WO2021063313A1 - Variante fc ayant une fonction effectrice modifiée et protéine de fusion de celle-ci - Google Patents

Variante fc ayant une fonction effectrice modifiée et protéine de fusion de celle-ci 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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Chinese (zh)
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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

Une variante Fc qui a une fonction effectrice modifiée et une protéine de fusion de celle-ci sont divulguées. Une région Fc de la variante Fc comprend un ou plusieurs changements d'acides aminés, et a une fonction effectrice modifiée ; en outre, l'invention concerne une protéine de fusion comprenant la variante Fc qui a une fonction effectrice modifiée, un procédé de préparation de la variante Fc ou de la protéine de fusion, un acide nucléique codant la variante Fc ou la protéine de fusion, un vecteur d'expression comprenant l'acide nucléique, une cellule hôte comprenant la variante Fc, la protéine de fusion, l'acide nucléique ou le vecteur d'expression, une composition pharmaceutique comprenant la variante Fc ou la protéine de fusion, et l'utilisation de la variante Fc ou de la protéine de fusion.
PCT/CN2020/118409 2019-09-30 2020-09-28 Variante fc ayant une fonction effectrice modifiée et protéine de fusion de celle-ci WO2021063313A1 (fr)

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CN108948207A (zh) * 2017-05-22 2018-12-07 杭州博虎生物科技有限公司 一种人白细胞介素10-Fc融合蛋白及其编码基因与应用

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