胞培养摇床(Adolf Kuhner；ISF4-XC)中37℃；8％CO 2；120rpm培养。
转染4h之后补加双抗(gibco；15140122)和抗凝剂(gibco；0010057)
连续培养7天，之后收样，先低速1000rpm；10min；4℃离心(湘仪，H2050R)，收集细胞上清再一次进行高速离心(湘仪，H2050R)，12000rpm；30min 4℃；收集上清；转交给纯化组进行纯化，方法如下。
2)IL-10融合蛋白R0354/R0355/R0356/R0359/R0330纯化方法
实验目的：从细胞发酵上清中分离纯化得到SE-HPLC纯度>95％的目标hIL-10-hFc融合蛋白；
纯化方法：采用MabSelect SuRe LX(Protein A)亲和捕获——pH5.0±0.1调节——Chromdex PG200分子筛精纯。
实验材料：Expi293细胞发酵上清液；缓冲液：缓冲液A：1*PBS(10mM PB,150mM NaCl,pH7.2，广州化学试剂厂，除非特殊说明，以下试剂均来源广州化学试剂厂)；缓冲液B(20mM NaAc，pH3.4)；CIP缓冲液(0.1M NaOH)；中和缓冲液(1M Tris，pH8.0(aMResCO,0497-500G))；缓冲液：0.5M HCitrate(国药，沪试，10007108)；超滤浓缩管(Millipore，
Figure PCTCN2020118409-appb-000001
Ultra 15mL Centrifugal Filters，30KDa)。
实验设备:层析填料1(GE Healthcare，MabSelect SuRe LX)，层析柱1(GE Healthcare，XK16/20，柱体积(CV)，26ml)；层析系统：GE Healthcare，AKTA pure150；便携式pH计(Horiba)；酶标仪(Epoch，BioTek)；离心机(湘仪，H2050R)；层析填料2：博格隆，Chromdex PG200，层析柱2：GE Healthcare，XK26/40，柱体积(Colunm volume,CV)188.91ml。
实验步骤：
以融合蛋白R0354为例的纯化流程如下:
1)目标分子捕获
在层析系统(GE Healthcare，AKTA pure150)中对装有层析填料1(GE Healthcare，MabSelect SuRe LX)的层析柱1(GE Healthcare，XK16/20，柱体积(CV)，26ml)再生后用缓冲液A平衡10CV，重设紫外检测器(UV Monitor)，以气泡感应方式载入Expi293细胞发酵上清液。用缓冲液A洗涤层析柱20CV，接着用100％缓冲液B步阶洗脱7CV，收集20mAu～20mAu280nm紫外吸收组分，并在收集管中预先加入～5％中和缓冲液使最终pH在6.0-7.0范围,接着在位清洗(Clean in place)(CIP，CIP缓冲液向上流动(up-flow)清洗3CV，保持3min，然后缓冲液A向下流动(down-flow)清洗5CV后结束。
按上述流程将细胞发酵后离心获得的上清液进行亲和捕获。并对各捕获后的洗脱样品分别采用SE-HPLC(分子筛-高效液相色谱)法进行纯度分析。其纯度结果如图3a所示。
2)pH调节
取该亲和洗脱组分，加入0.5M HCitrate调节pH至5.0±0.1，室温(RT)下静置>1小时后，在3500rpm，4℃下离心10min并以0.2um滤膜过滤，用SE-HPLC法进行纯度分析。其纯度结果如图3b所示。
3)精细纯化
将pH调节后样品采用超滤浓缩管(Millipore，
Figure PCTCN2020118409-appb-000002
Ultra 15mL Centrifugal Filters，30KDa)浓缩至目标体积以使每次上样样品体积<5％层析柱体积，再按下述流程进行分子筛精细纯化：
在层析系统中对装有层析填料2(博格隆，Chromdex PG200)的层析柱2(GE Healthcare，XK26/40，柱体积(Colunm volume,CV)188.91ml)再生后用缓冲液A平衡2CV至基线，重设紫外检测器，并以气泡感应方式载入样品，用缓冲液A洗涤层析柱1.5CV，收集20mAu～20mAu 280nm紫外吸收组分,在位清洗(CIP，CIP缓冲液向上流动(up-flow)清洗2CV，保持3min，然后缓冲液A向下流动(down-flow)清洗5CV后结束。层析柱最终保存在CIP缓冲液中。其层析图谱如图3c所示。
将收集的洗脱组分分别送检SE-HPLC，其中合并后的4.B.3-4.C.1洗脱组分纯度如图3d所示。从而得到纯度>95％的hIL-10-hFc融合蛋白。
实施例3.通过Fortebio法鉴定R0330、R0354、R0355、R0356融合蛋白与Fc受体的亲和力
实验材料
样品来源：融合蛋白R0330、R0354、R0355以及R0356来自实施例2中制备的融合蛋白。其分子量依次为90.24kDa，90.38kDa，90.34kDa以及90.62kDa。
Fc受体：FcR gamma I，FcR gamma IIa，FcR gamma IIb，FcR gamma IIIa以及FcRn重组蛋白购自Acro Biosystems；对照品hIgG1(403502，Biolegend)分以及hIgG4(403702，Biolegend)
缓冲液体系：1*磷酸缓冲盐溶液(SBJ-0032,森贝伽)，其中含0.02％吐温20(44112-100G-F，Merck)(PBST)
再生液体系：10mM甘氨酸(G8898-500G，Merck)
仪器设备
分子互相作用分析仪ForteBIO(Octet OK e)；96孔板(上海晶安生物科技，J09602)；Anti-Penta-HIS(HIS1K)(18-5120，Fortebio)探针
实验方法
FcR gamma I用1*PBST缓冲液稀释到2ug/ml，并以此浓度固化到分子互相作用分析仪ForteBIO(Octet OK e)的Anti-Penta-HIS(HIS1K)(18-5120，Fortebio)探针上，此受体对应的分析物R0330、R0354、R0355、R0356、hIgG1和hIgG4分别用1*PBST缓冲液稀释到66.67nM。FcRn，FcR gamma  IIa，FcR gamma IIb以及FcR gamma IIIa分别用1*PBST稀释到3ug/ml，并以此浓度固化到HIS1K探针上,每个受体对应的分析物R0330、R0354、R0355、R0356、hIgG1、hIgG4分别用1*PBST缓冲液稀释到2μM。用PBST平衡探针60s，分别结合各个受体180s，再次用PBST平衡探针90s后，再与对应的分析物R0330、R0354、R0355、R0356、hIgG1、hIgG4结合180s，然后于PBST中解离300s，探针于10mM甘氨酸中再生中和以后，再按照这个方法进行下个循环的平衡，结合，解离以及再生中和。所有循环转速都设定为1000rpm；实验温度为30℃。
结果和讨论
与野生型对照品hIgG1以及hIgG4相比较，R0330、R0354、R0355、R0356三种分子与FcR gamma I，FcR gamma IIa，FcR gamma IIb，FcR gamma IIIa四个受体几乎不结合，但是与FcRn的亲和力没有明显的减弱，与预期结果一致(见表1)。
表1：Fortebio法评价R0330、R0354、R0355、R0356、hIgG1、hIgG4分别与FcR gamma I(表1a)，FcR gamma IIa(表1b)，FcR gamma IIb(表1c)，FcR gamma IIIa(表1d)以及FcRn(表1e)受体的亲和力
表1a
样品ID 上样样品ID k a(1/Ms) k d(1/s) K D(M)
R0354 FcR gamma I 4.18E+05 4.87E-03 1.17E-08
R0355 FcR gamma I 8.89E+05 1.00E-02 1.13E-08
R0356 FcR gamma I 6.44E+04 1.67E-03 2.59E-08
R0330 FcR gamma I 1.07E+05 8.47E-04 7.93E-09
hIgG1 FcR gamma I 3.41E+05 9.18E-04 2.69E-09
hIgG4 FcR gamma I 9.45E+06 4.96E-02 5.24E-09
表1b
样品ID 上样样品ID k a(1/Ms) k d(1/s) K D(M)
R0354 FcR gamma IIa 1.06E+03 3.67E-03 3.45E-06
R0355 FcR gamma IIa 1.52E+03 7.39E-03 4.87E-06
R0356 FcR gamma IIa 2.03E+03 6.26E-03 3.08E-06
hIgG1 FcR gamma IIa 1.06E+04 3.67E-03 3.45E-07
HIgG4 FcR gamma IIa 4.44E+04 5.45E-03 1.26E-07
表1c
样品ID 上样样品ID k a(1/Ms) k d(1/s) K D(M)
R0354 FcR gamma IIb 1.63E+03 1.07E-02 6.56E-06
R0355 FcR gamma IIb 1.63E+04 2.25E-02 1.38E-06
R0356 FcR gamma IIb 1.53E+04 2.15E-02 1.41E-06
R0330 FcR gamma IIb 8.41E+03 7.78E-04 9.24E-08
hIgG1 FcR gamma IIb 3.47E+03 2.05E-03 5.91E-07
HIgG4 FcR gamma IIb 8.49E+03 2.15E-03 2.53E-07
表1d
样品ID 上样样品ID k a(1/Ms) k d(1/s) K D(M)
R0354 FcR gamma IIIa 2.01E+03 9.83E-03 4.89E-06
R0355 FcR gamma IIIa 1.76E+03 1.47E-02 8.35E-06
R0356 FcR gamma IIIa 3.13E+03 1.74E-03 5.56E-06
hIgG1 FcR gamma IIIa 1.17E+03 1.16E-03 9.91E-07
HIgG4 FcR gamma IIIa 5.92E+04 1.07E-03 5.53E-07
表1e
样品ID 上样样品ID k a(1/Ms) k d(1/s) K D(M)
R0354 FcRn 1.05E+04 4.47E-03 4.24E-07
R0355 FcRn 9.45E+03 1.87E-03 1.97E-07
R0356 FcRn 3.94E+05 5.72E-02 1.45E-07
hIgG1 FcRn 3.38E+04 5.93E-03 1.75E-07
HIgG4 FcRn 3.98E+04 5.53E-03 1.39E-07
实施例4：CD8+T细胞的活化以及INF-γ分泌检测
1)目的：探究CD8+T细胞活化程度与其IL-10受体(IL-10R)表达的关系
实验材料：正常人来源的PBMC(本实验中所用血液为公司员工自愿捐献血液，编号为80)，CD8+T分离试剂盒(Miltenyi，cat：130-096-495)，DPBS(Hyclone，cat：SH3002802)，胎牛血清(FBS)(Gibco，cat：10091-148)，X-VIVO(LONZA,cat：04-418Q)，抗-CD8荧光抗体(Biolegend，cat：344732)，抗-CD45RO荧光抗体(Biolegend，cat：304220)，抗-hIL-10R荧光抗体(Biolegend，cat：501404)，PE Rat IgG1,κ同种型对照抗体(Isotype Ctrl Antibody)(Biolegend，cat：400407)，抗-CD3鼠单抗(自产)，抗-CD28鼠单抗(自产)，24孔板、移液管等细胞培养常规耗材。
仪器设备：生物安全柜(ESCO,AC2-4S1)，离心机(湖南湘仪实验室开发有限公司，L530R)，流式细胞仪(Beckman Coulter,Cytoflex)，二氧化碳培养箱(日本松下健康医疗器械株式会社，MCO-18AC)
实验方法
CD8+T细胞分离与培养：取新鲜分离来自正常人PBMC约1×10 8个，按照CD8+T分离试剂盒的说明书步骤分离得到CD8+T细胞。提前在24孔板中以1μg/mL的浓度包被抗-CD3鼠单抗，用含10％FBS的X-VIVO培养基将细胞调整到3×10 6个/mL，加入抗-CD28鼠单抗使其浓度为1μg/mL。最终将细胞以1mL/孔的体积在二氧化碳培养箱中37℃下培养在24孔板中。
细胞检测：分别在第0天、第3天和第6天收取培养在24孔板中的CD8+T细胞，PBS清洗一遍后，加入抗-CD8荧光抗体，CD45RO荧光抗体，抗-hIL-10R荧光抗体，对照组加入抗-CD8荧光抗体，CD45RO荧光抗体，PE Rat IgG1,κ同种型对照抗体，4℃孵育30min后PBS再次清洗细胞一遍，流式细胞仪检测其IL-10R的表达。
实验结果：未活化的CD8+T细胞(CD8+,CD45RO-)表达IL-10R极少，经过刺激活化后的CD8+T细胞(CD8+,CD45RO+)在第三天就较高表达IL-10R，且在第6天还会增加(见图4),灰色线表示anti IL-10R抗体的同型对照，黑色线表示anti IL-10R。使用anti-CD3antibody(10μg/mL，提前包被于96孔板)和anti-CD28(1μg/mL，游离)培养从PBMC中分离出来的CD8+T 6天。图中表示的是在0天，3天和6天，用上述方法刺激的CD8+T细胞IL-10R的表达情况： 在第1天CD8+T细胞IL-10R较少，第3天CD8+T细胞IL-10R明显增加，第6天CD8+T细胞IL-10R持续显著增加。
实施例5：活化的CD8+T细胞的INF-γ分泌检测
2)目的：IL-10融合蛋白促进活化后CD8+T细胞INF-γ的分泌
实验材料：正常人来源的PBMC(本实验中所用血液为公司员工自愿捐献血液，编号为110)，CD8+T分离试剂盒(Miltenyi，cat：130-045-201)，DPBS(Hyclone，cat：SH3002802)，FBS(Gibco，cat：10091-148)，X-VIVO(LONZA,cat：04-418Q)，CD8荧光抗体(Biolegend，cat：344732)，抗-CD3鼠单抗(Merck,SAB4700048-100TST))，抗-CD28鼠单抗(Sino Biological,11524-R007)，INFγ检测试剂盒(invitrogen,88-7316-88)，24孔板、移液管等细胞培养常规耗材。
仪器设备：生物安全柜(ESCO,AC2-4S1)，离心机(湖南湘仪实验室开发有限公司，L530R)，流式细胞仪(Beckman Coulter,Cytoflex)，二氧化碳培养箱(日本松下健康医疗器械株式会社，MCO-18AC)，酶标仪(Molecular devices,spectramax i3x)
实验方法
CD8+T细胞分离与培养：取新鲜分离来自正常人PBMC约1×10 8个，按照CD8+T分离试剂盒的说明书步骤分离得到CD8+T细胞。提前在24孔板中以1μg/mL的浓度包被抗-CD3鼠单抗，用含10％FBS的X-VIVO培养基将细胞调整到3×10 6个/mL，加入抗-CD28鼠单抗使其浓度为1μg/mL。最终将细胞以1mL/孔的体积培养在24孔板中，二氧化碳培养箱37℃继续培养三天。
细胞检测：分离CD8+T细胞后取少量细胞，进行CD8表型鉴定，检测其分离细胞纯度。
IL-10融合蛋白刺激活化后CD8+T细胞：收集活化三天后的CD8+T，用含10％FBS的X-VIVO重悬细胞使之密度为2×10 6个/mL，以250μL/孔的体积培养在96孔板中，同时加入一定剂量的IL-10融合蛋白R0354、R0355、R0356以及同型对照蛋白R0359，二氧化碳培养箱37℃继续培养三天。在活化三天后再次加入抗-CD3鼠单抗，使之浓度为1μg/mL，继续培养4h。
INFγ检测：到达培养终点后收集细胞培养上清，使用INFγ检测试剂盒按照INFγ检测试剂盒说明书步骤检测其INFγ的含量。
实验结果：自产IL-10融合蛋白R0354、R0355、R0356均能促进活化后CD8+T细胞分泌INFγ，不同供体之间存在一定的差异(见图5),左图表示本次实验中所分离donor的CD8+T细胞纯度，为99.75％。右图是本次实验中该donor的CD8+T细胞经过anti-CD3 antibody(10μg/mL，提前包被于96孔板)和anti-CD28(1μg/mL，游离)活化培养3天后，再使用IL-10融合蛋白：R0354、R0355、R0356继续培养3天后细胞的INFγ的分泌情况(R0359为同型对照)。实验结果表明IL-10融合蛋白的三个浓度梯度(10/1/0.1μg/mL)均能刺激活化后的CD8+T细胞使其分泌INFγ，且活性R0355>R0354>R0356。
实施例6：肿瘤模型的建立
1)目的：用不同细胞密度的CT26WT小鼠结肠癌细胞在Balb/c小鼠皮下建立肿瘤模型，观察肿瘤生长、肿瘤出现的关系，以确定建立稳定的CT26WT小鼠结肠癌模型所需要的细胞密度。
实验材料：雌性，Balb/c小鼠；RPMI 1640培养基(Gibco,11875085)，FBS(Gibco,10091-148)，0.25％胰蛋白酶-EDTA(Gibco,25200056)，青霉素-链霉素(Gibco,15140122)，DMSO(Sigma,D2650)，DPBS(Hyclone,SH30028.02),CT26WT细胞(上海细胞库，TCM37)
仪器设备：电子天平(上海舜宇恒平科学仪器有限公司，JA12002)，游标卡尺(上海美耐特实业有限公司，MNT-150T)，显微镜(重庆奥特光学仪器有限公司，BDS200)，医用离心机(湖南湘仪实验室开发有限公司，L530R)，数显恒温水浴锅(普瑞斯机械有限公司，HH-S)，二氧化碳培养箱(日本松下健康医疗器械株式会社，MCO-18AC)，双人垂直型超净台(无锡易净化设备有限公司，SW-CJ-VS2)
实验方法：CT26WT小鼠结肠癌体内肿瘤模型的建立：用完全培养基：RPMI 1640培养基+10％胎牛血清(Gibco，cat：10091-148)+1％双抗生素(1：1的青霉素－链霉素)培养小鼠结肠癌细胞CT26WT，取对数生长期的细胞，用台盼蓝染色确定活细胞数。收集细胞后，用无血清RPMI 1640培养基洗涤两次，以除去完全培养基。用75％酒精消毒雌性Balb/c小鼠前肢腋下皮肤，取已调整好密度的CT26WT小鼠结肠癌细胞接种于前肢腋下皮肤下，每只小鼠接种体积 0.2mL。细胞密度设定：按细胞不同的接种数量，将Balb/c小鼠分成3组，每组5只，即：密度1(1×10 5个细胞/0.2mL/只)、密度2(5×10 5个细胞/0.2mL/只)、密度3(1×10 6个细胞/0.2mL/只)。
实施例7：肿瘤模型中IL-10融合蛋白的体内抗肿瘤评价
实施例7.1：R0330,R0354，R0355，R0356对CT26小鼠结肠癌的体内抗肿瘤评价
1)目的：观察R0330,R0354，R0355，R0356对CT26小鼠结肠癌的抗肿瘤活性，同时设置同型对照R0359组和溶媒对照PBS组。
实验材料：雌性，Balb/c小鼠；CT26WT细胞；RPMI 1640培养基(Gibco,11875085)，FBS(Gibco,10091-148)，0.25％胰蛋白酶-EDTA(Gibco,25200056)，青霉素-链霉素(Gibco,15140122)，DMSO(Sigma,D2650)，DPBS(Hyclone,SH30028.02)
仪器设备：电子天平(上海舜宇恒平科学仪器有限公司，JA12002)，游标卡尺(上海美耐特实业有限公司，MNT-150T)，显微镜(重庆奥特光学仪器有限公司，BDS200)，医用离心机(湖南湘仪实验室开发有限公司，L530R)，数显恒温水浴锅(普瑞斯机械有限公司，HH-S)，二氧化碳培养箱(日本松下健康医疗器械株式会社，MCO-18AC)，双人垂直型超净台(无锡易净化设备有限公司，SW-CJ-VS2)
实验方法
细胞培养：将小鼠结肠癌细胞(CT26WT，)培养在含有10％胎牛血清(Gibco)、1％谷氨酰胺与1％青霉素-链霉素(1:1)的RPMI 1640培养基(Gibco)中。
接种：收集对数生长期的CT26WT细胞，调节细胞浓度为1X10 5/mL。取雌性BALB/C小鼠70只，皮下接种CT26WT细胞，接种体积为0.1mL/小鼠，即1X10 5/小鼠。
给药：接种当天记为第0天(D0)，第9天，将小鼠按瘤体积随机分为6组，每组10只，开始给药(给药方案见下表2)。
表2 CT26WT肿瘤模型给药剂量，方式以及频率
Figure PCTCN2020118409-appb-000003
Figure PCTCN2020118409-appb-000004
记录：
D9开始测量肿瘤体积并记录，之后每周2次用游标卡尺测量肿瘤长径和短径。以公式：(1/2)X长径X(短径) 2计算肿瘤体积(见表3；图6a)。每只小鼠达到实验终点时(肿瘤体积超过2000mm 3达到仁慈终点)，颈椎脱臼法处死小鼠，记录生存曲线(见图6b)。
表3 R0330，R0354，R0355，R0356以及R0359CT26WT模型抗肿瘤活性评价
Figure PCTCN2020118409-appb-000005
实验结果：与溶媒对照PBS及同型对照R0359组相比，R0354和R0356只腹腔给药1次就对CT26WT模型肿瘤生长有显著的抑瘤作用(R0330组TGI＝70.35％，R0354组TGI＝66.0％,R0356组TGI＝82.71％)(见表3；图6a)。生存期曲线见图6b，R356生存期最长，其余依次是R0354,R355、R0330。
实施例7.2：R0356对B16-F1小鼠黑色素瘤的体内抗肿瘤评价
1)目的：观察R0356对B16-F1小鼠黑色素瘤的抗肿瘤活性，同时设置同型对照R0359组。
实验材料：雌性，C57BL/6小鼠；B16-F1细胞(国家实验细胞资源共享平台)；DMEM(Gibco,11965084)，FBS(Gibco,10091-148)，0.25％胰蛋白酶-EDTA(Gibco,25200056)，青霉素-链霉素(Gibco,15140122)，DMSO(Sigma,D2650)，DPBS(Hyclone,SH30028.02)
仪器设备：电子天平(上海舜宇恒平科学仪器有限公司，JA12002)，游标卡尺(上海美耐特实业有限公司，MNT-150T)，显微镜(重庆奥特光学仪器有限公司，BDS200)，医用离心机(湖南湘仪实验室开发有限公司，L530R)，数显恒温水浴锅(普瑞斯机械有限公司，HH-S)，二氧化碳培养箱(日本松下健康医疗器械株式会社，MCO-18AC)，双人垂直型超净台(无锡易净化设备有限公司，SW-CJ-VS2)
实验方法
细胞培养：小鼠黑色素瘤细胞(B16-F1)培养在含有10％FBS(Gibco)、1％谷氨酰胺与1％青霉素-链霉素的DMEM培养基(Gibco)中。
接种：收集对数生长期的B16-F1细胞，调节细胞浓度为2X10 6/mL。(1)取雌性C57BL/6小鼠20只，皮下接种B16-F1细胞，接种体积为0.1mL/小鼠，即2X10 5/小鼠。
给药：接种当天记为第0天(D0)，将小鼠按体重随机分为2组，每组10只，开始给药(给药方案见下表4)。
表4 B16-F1肿瘤模型给药剂量，方式以及频率
Figure PCTCN2020118409-appb-000006
记录：
D10开始测量肿瘤体积并记录，之后每周2次用游标卡尺测量肿瘤长径和短径。以公式：(1/2)X长径X(短径) 2计算肿瘤体积(见表5)。
表5 R0356以及R0359B16-F1模型抗肿瘤活性评价
植入后天数 R0359 R0356
  (10mg/kg) (10mg/kg)
10 210.42 66.57
13 815.32 188.40
17 2861.29 659.90
实验结果：与同型对照R0359组相比，R0356腹腔给药2次对B16-F1模型肿瘤生长有显著的抑瘤作用(TGI＝76.94％)(见表5；图7c)。
实施例7.3：R0354，R0355，R0356对MC38小鼠结肠癌的抗肿瘤活性评价
1)目的：观察R0354，R0355，R0356对MC38小鼠结肠癌的抗肿瘤活性，同时设置同型对照R0359组。
实验材料：雌性，C57BL/6小鼠；MC38细胞(国家实验细胞资源共享平台)；RPMI Medium 1640(Gibco,11875085)，FBS(Gibco,10091-148)，0.25％trypsin-EDTA(Gibco,25200056)，Penicillin Streptomycin(Gibco,15140122)，DMSO(Sigma,D2650)，DPBS(Hyclone,SH30028.02)
仪器设备：电子天平(上海舜宇恒平科学仪器有限公司，JA12002)，游标卡尺(上海美耐特实业有限公司，MNT-150T)，显微镜(重庆奥特光学仪器有限公司，BDS200)，医用离心机(湖南湘仪实验室开发有限公司，L530R)，数显恒温水浴锅(普瑞斯机械有限公司，HH-S)，二氧化碳培养箱(日本松下健康医疗器械株式会社，MCO-18AC)，双人垂直型超净台(无锡易净化设备有限公司，SW-CJ-VS2)
实验方法
细胞培养：小鼠结肠癌细胞(MC38)培养在含有10％胎牛血清(Gibco)、1％谷氨酰胺与1％青-链霉素的RPMI 1640培养基(Gibco)中。
接种：收集对数生长期的MC38细胞，调节细胞浓度为1X10 7/mL。(1)取雌性BALB/C小鼠30只，皮下接种MC38细胞，接种体积为0.1mL/小鼠，即1X10 6/小鼠。
给药：接种当天记为D0，在第5天，挑选肿瘤大小较均一的小鼠入组，将小鼠按瘤体积随机分为4组，每组5只，开始给药(给药方案见下表6)。
表6 MC38肿瘤模型给药剂量，方式以及频率
Figure PCTCN2020118409-appb-000007
记录：
D5开始测量肿瘤体积并记录，之后每周2次用游标卡尺测量肿瘤长径和短径。以公式：(1/2)X长径X(短径) 2计算肿瘤体积(见表7)。
表7 R0354，R0355，R0356以及R0359MC38模型抗肿瘤活性评价
Figure PCTCN2020118409-appb-000008
实验结果：与同型对照R0359组相比，低剂量1mg/kg的R0354、R0355和R0356仅腹腔给药1次对MC38模型肿瘤生长有显著的抑瘤作用(R0354组TGI＝65.32％,R0355组TGI＝54.28，R0356组TGI＝74.77％)(见表7；图7d)。
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明，应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种Fc变体，所述Fc变体依照EU编号在选自第223，235，237，238，265，297，327，434位的至少一处具有氨基酸取代；所述Fc为IgG Fc，选自IgG1 Fc，IgG2 Fc，IgG3Fc，IgG4 Fc中的一种，优选是IgG1 Fc。
  2. 根据权利要求1所述的Fc变体，所述氨基酸取代选自E223P，L235A，G237A，P238A，D265A，N297A，A327Q，和N434A中的至少一种；
    优选地，所述氨基酸取代选自E223P，L235A，G237A，P238A，D265A，N297A，A327Q，和N434A中的至少三种；
    更优选地，所述氨基酸取代为选自：E223P，P238A，D265A；N297A，L235A，N434A；和A327Q，G237A，L235A。
  3. 根据权利要求1或2所述的Fc变体，所述Fc变体减弱了与FcγR的亲和力。
  4. 一种融合蛋白，其由Fc变体与多肽融合形成，所述Fc变体依照EU编号在选自第223，235，237，238，265，297，327，434位的至少一处具有氨基酸取代；所述Fc为IgG Fc，选自IgG1 Fc，IgG2 Fc，IgG3Fc，IgG4 Fc中的一种，优选是IgG1 Fc。
  5. 根据权利要求4所述的融合蛋白，所述氨基酸取代选自E223P，L235A，G237A，P238A，D265A，N297A，A327Q，和N434A中的至少一种；
    优选地，所述氨基酸取代选自E223P，L235A，G237A，P238A，D265A，N297A，A327Q，和N434A中的至少三种；
    更优选地，所述氨基酸取代为选自：E223P，P238A，D265A；N297A，L235A，N434A；和A327Q，G237A，L235A。
  6. 根据权利要求4或5所述的融合蛋白，所述Fc变体减弱了所述融合蛋白与FcγR的亲和力。
  7. 根据权利要求4-6任一项所述的融合蛋白，其特征在于，所述融合蛋白具有式(I)所示结构：
    A-肽接头-B或B-肽接头-A(I)，
    其中，A为IL-10，IL-6或IL-12，肽接头为(GS) n、(G4S) n或不存在，其中n为1-10的整数，B为Fc变体；
    优选地，所述A是IL-10，更优选地，A是人IL-10，最优选地，所述IL-10的氨基酸序列如SEQ ID NO：17所示。
  8. 一种核酸，其编码权利要求1-3任一项所述的Fc变体或权利要求4-8任一项所述的融合蛋白；
    优选地，其中所述核酸的核苷酸序列为SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:20、SEQ ID NO:21、或SEQ ID NO:22。
  9. 一种药物组合物，其包含权利要求1-3任一项所述的Fc变体或权利要求4-8任一项所述的融合蛋白和药学上可接受的载体。
  10. 权利要求1-3任一项所述的Fc变体、权利要求4-8任一项所述的融合蛋白或权利要求9所述的药物组合物在制备用于治疗有此需要的个体中癌症的药物中的用途，
    优选地，其中所述癌症为实体瘤，
    更优选，其中所述实体瘤为结肠癌，黑色素瘤，结肠直癌，乳腺癌，
    进一步更优选地，其中所述个体是人。
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