WO2020097946A1 - 一种重组人白细胞介素10融合蛋白及其应用 - Google Patents

一种重组人白细胞介素10融合蛋白及其应用 Download PDF

Info

Publication number
WO2020097946A1
WO2020097946A1 PCT/CN2018/116074 CN2018116074W WO2020097946A1 WO 2020097946 A1 WO2020097946 A1 WO 2020097946A1 CN 2018116074 W CN2018116074 W CN 2018116074W WO 2020097946 A1 WO2020097946 A1 WO 2020097946A1
Authority
WO
WIPO (PCT)
Prior art keywords
fusion protein
ala
sequence
igg4
seq
Prior art date
Application number
PCT/CN2018/116074
Other languages
English (en)
French (fr)
Inventor
汤甜甜
许宝青
Original Assignee
杭州博虎生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 杭州博虎生物科技有限公司 filed Critical 杭州博虎生物科技有限公司
Priority to CN201880092332.3A priority Critical patent/CN111989340A/zh
Priority to PCT/CN2018/116074 priority patent/WO2020097946A1/zh
Publication of WO2020097946A1 publication Critical patent/WO2020097946A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • 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

Definitions

  • the invention relates to the field of genetic engineering medicines, in particular to a recombinant human interleukin 10-Fc fusion protein and its coding gene and application.
  • Interleukin-10 is a cytokine discovered in 1991, which can regulate the body's inflammation and immune response. It was initially reported that the cytokine can inhibit cytokine secretion, antigen presentation and CD4 + cell activation, and IL-10 can inhibit IL-1 ⁇ , IL-1 ⁇ , IL-6, IL by activating monocytes and activated macrophages -8, the expression of TNF- ⁇ , GM-CSF and G-CSF to suppress the immune response, and it also suppresses the IFN- ⁇ production of NK cells.
  • IL-10 is mainly expressed in macrophages, it has also been detected in activated T cells, B cells, mast cells, and monocytes. In addition to suppressing the immune response, IL-10 exhibits immunostimulatory properties, including stimulating the proliferation of IL-2 and IL-4 treated thymocytes, enhancing the viability of B cells, and stimulating MHC class II expression.
  • IL-10 can actually activate human immune system functions, especially CD8 + T cells with cancer cell killing effect.
  • IL-10 can co-stimulate B cell activation, prolong B cell survival, and help class switching in B cells.
  • IL-10 can co-stimulate natural killer (NK) cell proliferation and cytokine production and act as growth factors to stimulate the proliferation of certain subsets of CD8 + T cells (Mosser, DM & Yhang, X., Immunological Reviews 226, 205-218 (2008) , High doses of IL-10 (20 and 25 ⁇ g / kg, respectively) can cause increased INF ⁇ production in humans (Lauw, FNet, J. Immunol.
  • the immunostimulatory activity of IL-10 is reported to be determined by the single amino acid isoleucine at position 87 in the cell IL-10 (Ding, Y. et al. , J. Exp. Med. 191 (2), 213-223 (2000)).
  • Human IL-10 is a homodimeric protein, each monomer contains 178 amino acids, and its first 18 amino acids contain a signal peptide. Certain embodiments of the present disclosure include mature human IL-10 polypeptide lacking a signal peptide (see US Patent No. 6,217,857). Mature IL-10 has 160 amino acid residues (Seq ID No: 1), monomer molecular weight is 18.7KD, and contains 4 cysteine formed disulfide bonds (12-108, 62-114), which is naturally The active form is a 38KD homo-oligomeric dimer linked by non-covalent bonds, which becomes inactive after the non-covalent interaction between the two monomer subunits is disrupted.
  • IL-10 has been associated with numerous diseases, disorders, and conditions, including inflammatory conditions, immune-related disorders, fibrotic disorders, and cancer.
  • Immunoglobulins generally have a long circulating half-life in the body. For example, IgG molecules have a half-life of up to 23 days in the human body.
  • the immunoglobulin Fc portion is the main reason for this in vivo stability. While retaining the biological activity of the IL-10 molecule, the IL10-Fc fusion protein has the advantage of maintaining the stability provided by the Fc portion of the immunoglobulin while retaining the biological activity of the IL-10 molecule.
  • the present invention provides a fusion protein of human interleukin-10 (IL-10) and the human IgG Fc portion (IL10-Fc fusion protein).
  • IL-10 human interleukin-10
  • IL10-Fc fusion protein human IgG Fc portion
  • the present invention provides an IL10-Fc fusion protein, wherein the C-terminus of IL-10 is directly or through a connecting peptide connected to the N-terminus of human IgG Fc protein; wherein the IL-10 sequence is linked to Seq ID No: 1. Consistent; the general formula of the connecting peptide sequence is (G 4 S) n , (SG 4 ) n or G 4 (SG 4 ) n , n is a number between 1 and 10; human IgG Fc protein is selected from human IgG2 , IgG4 native sequence Fc region or variant Fc region.
  • the human IgG2 Fc sequence is shown in SEQ ID NO: 10.
  • the human IgG4 Fc sequence is shown in SEQ ID NO: 11.
  • the IL10-Fc fusion protein provided by the present invention, wherein the preferred linker peptide sequence formula is [GlyGlyGlyGlySer] n , n is an integer of 1-4; more preferably, n is 3, and its sequence is [ GlyGlyGlyGlySer] 3 .
  • the in vivo function and stability of the fusion protein of the present invention are optimized by adding small connecting peptides to prevent potential unnecessary domain interactions.
  • the glycine-rich linker peptide provides a certain degree of structural flexibility so that the IL-10 moiety can effectively interact with the IL-10 receptor on the target cell.
  • the present invention provides that the IL10-Fc fusion protein is the C-terminus of IL-10 connected to the N-terminus of the human IgG Fc protein through a connecting peptide; wherein the IL-10 sequence is consistent with that shown in SeqID No: 1
  • the connecting peptide sequence is [GlyGlyGlyGlySer] 3
  • the human IgG Fc protein is selected from the natural sequence Fc region or variant Fc region of human IgG2 and IgG4.
  • the IL10-Fc fusion protein provided by the present invention is IL10-human IgG2 Fc fusion protein, and its sequence is shown in SEQ ID NO: 12.
  • the IL10-Fc fusion protein provided by the present invention is IL10-human IgG4 Fc fusion protein, the sequence of which is shown in SEQ ID NO: 13.
  • the present invention provides that the IL10-Fc fusion protein is linked to the N-terminus of human IgG4 Fc variant protein directly or via a linker peptide at the C-terminus of IL-10; wherein the IL-10 sequence is linked to Seq ID No: 1 is identical; the general formula of the connecting peptide sequence is (G 4 S) n , (SG 4 ) n or G 4 (SG 4 ) n , n is a number between 1 and 10; human IgG4 Fc variant protein Contains the sequence of SEQ ID NO: 2, where:
  • 16-bit X1 is Pro or Glu
  • the X2 of 17 is Phe, Val, or Ala;
  • X3 at position 18 is Leu, Glu, or Ala
  • 80-bit X4 is Asn or Ala
  • X5 at 230 is Lys or does not exist.
  • the preferred linker peptide sequence in the IL10-IgG4 Fc fusion protein provided by the present invention is [GlyGlyGlyGlySer] n , where n is an integer of 1-4.
  • the preferred linker peptide sequence in the IL10-IgG4 Fc fusion protein provided by the present invention is [GlyGlyGlyGlySer] 3 .
  • the in vivo function and stability of the fusion protein of the present invention are optimized by adding small connecting peptides to prevent potential unnecessary domain interactions.
  • the glycine-rich linker peptide provides a certain degree of structural flexibility so that the IL-10 moiety can effectively interact with the IL-10 receptor on the target cell.
  • the present invention further modifies the wild-type IgG4 Fc sequence.
  • the IgG4 Fc portion of the fusion protein of the present invention may contain one or more of the following substitutions: corresponding to SEQ ID NO: 2 at position 16 with proline (Pro) or glutamate (Glu) in place of glutamine (Gln ), which corresponds to replacing phenylalanine (Phe) with alanine (Ala) or valine (Val) at position 17 in SEQ ID NO: 2, corresponding to position 18 at SEQ ID NO: 2 Alanine (Ala) or glutamate (Glu) replaces leucine (Leu).
  • the preferred IL10-IgG4 Fc fusion protein of the present invention includes the following proteins:
  • IL10- [GlyGlyGlyGlySer] 3 -IgG4 Fc where X1 at Fc position 16 is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Asn, and X5 at position 230 does not exist, which The sequence is shown in SEQ ID NO: 3.
  • IL10- [GlyGlyGlyGlySer] 3 -IgG4 Fc where X1 at Fc position 16 is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Ala, and X5 at position 230 does not exist, which The sequence is shown in SEQ ID NO: 4.
  • IL10-GlyGlyGlyGlySer-IgG4 Fc where X1 at Fc position 16 is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Asn, and X5 at position 230 is Lys, the sequence of which is SEQ ID NO: 5 shows.
  • IL10-IgG4 Fc where X1 at Fc position 16 is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Ala, and X5 at position 230 does not exist, the sequence SEQ ID NO: 6 shown.
  • IL10- [GlyGlyGlyGlySer] 3 -IgG4 Fc wherein X1 at Fc position 16 is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Asn, and X5 at position 230 is Lys.
  • IL10-GlyGlyGlyGlySer-IgG4 Fc where X1 at Fc position 16 is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Asn, and X5 at position 230 does not exist.
  • IL10- [GlyGlyGlyGlySer] 3 -IgG4 Fc where X1 at position 16 of the Fc is Pro, X2 at position 17 is Phe, X3 at position 18 is Ala, X4 at position 80 is Ala, and X5 at position 230 is absent.
  • IL10- [GlyGlyGlyGlySer] 3 -IgG4 Fc where X1 at Fc position 16 is Pro, X2 at position 17 is Val, X3 at position 18 is Ala, X4 at position 80 is Ala, and X5 at position 230 is absent.
  • IL10-IgG4 Fc where X1 at Fc position 16 is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Ala, and X5 at position 230 is Lys.
  • IL10- [GlyGlyGlyGlySer] 2 -IgG4 Fc wherein X1 at position 16 of the Fc is Pro, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Asn, and X5 at position 230 is Lys.
  • the invention further provides a polynucleotide encoding the IL10-Fc fusion protein of the invention. Further provided is a vector containing the polynucleotide of the present invention, especially an expression vector. In another aspect, the invention provides a host cell comprising the polynucleotide or vector of the invention. The present invention also provides a method for producing the IL10-Fc fusion protein of the present invention, which includes the following steps: (i) cultivating the host cell of the present invention under conditions suitable for expressing the IL10-Fc fusion protein, and (ii ) Recovery of the fusion protein.
  • the invention provides a pharmaceutical composition comprising an effective amount of the IL10-Fc fusion protein of the invention and a pharmaceutically acceptable carrier. Also provided is the IL10-Fc fusion protein or pharmaceutical composition of the present invention for use as a medicament and for the treatment or prevention of diseases in an individual in need thereof, said diseases including viral diseases, inflammatory diseases, immune-related disorders , Fibrosis disorders and proliferative conditions, etc.
  • the IL10-Fc fusion protein provided by the present invention is used to treat or prevent proliferative conditions or disorders, including cancer, such as uterus, cervix, breast, prostate, testis, gastrointestinal tract, kidney, bladder Cancers of bone, bone marrow, skin, head or neck, skin, liver, gallbladder, heart, lung, pancreas, salivary glands, adrenal gland, thyroid, brain, ganglia, central nervous system (CNS) and peripheral nervous system (PNS), and Cancer of the hematopoietic system and immune system.
  • the tumor or cancer is colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, pancreatic cancer, glioblastoma, leukemia, or the like.
  • a method of using the IL10-Fc fusion protein drug for treating a disease is disclosed.
  • a serum trough concentration of IL10- greater than about 0.1 ng / mL (eg, 0.1-2 ng / mL, 0.1-1 ng / mL, 0.5-1.5 ng / mL, or 1.1-2.1 ng / mL)
  • the Fc fusion protein may be the initial candidate dose for administration to the patient, whether for example by one or more separate administrations, or by continuous infusion.
  • Figure 1 Schematic diagram of human interleukin 10-Fc fusion protein structure.
  • the C-terminus of IL-10 is directly or through a connecting peptide connected to the N-terminus of human IgG Fc protein, and forms a dimer through the disulfide bond of human IgG Fc region.
  • Figure 2 shows the cell growth curves of different clones cultured in 7L tanks for 0-13 days.
  • the unit of ordinate is 10 6 / ml.
  • Figure 3 The expression levels of different clones in 7L tanks on days 10, 12 and 13 in mg / ml.
  • Fig. 4 is an electrophoresis diagram after IL10-Fc fusion protein affinity chromatography purification.
  • 1 is cell line 1 culture supernatant; 2 is cell line 1 flow-through; 3 is cell line 1 purification; 4 is cell line 2 culture supernatant; 5 is cell line 2 flow-through; 6 is cell line 2 purification, 7 is Cell 3 culture supernatant; 8 cell 3 flow through; 9 cell 3 purification; 10 commercial marker; IL10-Fc fusion protein molecular weight is about 90kD.
  • IL10-Fc stimulates CD8 + cells to produce cytotoxic factors.
  • CD8 + cells were isolated from the mouse spleen, and after in vitro cultivation and activation, adding different concentrations of IL10-Fc (using IL-10 as a control) can stimulate the cells to produce cytotoxicity (elevated granzyme / perforin expression) and Stimulates IFN ⁇ expression.
  • Figure 6 Changes of tumor proliferation volume in different groups after 6 doses.
  • different antibodies are given, intraperitoneal injection, 250ug / head for the first administration, then 200ug / head / time (10mg / kg), q3dx6 times; IL10-Fc is administered subcutaneously around the tumor, each tumor
  • fusion protein refers to a fusion polypeptide molecule comprising an IL-10 molecule and a human IgG Fc portion, where the components of the fusion protein are connected to each other by peptide bonds, either directly or via a connecting peptide.
  • the individual peptide chains of the human IgG Fc portion of the fusion protein may be non-covalently linked, for example, by disulfide bonds.
  • Fusion means that the components are connected directly or via one or more connecting peptides by peptide bonds.
  • Native IL-10 is a homodimer composed of two ⁇ -helix monomer domains.
  • the sequence of the native human IL-10 monomer domain is shown in SEQ ID NO: 1.
  • Fc domain or "Fc region” is used herein to define the C-terminal region of the antibody heavy chain that contains at least a portion 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 Fc region of human IgG generally extends from the amino acid residue at approximately position 231 to the amino acid residue at approximately position 340.
  • the carbohydrate chain is attached to the CH2 domain.
  • the CH2 domain herein may be a natural sequence CH2 domain or a variant CH2 domain.
  • the "CH3 domain” comprises a stretch of residues at the C-terminus of the CH2 domain in the Fc region (ie, from the amino acid residue at about position 341 to the amino acid residue at about position 447 in IgG).
  • the CH3 region herein may be a natural sequence CH3 domain or a variant CH3 domain, see US Patent No. 5,821,333, expressly incorporated herein by reference).
  • 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.
  • variant encompasses naturally-occurring variants and non-naturally-occurring variants, and broadly refers to mutated recombinant proteins, which usually carry single or multiple amino acid substitutions and are often derived from clones that have undergone site-directed or random mutagenesis Genes or genes from complete synthesis.
  • DNA DNA
  • nucleic acid nucleic acid molecule
  • polynucleotide polynucleotide
  • mRNA messenger RNA
  • cDNA complementary DNA
  • recombinant polynucleotides vectors, probes, primers, and the like.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures and vectors that are integrated into the genome of the host cell to which they are 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.
  • composition refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and does not contain other ingredients that have unacceptable toxicity to a subject who will receive the administration of the formulation.
  • pharmaceutically acceptable carrier refers to an ingredient other than the active ingredient in the pharmaceutical composition that is not toxic to the subject.
  • Pharmaceutically acceptable carriers include but are not limited to buffers, excipients, stabilizers or preservatives, and the like.
  • 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 (at the necessary dose and for the necessary time).
  • patient or “subject” are used interchangeably to refer to human or non-human animals (eg, mammals).
  • treatment means that after the disease, disorder or condition or its symptoms have been diagnosed, observed, etc., for the purpose of temporarily or permanently eliminating, reducing, suppressing, alleviating or improving the disease, disorder or condition that afflicted the subject Clinical intervention (eg administration of IL10-Fc fusion protein or a pharmaceutical composition containing IL10-Fc fusion protein).
  • the present invention provides an IL10-Fc fusion protein, wherein the C-terminus of IL-10 is directly or through a connecting peptide connected to the N-terminus of human IgG Fc protein; wherein the IL-10 sequence is linked to Seq ID No: 1. Consistent; the general formula of the connecting peptide sequence is (G 4 S) n , (SG 4 ) n or G 4 (SG 4 ) n , n is a number between 1 and 10; human IgG Fc protein is selected from human IgG2 , IgG4 native sequence Fc region or variant Fc region.
  • the human body has five types of human immunoglobulins with different effector functions and pharmacokinetic properties.
  • IgG is the most stable of the five types and has a serum half-life of about 23 days in humans.
  • These effector functions are usually mediated by interaction with Fc receptors (Fc ⁇ R) or by binding Clq and immobilizing complement. Binding to FcyR can result in antibody-dependent cell-mediated cell lysis, while binding to complement factor can result in complement-mediated cell lysis.
  • Fc fusion proteins that use only the Fc portion to extend half-life, it is important to minimize effector function.
  • antagonistic antibodies such as soluble cytokines such as TNF ⁇ , IL17A, etc., or immune checkpoints such as PD-1 antibodies
  • the effect of Fc ⁇ Rs is not required, and it can prevent the cells brought by ADCC.
  • Toxicity, IgG2 and IgG4 with weak Fc effects were selected as the backbone.
  • 4 IgG2 and 6 IgG4 monoclonal antibodies have been approved for marketing.
  • anti-PD1 monoclonal antibodies nivolumab and pembrolizumab, anti-IL17A monoclonal antibody ixekizumab, and anti-PCSK9 monoclonal antibody evolocumab have all adopted IgG2 or IgG4 subtypes.
  • An object of the present invention is to extend the half-life of recombinant human IL-10 in vivo, improve circulation time, exposure, efficacy, and reduce renal uptake, while reducing unnecessary immune effects such as ADCC, CDC, etc. Therefore, the structure of the Fc fusion protein of the present invention
  • the Fc portion is preferably derived from the human IgG2 Fc sequence or IgG4 Fc sequence because of its reduced ability to bind Fc ⁇ R and complement factors compared to other IgG subtypes.
  • the present invention provides that the IL10-Fc fusion protein is a C-terminal linking peptide of IL-10 connected to the N-terminus of the Fc protein; wherein, the IL-10 sequence is consistent with that shown in SeqID No: 1, the linking peptide The sequence is [GlyGlyGlyGlySer] 3, and the human IgG Fc protein is selected from the natural sequence Fc region or variant Fc region of human IgG2 or IgG4.
  • the IL10-Fc fusion protein provided by the present invention is IL10-human IgG2 Fc fusion protein, and its sequence is shown in SEQ ID NO: 12.
  • the IL10-Fc fusion protein provided by the present invention is IL10-human IgG4 Fc fusion protein, the sequence of which is shown in SEQ ID NO: 13.
  • the present invention provides that the IL10-Fc fusion protein is connected to the C-terminus of IL-10 directly or through a connecting peptide to the N-terminus of the human IgG4 Fc variant protein; wherein the IL-10 sequence is linked to Seq ID No: 1 is identical; the general formula of the connecting peptide sequence is (G 4 S) n , (SG 4 ) n or G 4 (SG 4 ) n , where n is a number between 1 and 10; human IgG4 Fc region or variable
  • the body protein contains the sequence of SEQ ID NO: 2, where:
  • 16-bit X1 is Pro or Glu
  • the X2 of 17 is Phe, Val, or Ala;
  • X3 at position 18 is Leu, Glu, or Ala
  • 80-bit X4 is Asn or Ala
  • X5 at 230 is Lys or does not exist.
  • the preferred linker peptide sequence in the IL10-Fc fusion protein provided by the present invention is [GlyGlyGlyGlySer] n, and n is an integer of 1-5.
  • the preferred linker peptide sequence in the IL10-Fc fusion protein provided by the present invention is [GlyGlyGlyGlySer] 3.
  • the in vivo function and stability of the fusion protein of the present invention are optimized by adding small connecting peptides to prevent potential unnecessary domain interactions.
  • the glycine-rich linker peptide provides a certain degree of structural flexibility so that the IL-10 moiety can effectively interact with the IL-10 receptor on the target cell.
  • the present invention further modifies the wild-type IgG4 Fc sequence.
  • the present invention obtains a novel human interleukin 10-IgG4 Fc fusion protein (IL10-IgG4 Fc fusion protein) by modifying the IgG4 Fc protein sequence. By replacing amino acids in multiple positions in the Fc part, the modified The IL10-Fc fusion protein has more superior properties than existing Fc fusion proteins, such as increasing in vivo stability, eliminating unnecessary effector functions, and reducing the immunogenicity of the fusion protein in vivo.
  • the IL10-IgG4 Fc fusion protein of the present invention wherein the C-terminus of IL-10 is directly or through a connecting peptide connected to the N-terminus of the Fc protein; wherein the IL-10 sequence is consistent with that shown in SeqID No: 1; the connecting peptide
  • the general formula of the sequence is [GlyGlyGlyGlySer] n, n is an integer of 1-5; the Fc protein part contains the sequence of SEQ ID NO: 2, where:
  • 16-bit X1 is Pro or Glu
  • the X2 of 17 is Phe, Val, or Ala;
  • X3 at position 18 is Leu, Glu, or Ala
  • 80-bit X4 is Asn or Ala
  • X5 at 230 is Lys or does not exist.
  • the IL10-IgG4 Fc fusion protein according to the present invention wherein the preferred connecting peptide sequence has the general formula [GlyGlyGlyGlySer] n, n is an integer of 1-3; more preferably, n is 3, and its sequence is Gly-Gly-Gly -Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser.
  • the in vivo function and stability of the fusion protein of the present invention are optimized by adding small connecting peptides to prevent potential unnecessary domain interactions.
  • the glycine-rich linker peptide provides a certain degree of structural flexibility so that the IL-10 moiety can effectively interact with the IL-10 receptor on the target cell.
  • the Fc protein portion of the present invention is derived from human IgG4, but includes one or more amino acid substituted Fc portions compared to wild-type human sequences.
  • the Fc part consists of two heavy chain constant regions of the antibody that are bound by non-covalent interactions and disulfide bonds.
  • the Fc portion may contain a hinge region and extend to the C-terminus of the antibody via the CH2 and CH3 domains.
  • the Fc portion may also contain one or more glycosylation sites.
  • the IgG4 Fc portion of the fusion protein of the present invention may contain one or more of the following substitutions: corresponding to SEQ ID NO: 2 at position 16 with proline (Pro) or glutamate (Glu) in place of glutamine (Gln ), which corresponds to replacing phenylalanine (Phe) with alanine (Ala) or valine (Val) at position 17 in SEQ ID NO: 2, corresponding to position 18 at SEQ ID NO: 2 Alanine (Ala) or glutamate (Glu) replaces leucine (Leu).
  • the N297 position (EU numbering system) of the Fc portion of the human IgG molecule can be glycosylated, and this glycosylation has a great influence on the activity of IgG. If glycosylation at this site is removed, it will affect the conformation of the upper half of CH2, thereby losing the ability to bind Fc ⁇ Rs and affecting antibody-related biological activity.
  • the fusion protein constructed in the present invention since the effect effect by Fc ⁇ Rs is not required, and the cytotoxicity caused by ADCC of the fusion protein needs to be prevented, it is necessary to modify the Fc portion without glycosylation.
  • replacing Asn with Ala at position 80 corresponding to SEQ ID NO: 2 can remove the N-linked glycosylation site in the IgG4 Fc region, and this aglycosylation modification can reduce the fusion protein Biological effects such as ADCC.
  • the IgG4-derived Fc portion of the IL10-Fc fusion protein discussed here may have the C-terminal lysine residue (Seq ID NO: 2 position 230) present in the natural molecule deleted; the deleted lysine is called For des-K).
  • Some cells such as NS0 cells, express an Fc fusion protein whose C-terminal is lysine is heterogeneous, and the C-terminal amino acid of some fusion proteins is lysine, and part of the fusion protein will lack lysine at the C-terminal.
  • the deletion is due to the action of proteases in the expression of certain types of mammalian cells. Therefore, in order to avoid such heterogeneity, it is preferable to delete lysine at the C-terminus when constructing the Fc fusion protein.
  • Preferred IL10-Fc fusion proteins of the present invention include the following proteins:
  • IL10- [GlyGlyGlyGlySer] 3 -IgG4 Fc where X1 at Fc position 16 is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Asn, and X5 at position 230 does not exist, which The sequence is shown in SEQ ID NO: 3.
  • IL10- [GlyGlyGlyGlySer] 3 -IgG4 Fc where X1 at Fc position 16 is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Ala, and X5 at position 230 does not exist, which The sequence is shown in SEQ ID NO: 4.
  • IL10-GlyGlyGlyGlySer-IgG4 Fc where X1 at Fc position 16 is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Asn, and X5 at position 230 is Lys, the sequence of which is SEQ ID NO: 5 shows.
  • IL10-IgG4 Fc where X1 at Fc position 16 is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Ala, and X5 at position 230 does not exist, the sequence SEQ ID NO: 6 shown.
  • IL10- [GlyGlyGlyGlySer] 3 -IgG4 Fc wherein X1 at Fc position 16 is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Asn, and X5 at position 230 is Lys.
  • IL10-GlyGlyGlyGlySer-IgG4 Fc where X1 at Fc position 16 is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Asn, and X5 at position 230 does not exist.
  • IL10- [GlyGlyGlyGlySer] 3 -IgG4 Fc where X1 at position 16 of the Fc is Pro, X2 at position 17 is Phe, X3 at position 18 is Ala, X4 at position 80 is Ala, and X5 at position 230 is absent.
  • IL10- [GlyGlyGlyGlySer] 3 -IgG4 Fc where X1 at Fc position 16 is Pro, X2 at position 17 is Val, X3 at position 18 is Ala, X4 at position 80 is Ala, and X5 at position 230 is absent.
  • IL10-IgG4 Fc where X1 at Fc position 16 is Glu, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Ala, and X5 at position 230 is Lys.
  • IL10- [GlyGlyGlyGlySer] 2 -IgG4 Fc wherein X1 at position 16 of the Fc is Pro, X2 at position 17 is Ala, X3 at position 18 is Ala, X4 at position 80 is Asn, and X5 at position 230 is Lys.
  • Wild-type human IgG2 or IgG4 protein can be obtained from a variety of sources.
  • a cDNA library can be prepared from cells that express the mRNA of interest at a detectable level to obtain these proteins.
  • the library can be screened using probes designed using published DNA or protein sequences of specific proteins of interest. For example, in Adams et al. (1980) Biochemistry 19: 2711-2719; Goughet et al. (1980) Biochemistry 19: 2702-2710; Dolby et al. (1980) Proc. Natl. Acad. Sci. USA 77: 6027- 6031; Rice et al. (1982) Proc. Natl. Acad. Sci.
  • the invention also provides polynucleotides that encode the IL10-Fc fusion protein as described herein.
  • the polynucleotides of the invention also include those having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% of the polynucleotide sequence encoding the IL10-Fc fusion protein as described herein , 99% or 100% homologous polynucleotides.
  • the DNA encoding IL-10 and IgG (IgG2 Fc or IgG4 Fc) of the present invention can be produced by a variety of different methods, including molecular cloning methods of standard procedures and chemically synthesized DNA.
  • the gene encoding the fusion protein can then be constructed by ligating the DNA encoding IL-10 in frame with the DNA encoding the IgG Fc protein described herein.
  • the DNA encoding the wild-type IgG Fc fragment can be mutated before ligation or in the cDNA encoding the entire fusion protein.
  • Various mutagenesis techniques are widely known in the art.
  • Genes encoding the IL-10 gene and IgG and Fc analog protein can also be linked in frame by DNA encoding a G-rich linker peptide.
  • the present invention provides genes encoding IL10-Fc fusion protein, for example, the gene sequence encoding IL10- [GlyGlyGlyGlySer] 3-IgG4 shown in SEQ ID NO: 3 is shown in SEQ ID NO: 7.
  • the fusion protein of the present invention can be obtained, for example, by recombinant production.
  • one or more polynucleotides encoding the IL10-Fc fusion protein are isolated and inserted into one or more vectors for further cloning in the host cell and / Or expression.
  • Such polynucleotides can be easily isolated and sequenced using conventional procedures.
  • a vector preferably an expression vector
  • the expression vector containing the coding sequence of the IL10-Fc fusion protein and appropriate transcription / translation control signals can be constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA technology, synthetic technology, and in vivo recombination / genetic recombination.
  • the present invention also provides a method for producing the IL10-Fc fusion protein of the present invention, which includes the following steps:
  • Suitable vectors are various commercially available prokaryotic or eukaryotic expression vectors known to those skilled in the art.
  • Prokaryotic expression vectors such as pET series vectors and pQE series vectors; yeast expression vectors pPICZ- ⁇ -A, pHIL-D2 pPIC9, pHIL-S1 (Invitrogen Corp. San Diego. California. USA); animal cell expression vectors pIRES plasmid, pSVK3, pMSG (Amersham Pharmacia Biotech Inc. USA), etc.
  • Suitable host cells include but are not limited to bacterial, yeast, insect and mammalian cells.
  • Recombinant cells containing exogenous nucleic acids encoding IL10-Fc fusion proteins can be prepared by any suitable technique, for example, transfection / transformation with naked DNA plasmid vectors, viral vectors, invasive bacterial cell vectors, or other whole cell vectors, including Transfection by calcium phosphate precipitation, receptor-mediated localization and transfection, bioprojectile delivery, electroporation, dextran-mediated transfection, liposome-mediated transformation, protoplast fusion, direct microinjection And other methods to deliver the IL10-Fc fusion protein coding sequence to the cell for preparation.
  • Methods for transforming / transfecting cells are known in the art, see Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2d Edition, 1989 or 3rd Edition, 2001).
  • the expression of the nucleotide molecule of the present invention can be regulated by another nucleotide sequence, so that the molecule can be expressed in the host transformed with the recombinant DNA molecule.
  • expression can be controlled by any promoter / enhancer element known in the art.
  • Promoters that can be used to control the expression of chimeric polypeptide molecules include but are not limited to long terminal repeats (Squinto et al., 1991, Cell, 65: 1-20); SV40 early promoter region, CMV, M-MuLV, thymidine kinase promoter Regulatory sequences of metallothionine genes; prokaryotic expression vectors such as the b-lactamase promoter or tac promoter (see Scientific American (1980), 242: 74-94); promoters from yeast or other fungi Subelements such as the Gal 4 promoter, ADH, PGK, alkaline phosphatase, and tissue-specific transcription control regions derived from genes such as elastase I gene.
  • Cell lines used as hosts for recombinant proteins are well known in the art and include a variety of immortalized cell lines available from the American Type Culture Collection (ATCC). These cell lines include Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human liver cancer cells (such as Hep G2), A549 cells, and many others Other cell lines.
  • ATCC American Type Culture Collection
  • CHO Chinese hamster ovary
  • NSO Chinese hamster ovary
  • SP2 cells HeLa cells
  • BHK baby hamster kidney cells
  • COS monkey kidney cells
  • human liver cancer cells such as Hep G2
  • A549 cells and many others Other cell lines.
  • the fusion protein antibody is expressed in CHO cells (dhfr-CHO cells, with DHFR as a selection marker).
  • GS glutase gene expression system.
  • nucleic acid or nucleic acid-containing vector
  • the host cell can be cultured enough to express the IL10-Fc fusion protein in the host cell or secrete the fusion protein to the host Cell growth medium.
  • the IL10-Fc fusion protein can be recovered from the culture medium using any standard protein purification methods known in the art, such as immunoaffinity column purification, sulfate precipitation, ion exchange chromatography, hydrophobic interaction chromatography, reverse chromatography, or gel filtration, etc. Technology or any combination thereof.
  • the actual conditions used to purify specific proteins will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, etc.
  • a matrix with protein A or protein G can be used.
  • the invention provides a pharmaceutical composition comprising any IL10-Fc fusion protein provided herein.
  • the pharmaceutical composition of the invention comprises a therapeutically effective amount of the IL10-Fc fusion protein and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers refer to molecular entities and compositions that do not generally cause toxicity to the recipient at the dosage and concentration employed, ie, do not cause adverse, allergic, or other inappropriate reactions when administered to animals (eg, humans) when appropriate .
  • Pharmaceutically acceptable carriers include any and all solvents, buffers, dispersion media, coating materials, surfactants, antioxidants, preservatives (such as antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts , Preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, fragrances, dyes, and similar materials And combinations.
  • the pharmaceutical composition of the present invention can be administered intravenously, intradermally, intra-arterially, intraperitoneally, intracranially, intra-articularly, etc.
  • the fusion protein of the present invention is particularly suitable for parenteral administration, especially by injection, such as subcutaneous, intradermal, intravenous, intraarterial, intramuscular, intrathecal or intraperitoneal injection.
  • the fusion protein of the invention can be formulated in an aqueous solution, preferably in a physiologically compatible buffer.
  • the fusion protein may be in powder form for dissolution with a suitable vehicle, such as sterile water, before use.
  • IL10-Fc fusion proteins Any of the IL10-Fc fusion proteins provided herein can be used in therapeutic methods.
  • the fusion protein of the invention will be formulated, administered and administered in a manner consistent with clinical 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 site of delivery of the agent, the method of administration, the timing of administration, and other factors known to medical practitioners.
  • the IL10-Fc fusion protein or pharmaceutical composition of the present invention is provided for use as a medicament and for the treatment or prevention of diseases in an individual in need thereof, said diseases including viral diseases, inflammatory diseases , Immune-related disorders, fibrotic disorders and proliferative conditions.
  • IL-10 is a cytokine with multiple effects in immune regulation and inflammation. It is produced by mast cells, which eliminates the inflammatory effects of these cells at the site of allergic reactions. Although it can inhibit the synthesis of pro-inflammatory cytokines such as IFN- ⁇ , IL-2, IL-3, TNF ⁇ and GM-CSF, IL-10 is also irritating to certain T cells and mast cells and stimulates B cells Maturation, proliferation and antibody production. IL-10 can block NF- ⁇ B activity and participate in the regulation of JAK-STAT signal transduction pathway. It also induces CD8 + T cell cytotoxic activity and B cell antibody production, and it inhibits macrophage activity and promotes tumor inflammation. The regulation of CD8 + T cells is dose-dependent, where higher doses induce stronger cytotoxic responses.
  • IL-10 plays multiple roles in the activation of CD8 + T cells.
  • IL-10 induces effector molecules (IFN ⁇ , perforin, and granzyme B) in memory CD8 + T cells.
  • IFN ⁇ effector molecules
  • memory CD8 + T cells are responsible for providing long-term antiviral protection of the subject.
  • IL-10 directly The fact that such cells are activated provides unique and alternative treatments.
  • IL10-Fc fusion protein has similar biological activity as IL-10.
  • the embodiments of the present disclosure are based on the connection between CD8 + T cells and cancer and viral infections. Therefore, certain methods of treating and / or preventing cancer-related diseases, disorders, and conditions, such as maintaining an average IL10-Fc fusion protein serum concentration of, for example, greater than 0.5 ng / mL, greater than 1 ng / mL, or greater than 0.1 ng / mL, should also be applicable For the treatment of such diseases.
  • the present disclosure covers the use of the IL10-Fc fusion protein described herein in the treatment or prevention of a wide range of diseases, disorders or conditions and / or symptoms thereof.
  • the IL10-Fc fusion protein is used to treat or prevent proliferative conditions or disorders, including cancer, such as uterus, cervix, breast, prostate, testis, gastrointestinal tract, kidney, bladder, bone, bone marrow, skin, head or Cancers of the neck, skin, liver, gallbladder, heart, lung, pancreas, salivary glands, adrenal gland, thyroid, brain, ganglia, central nervous system (CNS) and peripheral nervous system (PNS), as well as cancers of the hematopoietic system and immune system.
  • the tumor or cancer is colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, pancreatic cancer, glioblastoma, leukemia, or the like.
  • a method of using the IL10-Fc fusion protein drug for treating a disease comprising administering a therapeutically effective amount of the drug to an individual suffering from a disease, the disease including an inflammatory condition, an immune-related disorder , Fibrosis and cancer.
  • the individual is a mammal, preferably a human.
  • a serum trough concentration of IL10- greater than about 0.1 ng / mL eg, 0.1-2 ng / mL, 0.1-1 ng / mL, 0.5-1.5 ng / mL, or 1.1-2.1 ng / mL
  • the Fc fusion protein may be the initial candidate dose for administration to the patient, whether for example by one or more separate administrations, or by continuous infusion.
  • the subject when it is a human, it may be greater than 2.0 ⁇ g / kg / day, greater than 2.5 ⁇ g / kg / day, greater than 3.0 ⁇ g / kg / day, greater than 5 ⁇ g / kg / day, greater than 8 ⁇ g / kg / day, Greater than 10 ⁇ g / kg / day, greater than 12 ⁇ g / kg / day, 15 ⁇ g / kg / day, greater than 18 ⁇ g / kg / day, greater than 20 ⁇ g / kg / day, greater than 21 ⁇ g / kg / day, greater than 22 ⁇ g / kg / day, greater than 23 ⁇ g
  • the IL10-Fc fusion protein is administered at a dose of / kg / day, greater than 24 ⁇ g / kg / day or greater than 25 ⁇ g / kg / day.
  • the initial dose can be estimated from in vitro data such as animal models.
  • the initial dose can be estimated from in vitro data such as animal models.
  • Example 1 IL10-IgG4 Fc fusion protein gene construction
  • Example 2 Expression and purification of IL10-IgG4 Fc fusion protein
  • the supernatant was first ultrafiltered and concentrated with a 10KDa ultrafiltration membrane bag; then Mabselect Sure was used for preliminary affinity chromatography to collect the fusion protein.
  • the mobile phase of affinity chromatography is: A1: 25mM PB + 50mM Nacl, pH 7.0, B1: 20mM Gly, pH 3.0, B2: 20mM citrate buffer, pH 3.0.
  • the chromatographic column is first equilibrated with mobile phase A1, after loading the sample, the mobile phase B1 is used to elute impurities, and then the mobile phase B2 is used to elute the fusion protein, and the collected elution is also adjusted to pH with 1M Tris-His pH8.0 Sex.
  • the crude sample collected in this step is purified through Captoadhere chromatography column. After the sample was loaded and combined at pH 7.0, it was eluted with pH 4.0 to obtain a more pure sample (over 95%).
  • the electropherogram after affinity chromatography purification is shown in Figure 4. The molecular weight of IL10-IgG4 Fc fusion protein was determined to be about 90kD.
  • rhIL-10 (Rochy Hill Company) and the IL10-Fc fusion protein obtained in Example 2 were injected into SD rats with an average body weight of about 200 g at a dose of 200 ng / Kg body weight).
  • blood samples were collected at different time points (0,1,2,4,6,8,12,24,36,48,60,72,96 hours) by tail blood collection, heparin sodium was anticoagulated, and The collected blood sample was centrifuged at 12000g for 5 minutes to collect serum.
  • the blood samples were tested with human IL-10 ELISA kit (purchased from Bender Medsystem) and the content of fusion protein in the serum was checked according to the instructions, and the results were averaged.
  • the results show that the in vivo elimination half-life of the IL10-Fc fusion protein prepared by the present invention is greater than 22.6 hours, and the elimination half-life of the human rhIL-10 after tail vein injection is 2-4 hours, indicating that the IL10-Fc fusion protein prepared by the present invention has an in vivo The half-life is 5-6 times longer than that of rhIL-10 control.
  • IL-10 can not only exert anti-tumor effects by activating NK cells, but also inhibit tumor development by activating T cells. Studies have shown that tumor-bearing mice treated with IL-10 can induce the expression of IFN- ⁇ and granzyme.
  • IL-10 activates phosphorylated STAT1 and STAT3 in CD8 + T cells, thereby inducing the proliferation of CD8 + T cells and Expression of IFN- ⁇ , cytotoxic protein perforin and granule protease; IFN- ⁇ can induce the expression of MHC class I antigen presenting molecules in tumor cells and monocyte macrophages, helping CD8 + T cells to kill most antigens Specific tumor cells; activation of TCR in CD8 + T cells can effectively induce anti-apoptotic signals and cell proliferation signals.
  • IL-10 can not only enhance the tumor killing effect by increasing the cytotoxic activity of NK cells, but also can mediate the infiltration and activation of specific cytotoxic CD8 + T cells in tumors, and the expression and enhancement of IFN- ⁇ and granule protease
  • the presentation of tumor antigens etc. improves the tumor killing capacity of intratumoral CD8 + T cells and the antigen presentation capacity induced by IFN- ⁇ , thereby enhancing the anti-tumor immune escape function.
  • CD8 + cells were isolated from the spleen of the mouse, and after in vitro cultivation and activation, the cells were stimulated by adding different concentrations of IL10-Fc (using IL-10 as a control) It produces cytotoxicity (elevated granzyme / perforin expression) and stimulates IFN ⁇ expression (see Figure 5).
  • IL10-Fc is only about 30% -40% of IL10, but considering the significantly prolonged half-life of IL10-Fc in vivo, its biological activity in vivo has not decreased significantly.
  • Example 5 IL10-Fc in vivo drug efficacy test experiment
  • mice build an animal model of SPC immune group tolerance, and inoculate SPC-PDL1 lung cancer cell line and PBMC extracted from human peripheral blood into the right armpit of mice, inoculate 3x10 6 at each place. After about 7-9 days, wait for most When the tumor proliferated to 90-250 mm 3 (average value was 120-140 mm 3 ). Then exclude the mice with tumors less than 90mm 3 or greater than 250mm 3 or other abnormal conditions. The remaining mice are equally allocated to the human IgG4 antibody (HuIgG4) blank group and the PD-1 antibody positive control according to the random allocation method. Group and IL10-Fc group (10 mice in each group), then 6 times of antibody treatment or IL10-Fc according to different group requirements.
  • Human IgG4 antibody Human IgG4 antibody
  • the dosage regimen for mice is as follows: different antibodies are given according to the group, intraperitoneal injection, 250ug / head for the first administration, then 200ug / head / time (10mg / kg), q3dx6 times; Administration, each tumor is administered 5ug (100ul solution), q3dx6 times; observe the tumor proliferation and the state of mice after the administration, continue to observe and measure the tumor after 1-2 weeks of administration Proliferation. According to the design of the experimental protocol, each group was administered 6 times. During the administration period, the tumor volume and proliferation rate of the PD-1 antibody and IL10-Fc groups were almost the same, and they were significantly slower than the control HuIgG4 group. Sex difference (p ⁇ 0.05), as shown in Figure 6. Preliminary animal experiments have shown that the effect of IL10-Fc in inhibiting tumor cell growth in mice is close to that of PD-1 antibody, showing the potential medical value of the drug in tumor treatment.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

一种人白细胞介素10-Fc融合蛋白及其编码基因与应用。提供IL10-Fc融合蛋白，其中IL-10的C末端直接或通过连接肽与人IgG2或人IgG4的Fc蛋白N端相连。同时提供所述IL10-Fc融合蛋白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，所述的疾病包括炎性病状、免疫相关病症、纤维化病症和癌症等。

Description

[根据细则26改正21.12.2018]　一种重组人白细胞介素10融合蛋白及其应用 技术领域
本发明涉及基因工程药物领域，具体涉及一种重组人白细胞介素10-Fc融合蛋白及其编码基因与应用。
背景技术
白细胞介素-10(Interleukin-10，IL-10)是于1991年被发现的细胞因子，可以调节机体炎症及免疫反应。起初报道该细胞因子能抑制细胞因子分泌、抗原提呈和CD4+细胞活化，IL-10可通过抑制活化的单核细胞和活化的巨噬细胞的IL-1α、IL-1β、IL-6、IL-8、TNF-α、GM-CSF和G-CSF的表达来抑制免疫应答，并且其还抑制NK细胞的IFN-γ产生。虽然IL-10主要在巨噬细胞中表达，但也已在活化的T细胞、B细胞、肥大细胞和单核细胞中检测到表达。除了抑制免疫应答以外，IL-10还展现免疫刺激性质，包括刺激IL-2和IL-4处理的胸腺细胞的增殖，增强B细胞的活力，和刺激MHCII类的表达。
然而，最新的临床研究显示聚乙二醇修饰的IL-10事实上可强有力地激活人体免疫***功能，尤其是能激活具有杀灭癌细胞作用的CD8+T细胞。IL-10能共刺激B细胞激活、延长B细胞存活、及帮助B细胞中的类别转换。此外，它能共刺激天然杀伤(NK)细胞增殖和细胞因子生成且起生长因子的作用刺激某些CD8+T细胞子集增殖(Mosser,D.M.&Yhang,X.,Immunological Reviews 226,205-218(2008)，高剂 量的IL-10(分别为20和25μg/kg)在人中能引起INFγ生成升高(Lauw,F.N.et al.,J.Immunol.165,2783-2789(2000)；Tilg,H.et al.,Gut 50,191-195(2002))。IL-10的免疫刺激活性据报告是由细胞IL-10中第87位处的单一氨基酸异亮氨酸决定的(Ding,Y.et al.,J.Exp.Med.191(2),213-223(2000))。
人IL-10是同型二聚体蛋白，每一个单体包含178个氨基酸，其前18个氨基酸包含信号肽。本公开的特定实施方案包含缺乏信号肽的成熟人IL-10多肽(参见美国专利第6,217,857号)。成熟的IL-10有160个氨基酸残基(Seq ID No：1)，单体分子量为18.7KD，含有4个半胱氨酸形成的二硫键(12-108，62-114)，其天然活性形式是由非共价键连接的分子量为38KD的同源寡二聚体，其在两个单体亚单位之间的非共价相互作用受到破坏后变得无生物活性。获自IL-10的公开晶体结构的数据表明功能二聚体展现与IFN-γ的一定相似性(Zdanov等，1995，Structure(Lond)，3:591-601)。作为其多效性活性的结果，IL-10已与众多种疾病、病症和病状，包括炎性病状、免疫相关病症、纤维化病症和癌症关联。
重组人IL-10在体内的半衰期仅仅2-3h，蛋白很快被清除，这限制了IL-10的生物利用度(Braat,H.et al.,Expert Opin.Biol.Ther.3(5),725-731(2003))。为了改进循环时间、暴露、功效及降低肾摄取，已有文献公开可采用PEG化修饰方法延长其体内半衰期(Mattos,A.et al.,J.Control Release 162,84-91(2012)；Mumm,J.B.et al.,Cancer Cell 20(6),781-796(2011)；Alvarez,H.M.et al.,Drug Metab.Dispos.，40(2),360-373(2012)；CN 201480024021.5等)。但是，由于PEG修饰位 点有多个，因此采用PEG化修饰后的产物是不均一的，这给后续的药物质量控制带来麻烦。
另一条途径涉及将IL-10肽与免疫球蛋白Fc部分融合。免疫球蛋白一般在体内具有长循环半寿期。例如，IgG分子在人体具有高达23天的半寿期。免疫球蛋白Fc部分是这种体内稳定性的主要原因。在保留IL-10分子生物学活性的同时，IL10-Fc融合蛋白质具有由免疫球蛋白Fc部分提供的稳定性而保留IL-10分子的生物活性的优点。
尽管此途径对于IL-10疗法是可行的，但是Fc融合蛋白质长时期反复施用时人体会产生免疫原性是该类药物潜在的隐患。此外，如果Fc部分保留不需要的生物效应功能，会导致额外的治疗副反应，这也是Fc融合蛋白疗法的潜在顾虑。
发明概述
一方面，本发明提供一种人白细胞介素-10(IL-10)与人IgG Fc部分的融合蛋白(IL10-Fc融合蛋白)。
在一个实施方案中，本发明提供IL10-Fc融合蛋白，其中IL-10的C末端直接或通过连接肽与人IgG Fc蛋白的N端相连；其中，IL-10序列与Seq ID No：1所示一致；连接肽序列通式为(G 4S) n、(SG 4) n或G 4(SG 4) n，n是介于1和10之间的数字；人IgG Fc蛋白选自人类IgG2、IgG4的天然序列Fc区或变体Fc区。
本发明的一个优选实施方案中，人类IgG2 Fc序列如SEQ ID NO：10所示。
本发明的另一个优选实施方案中，人类IgG4 Fc序列如SEQ ID NO：11所示。
在一个实施方案中，本发明提供的IL10-Fc融合蛋白，其中优选的连接肽序列通式为[GlyGlyGlyGlySer] n，n为1-4的整数；更优选的，n为3，其序列为[GlyGlyGlyGlySer] 3。通过加入小的连接肽以防止潜在的不必要的结构域相互作用来优化本发明的融合蛋白的体内功能和稳定性。另外，富含甘氨酸的连接肽提供了一定的结构柔性，使IL-10部分可以与靶细胞上IL-10受体有效地相互作用。
在一个优选实施方案中，本发明提供IL10-Fc融合蛋白为IL-10的C末端通过连接肽与人IgG Fc蛋白的N端相连；其中，IL-10序列与Seq ID No：1所示一致，连接肽序列为[GlyGlyGlyGlySer]3，人IgG Fc蛋白选自人类IgG2、IgG4的天然序列Fc区或变体Fc区。
在另一个优选实施方案中，本发明提供的IL10-Fc融合蛋白为IL10-人IgG2 Fc融合蛋白，其序列如SEQ ID NO：12所示。
在另一个优选实施方案中，本发明提供的IL10-Fc融合蛋白为IL10-人IgG4 Fc融合蛋白，其序列如SEQ ID NO:13所示。
在另一个实施方案中，本发明提供IL10-Fc融合蛋白为IL-10的C末端直接或通过连接肽与人IgG4 Fc变体蛋白的N端相连；其中，IL-10序列与Seq ID No：1所示一致；连接肽序列通式为(G 4S) n、(SG 4) n或G 4(SG 4) n，n是介于1和10之间的数字；人IgG4 Fc变体蛋白包含SEQ ID NO：2的序列,其中:
16位的X1为Pro或Glu；
17位的X2为Phe、Val、或Ala；
18位的X3为Leu、Glu、或Ala；
80位的X4为Asn或Ala；以及
230位的X5为Lys或不存在。
在另一个优选实施方案中，本发明提供的IL10-IgG4 Fc融合蛋白中优选的连接肽序列通式为[GlyGlyGlyGlySer] n，n为1-4的整数。
在另一个优选实施方案中，本发明提供的IL10-IgG4 Fc融合蛋白中优选的连接肽序列通式为[GlyGlyGlyGlySer] 3。通过加入小的连接肽以防止潜在的不必要的结构域相互作用来优化本发明的融合蛋白的体内功能和稳定性。另外，富含甘氨酸的连接肽提供了一定的结构柔性，使IL-10部分可以与靶细胞上IL-10受体有效地相互作用。
在另一个优选实施方案中，本发明对野生型的IgG4 Fc序列进行进一步的改造。本发明的融合蛋白的IgG4 Fc部分可以含有一个或多个下列替换：对应于SEQ ID NO：2中的第16位处以脯氨酸(Pro)或谷氨酸(Glu)替换谷氨酰胺(Gln)，对应于SEQ ID NO：2中的第17位处以丙氨酸(Ala)或缬氨酸(Val)替换苯丙氨酸(Phe)，对应于SEQ ID NO：2中的第18位处以丙氨酸(Ala)或谷氨酸(Glu)替换亮氨酸(Leu)。
在另一个优选实施方案中，本发明优选的IL10-IgG4 Fc融合蛋白包括下列蛋白质：
IL10-[GlyGlyGlyGlySer] 3-IgG4 Fc，其中Fc第16位的X1为Glu，17位的X2为Ala，18位的X3为Ala，80位的X4为Asn，以及230位的X5不存在，其序列如SEQ ID NO：3所示。
IL10-[GlyGlyGlyGlySer] 3-IgG4 Fc，其中Fc第16位的X1为Glu，17位的X2为Ala，18位的X3为Ala，80位的X4为Ala，以及230位的X5不存在，其序列SEQ ID NO：4所示。
IL10-GlyGlyGlyGlySer-IgG4 Fc，其中Fc第16位的X1为Glu，17位的X2为Ala，18位的X3为Ala，80位的X4为Asn，以及230位的X5为Lys，其序列SEQ ID NO：5所示。
IL10-IgG4 Fc，其中Fc第16位的X1为Glu，17位的X2为Ala，18位的X3为Ala，80位的X4为Ala，以及230位的X5不存在，其序列SEQ ID NO：6所示。
IL10-[GlyGlyGlyGlySer] 3-IgG4 Fc，其中Fc第16位的X1为Glu，17位的X2为Ala，18位的X3为Ala，80位的X4为Asn，以及230位的X5为Lys。
IL10-GlyGlyGlyGlySer-IgG4 Fc，其中Fc第16位的X1为Glu，17位的X2为Ala，18位的X3为Ala，80位的X4为Asn，以及230位的X5不存在。
IL10-[GlyGlyGlyGlySer] 3-IgG4 Fc，其中Fc第16位的X1为Pro，17位的X2为Phe，18位的X3为Ala，80位的X4为Ala，以及230位的X5不存在。
IL10-[GlyGlyGlyGlySer] 3-IgG4 Fc，其中Fc第16位的X1为Pro，17位的X2为Val，18位的X3为Ala，80位的X4为Ala，以及230位的X5不存在。
IL10-IgG4 Fc，其中Fc第16位的X1为Glu，17位的X2为Ala，18 位的X3为Ala，80位的X4为Ala，以及230位的X5为Lys。
IL10-[GlyGlyGlyGlySer] 2-IgG4 Fc，其中Fc第16位的X1为Pro，17位的X2为Ala，18位的X3为Ala，80位的X4为Asn，以及230位的X5为Lys。
本发明进一步提供一种多核苷酸，其编码本发明的IL10-Fc融合蛋白。进一步提供一种包含本发明的多核苷酸的载体，特别是表达载体。另一方面，本发明提供一种宿主细胞，其包含本发明的多核苷酸或载体。本发明还提供一种用于生成本发明的IL10-Fc融合蛋白的方法，其包括以下步骤：(i)在适合于表达IL10-Fc融合蛋白的条件下培养本发明的宿主细胞，并(ii)回收该融合蛋白。
另一方面，本发明提供一种药物组合物，其包含有效量的本发明的IL10-Fc融合蛋白和药学可接受载体。还提供本发明的IL10-Fc融合蛋白或药物组合物，其用作药物及用于治疗或预防有此需要的个体中的疾病，所述的疾病包括病毒性疾病、炎性疾病、免疫相关病症、纤维化病症和增殖性病状等。
在一个优选的实施方案中，本发明提供的IL10-Fc融合蛋白用于治疗或预防增殖性病状或病症，包括癌症，例如子宫、宫颈、乳腺、***、睾丸、胃肠道、肾、膀胱、骨、骨髓、皮肤、头或颈、皮肤、肝脏、胆囊、心脏、肺、胰腺、唾液腺、肾上腺、甲状腺、脑、神经节、中枢神经***(CNS)和周围神经***(PNS)的癌症，以及造血***和免疫***的癌症。在特定实施方案中，肿瘤或癌症是结肠癌、卵巢癌、乳腺癌、黑色素瘤、肺癌、胰腺癌、胶质母细胞瘤或白血病等。
在一个实施方案中，公开了所述IL10-Fc融合蛋白药物用于治疗疾病的方法。根据疾病的类型和严重程度，大于约0.1ng/mL(例如，0.1-2ng/mL、0.1-1ng/mL、0.5-1.5ng/mL或1.1-2.1ng/mL)的血清谷浓度的IL10-Fc融合蛋白可以是用于对患者施用的起始候选剂量，不管是例如通过一次或多次分开的施用，还是通过连续输注进行。
附图简述
附图1:人白细胞介素10-Fc融合蛋白结构示意图。IL-10的C末端直接或通过连接肽与人IgG Fc蛋白的N端相连，并通过人IgG Fc区域的二硫键形成二聚体。
图2为不同克隆在7L罐培养0-13天的细胞生长曲线。纵坐标单位为10 6/ml。
图3不同克隆在7L罐培养时第10,12和13天的表达量，纵坐标的单位为mg/ml。
图4为IL10-Fc融合蛋白亲和层析纯化后电泳图。1为cell line 1培养上清；2为cell line 1流穿；3为cell line 1纯化；4为cell line 2培养上清；5为cell line 2流穿；6为cell line 2纯化，7为cell line 3培养上清；8为cell line 3流穿；9为cell line 3纯化；10为商业化标准Marker；IL10-Fc融合蛋白分子量约为90kD。
附图5：IL10-Fc刺激CD8+细胞产生细胞毒因子。从小鼠脾脏分离出CD8+细胞，在体外培养并激活后，加入不同浓度的IL10-Fc(以IL-10为对照)作用后，可刺激细胞产生细胞毒作用(颗粒酶/穿孔素表达提升) 并刺激IFNγ表达。
附图6：6次给药不同组别瘤体增殖体积变化情况。根据组别给予不同抗体，腹腔注射，首次给药按250ug/只，之后按200ug/只/次(10mg/kg)，q3dx6次；IL10-Fc采用在瘤体周围皮下给药，每个瘤体每次给药5ug(100ul溶液)，q3dx6次；观察给药治疗后瘤体增殖变化情况和小鼠状态，在给药结束后继续观察和测量1-2周瘤体增殖情况。
发明详述
为使本发明更易于理解，首先定义某些术语。别的定义将在整个详述中阐明。
除非另有说明，本发明的实施将采用分子生物学(包括重组技术)、微生物学、细胞生物学、生物化学和免疫学的常规技术，这些都在本领域的技术范围内。这些技术在文献中有充分解释，诸如Molecular Cloning：A Laboratory Manual(分子克隆：实验室手册)，第二版(Sambrook等，1989)；Oligonucleotide Synthesis(寡核苷酸合成)(M.J.Gait，1984)；Animal Cell Culture(动物细胞培养)(R.I.Freshney，1987)；Methods in Enzymology(酶学中的方法)(Academic Press，Inc.)；.Current Protocols in Molecular Biology(当代分子生物学方案)(F.M.Ausubel等，1987)；PCR：The Polymerase Chain Reaction(PCR：聚合酶链反应)，(Mullis等，1994)；A Practical Guide to Molecular Cloning (分子克隆实用指南)(Perbal Bernard V.，1988)；Phage Display：A Laboratory Manual(噬菌体展示：实验室手册)(Barbas等，2001)等。
如本文中使用的，术语“融合蛋白”指包含IL-10分子和人IgG Fc部分的融合多肽分子，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连接肽相连。为清楚起见，融合蛋白的人IgG Fc部分的各个肽链可以是非共价连接的，例如通过二硫键。
“融合”意指各组分直接地或经由一种或多种连接肽通过肽键连接。
天然IL-10是由两个α-螺旋单体域构成的同二聚体。天然人IL-10单体域的序列显示于SEQ ID NO：1。
本文中术语“Fc域”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。在一个实施方案中，碳水化合物链附着至CH2域。本文中的CH2域可以是天然序列CH2域或变体CH2域。“CH3域”包含Fc区中CH2域C端的一段残基(即IgG中自大约位置341处的氨基酸残基至大约位置447处的氨基酸残基)。本文中的CH3区可以是天然序列CH3域或变体CH3域，参见美国专利No.5,821,333，通过引用明确收入本文)。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***，也称为EU索引，如记载于Kabat et al.,Sequences of Proteins of  Immunological Interest,5th Ed.Public Health Service,Nation al Institutes of Health,Bethesda,MD,1991。
术语“变体”涵盖天然存在的变体和非天然存在的变体，广泛地指突变的重组蛋白，其通常携带单个或多个氨基酸取代并且经常源自已经进行了定点或随机诱变的克隆基因或来自完全合成的基因。
术语“DNA”、“核酸”、“核酸分子”、“多核苷酸”等在本文中可互换使用，是指任何长度的核苷酸(脱氧核糖核苷酸或核糖核苷酸或其类似物)的聚合形式。多核苷酸的非限制性实例包括线性和环状核酸、信使RNA(mRNA)、互补DNA(cDNA)、重组多核苷酸、载体、探针、引物等。
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。
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。
术语“药学可接受载体”指药物组合物中活性成分以外对受试者无毒的成分。药学可接受载体包括但不限于缓冲剂、赋形剂、稳定剂或防腐剂等。
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)。
术语“患者”或“受试者”可互换用于指人或非人动物(例如，哺乳动物)。
术语“治疗”等是指在疾病、病症或病状或其症状已被诊断、观察等之后，为了暂时或永久性消除、减轻、阻遏、缓和或改善折磨受试者的疾病、病症或病状而实施的临床干预(例如施用IL10-Fc融合蛋白或包含IL10-Fc融合蛋白的药物组合物)。
本发明的IL10-Fc融合蛋白
在一个实施方案中，本发明提供IL10-Fc融合蛋白，其中IL-10的C末端直接或通过连接肽与人IgG Fc蛋白的N端相连；其中，IL-10序列与Seq ID No：1所示一致；连接肽序列通式为(G 4S) n、(SG 4) n或G 4(SG 4) n，n是介于1和10之间的数字；人IgG Fc蛋白选自人类IgG2、IgG4的天然序列Fc区或变体Fc区。
人体有五种类型的具有不同效应子功能和药物动力学特性的人类免疫球蛋白。IgG是五种类型中最稳定的，在人体具有约23天的血清半衰期。人体IgG有四个亚类：IgG1、IgG2、IgG3和IgG4，各亚类具有称作效应子功能的不同生物学功能。这些效应子功能通常由与Fc受体(FcγR)的相互作用或通过结合Clq和固定补体介导。与FcγR的结合可以导致抗体依赖细胞介导的细胞裂解，而与补体因子的结合可以导致补体介导的细胞裂解。
在仅利用Fc部分延长半衰期的Fc融合蛋白的设计中，将效应子功能最小化是重要的。对于一些纯拮抗作用的抗体，如可溶性细胞激素如TNFα，IL17A等，或者免疫检查点如PD-1的抗体来说，并不需要FcγRs带来的效应作用，且能够防止ADCC等带来的细胞毒性，因此Fc效应弱的IgG2和IgG4被选择用来作为骨架。现在已有4个IgG2和6个IgG4单抗被批准上市，例如anti-PD1单抗nivolumab和pembrolizumab，anti-IL17A单抗ixekizumab以及anti-PCSK9单抗evolocumab等都采用了IgG2或IgG4亚型。
本发明的一个目的是延长重组人IL-10在体内的半衰期，改进循环时间、暴露、功效及降低肾摄取，同时降低不必要的ADCC、CDC等免疫效应，因此本发明的Fc融合蛋白结构中Fc部分优选来自人类IgG2 Fc序列或IgG4 Fc序列，因其与其他IgG亚型相比结合FcγR和补体因子的能力降低。
在一个优选实施方案中，本发明提供IL10-Fc融合蛋白为IL-10的C末端连接肽与Fc蛋白的N端相连；其中，IL-10序列与Seq ID No：1所示一致，连接肽序列为[GlyGlyGlyGlySer]3，人IgG Fc蛋白选自人类IgG2或IgG4的天然序列Fc区或变体Fc区。在另一个优选实施方案中，本发明提供的IL10-Fc融合蛋白为IL10-人IgG2 Fc融合蛋白，其序列如SEQ ID NO:12所示。在另一个优选实施方案中，本发明提供的IL10-Fc融合蛋白为IL10-人IgG4 Fc融合蛋白，其序列如SEQ ID NO:13所示。
在另一个实施方案中，本发明提供IL10-Fc融合蛋白为IL-10的C末端直接或通过连接肽与人IgG4 Fc变体蛋白的N端相连；其中，IL-10 序列与Seq ID No：1所示一致；连接肽序列通式为(G 4S) n、(SG 4) n或G 4(SG 4) n，n是介于1和10之间的数字；人IgG4 Fc区或变体蛋白包含SEQ ID NO：2的序列,其中:
16位的X1为Pro或Glu；
17位的X2为Phe、Val、或Ala；
18位的X3为Leu、Glu、或Ala；
80位的X4为Asn或Ala；以及
230位的X5为Lys或不存在。
在另一个优选实施方案中，本发明提供的IL10-Fc融合蛋白中优选的连接肽序列通式为[GlyGlyGlyGlySer]n，n为1-5的整数。
在另一个优选实施方案中，本发明提供的IL10-Fc融合蛋白中优选的连接肽序列通式为[GlyGlyGlyGlySer]3。通过加入小的连接肽以防止潜在的不必要的结构域相互作用来优化本发明的融合蛋白的体内功能和稳定性。另外，富含甘氨酸的连接肽提供了一定的结构柔性，使IL-10部分可以与靶细胞上IL-10受体有效地相互作用。
在另一个优选实施方案中，本发明对野生型的IgG4 Fc序列进行进一步的改造。本发明通过对IgG4 Fc蛋白序列的改造得到一种新型的人白细胞介素10-IgG4 Fc融合蛋白(IL10-IgG4 Fc融合蛋白)，通过对Fc部分中多个位置氨基酸的替换，使得改造后的IL10-Fc融合蛋白具有比现有Fc融合蛋白更优越的性能，例如增加体内稳定性、消除不必要的效应子功能以及降低该融合蛋白在生物体内的免疫原性。
本发明所述的IL10-IgG4 Fc融合蛋白，其中IL-10的C末端直接或通过连接肽与Fc蛋白的N端相连；其中，IL-10序列与Seq ID No：1所示一致；连接肽序列通式为[GlyGlyGlyGlySer]n，n为1-5的整数；Fc蛋白部分包含SEQ ID NO：2的序列,其中:
16位的X1为Pro或Glu；
17位的X2为Phe、Val、或Ala；
18位的X3为Leu、Glu、或Ala；
80位的X4为Asn或Ala；以及
230位的X5为Lys或不存在。
本发明所述的IL10-IgG4 Fc融合蛋白，其中优选的连接肽序列通式为[GlyGlyGlyGlySer]n，n为1-3的整数；更优选的，n为3，其序列为Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser。通过加入小的连接肽以防止潜在的不必要的结构域相互作用来优化本发明的融合蛋白的体内功能和稳定性。另外，富含甘氨酸的连接肽提供了一定的结构柔性，使IL-10部分可以与靶细胞上IL-10受体有效地相互作用。
本发明的Fc蛋白部分来自人类IgG4，但是与野生型人类序列相比包括一个或多个氨基酸替换的Fc部分。Fc部分由通过非共价相互作用和二硫键结合的抗体的两个重链恒定区组成。Fc部分可以包含铰链区，并经CH2和CH3结构域延伸到抗体C末端。Fc部分还可以包含一个或多个糖基化位点。
为进一步降低其效应子功能，本发明对野生型的IgG4 Fc区域进行进一步的改造。本发明的融合蛋白的IgG4 Fc部分可以含有一个或多个 下列替换：对应于SEQ ID NO：2中的第16位处以脯氨酸(Pro)或谷氨酸(Glu)替换谷氨酰胺(Gln)，对应于SEQ ID NO：2中的第17位处以丙氨酸(Ala)或缬氨酸(Val)替换苯丙氨酸(Phe)，对应于SEQ ID NO：2中的第18位处以丙氨酸(Ala)或谷氨酸(Glu)替换亮氨酸(Leu)。
人IgG分子Fc部分的N297位(EU编号***)能够发生糖基化，该糖基化对IgG的活性有很大影响。如果该位点糖基化被移除，则会影响CH2上半部分的构象，从而丧失对FcγRs的结合能力，影响抗体相关的生物活性。但是对于本发明构造的融合蛋白来说，由于并不需要FcγRs带来的效应作用，且需要防止融合蛋白的ADCC等带来的细胞毒性，因此需要对Fc部分进行无糖基化改造。基于此考虑，发明人发现在对应于SEQ ID NO：2第80位处用Ala替换Asn，能够去除IgG4 Fc区域中N-连接的糖基化位点，该无糖基化改造能够降低融合蛋白的ADCC等生物学效应。
此外，在此讨论的IL10-Fc融合蛋白的IgG4衍生的Fc部分中可以缺失存在于天然分子中的C末端赖氨酸残基(Seq ID NO：2第230位；将缺失的赖氨酸称为des-K)。某些细胞例如NS0细胞表达的C末端为赖氨酸的Fc融合蛋白是不均一的，某些融合蛋白的C末端氨基酸为赖氨酸，而一部分融合蛋白的C末端将缺失赖氨酸，该缺失是由于某些类型的哺乳动物细胞表达过程中蛋白酶的作用。因此，为了避免这种异质性，Fc融合蛋白构建时优选C末端缺失赖氨酸。
为方便理解，提供了常见氨基酸单字母和三字母代码对应表，如下所示。
Figure PCTCN2018116074-appb-000001
本发明优选的IL10-Fc融合蛋白包括下列蛋白质：
IL10-[GlyGlyGlyGlySer] 3-IgG4 Fc，其中Fc第16位的X1为Glu，17位的X2为Ala，18位的X3为Ala，80位的X4为Asn，以及230位的X5不存在，其序列如SEQ ID NO：3所示。
IL10-[GlyGlyGlyGlySer] 3-IgG4 Fc，其中Fc第16位的X1为Glu，17位的X2为Ala，18位的X3为Ala，80位的X4为Ala，以及230位的X5不存在，其序列SEQ ID NO：4所示。
IL10-GlyGlyGlyGlySer-IgG4 Fc，其中Fc第16位的X1为Glu，17位的X2为Ala，18位的X3为Ala，80位的X4为Asn，以及230位的X5为Lys，其序列SEQ ID NO：5所示。
IL10-IgG4 Fc，其中Fc第16位的X1为Glu，17位的X2为Ala，18位的X3为Ala，80位的X4为Ala，以及230位的X5不存在，其序列SEQ ID NO：6所示。
IL10-[GlyGlyGlyGlySer] 3-IgG4 Fc，其中Fc第16位的X1为Glu，17位的X2为Ala，18位的X3为Ala，80位的X4为Asn，以及230位的 X5为Lys。
IL10-GlyGlyGlyGlySer-IgG4 Fc，其中Fc第16位的X1为Glu，17位的X2为Ala，18位的X3为Ala，80位的X4为Asn，以及230位的X5不存在。
IL10-[GlyGlyGlyGlySer] 3-IgG4 Fc，其中Fc第16位的X1为Pro，17位的X2为Phe，18位的X3为Ala，80位的X4为Ala，以及230位的X5不存在。
IL10-[GlyGlyGlyGlySer] 3-IgG4 Fc，其中Fc第16位的X1为Pro，17位的X2为Val，18位的X3为Ala，80位的X4为Ala，以及230位的X5不存在。
IL10-IgG4 Fc，其中Fc第16位的X1为Glu，17位的X2为Ala，18位的X3为Ala，80位的X4为Ala，以及230位的X5为Lys。
IL10-[GlyGlyGlyGlySer] 2-IgG4 Fc，其中Fc第16位的X1为Pro，17位的X2为Ala，18位的X3为Ala，80位的X4为Asn，以及230位的X5为Lys。
IL10-Fc融合蛋白的结构如图1所示。
可以从多种来源获得野生型人类IgG2或IgG4蛋白质。例如，可以从以可检测水平表达目的mRNA的细胞制备cDNA文库以获得这些蛋白质。使用特定目的蛋白质的公开的DNA或蛋白质序列设计的探针可以对文库进行筛选。例如，在Adams等人，(1980)Biochemistry 19：2711-2719；Goughet等人，(1980)Biochemistry 19：2702-2710；Dolby等人，(1980)Proc.Natl.Acad.Sci.USA77：6027-6031；Rice等人， (1982)Proc.Natl.Acad.Sci.USA79：7862-7862；Falkner等人，(1982)Nature 298：286-288；以及Morrison等人，(1984)Ann.Rev.Immunol.2：239-256中描述了免疫球蛋白轻或重链恒定区。
多核苷酸
本发明还提供多核苷酸，其编码如本文中描述的IL10-Fc融合蛋白。
本发明的多核苷酸还包括那些与编码如本文中描述的编码IL10-Fc融合蛋白的多核苷酸序列具有至少约80％、85％、90％、95％、96％、97％、98％、99％或100％同源性的多核苷酸。
可以通过多种不同的方法产生编码本发明的IL-10和IgG Fc(IgG2 Fc或IgG4 Fc)的DNA，所述方法包括标准程序的分子克隆方法和化学合成的DNA。然后可以通过将编码IL-10的DNA在框内与此处所述的编码IgG Fc蛋白质的DNA连接而构建编码融合蛋白的基因。可以在连接前或在编码整个融合蛋白质的cDNA中突变编码野生型IgG Fc片段的DNA。多种诱变技术在本领域广为人知。编码IL-10基因和编码IgG Fc类似物蛋白质的基因也可以通过编码富含G的连接肽的DNA在框内连接。
本发明提供能编码IL10-Fc融合蛋白的基因，例如编码SEQ ID NO：3所示的IL10-[GlyGlyGlyGlySer]3-IgG4 Fc融合蛋白的基因序列如SEQ ID NO：7所示。
重组方法
可以例如通过重组生成来获得本发明的融合蛋白。对于重组生成，分离一种或多种编码所述IL10-Fc融合蛋白的多核苷酸(例如如上文描述 的)，并将其***一种或多种载体中用于在宿主细胞中进一步克隆和/或表达。可以使用常规规程容易分离并测序此类多核苷酸。在一个实施方案中，提供包含一种或多种本发明的多核苷酸的载体(优选为表达载体)。可以使用本领域技术人员公知的方法来构建含有IL10-Fc融合蛋白的编码序列以及适宜的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、合成技术和体内重组/遗传重组。
本发明还提供一种用于生成本发明的IL10-Fc融合蛋白的方法，其包括以下步骤：
(i)在适合于表达IL10-Fc融合蛋白的条件下培养本发明的宿主细胞；
(ii)回收该融合蛋白。
当把编码IL10-Fc融合蛋白的核酸分子***合适载体中，当将所述的载体引入合适的宿主细胞时能表达IL10-Fc融合蛋白。合适的载体为本领域技术人员所熟知的各种可商业化购买的原核或真核表达载体，原核表达载体如pET系列载体，pQE系列载体；酵母表达载体pPICZ-α-A，pHIL-D2，pPIC9，pHIL-S1(Invitrogen Corp.San Diego.California.USA)；动物细胞表达载体pIRES质粒、pSVK3、pMSG(Amersham Pharmacia Biotech Inc.USA)等。
合适的宿主细胞包括但不限于细菌、酵母、昆虫和哺乳动物细胞。含编码IL10-Fc融合蛋白的外源核酸的重组细胞可通过任何合适的技术制备，例如，用裸露DNA质粒载体、病毒载体、侵入性细菌细胞载体或其它全细胞载体等转染/转化，包括通过磷酸钙沉淀的转染、受体介 导的定位和转染、生物弹射击递送、电穿孔、葡聚糖介导的转染、脂质体介导的转化、原生质体融合、直接微注射等方法将IL10-Fc融合蛋白编码序列递送到细胞中制备。转化/转染细胞的方法是本领域已知的，参见Sambrook et al.Molecular Cloning：ALaboratory Manual，Cold Spring Harbor Laboratory Press(2d Edition，1989或3rd Edition，2001)。
本发明核苷酸分子的表达可通过另一核苷酸序列调控，因而该分子在用重组DNA分子转化的宿主中得以表达。例如，表达可通过本领域已知的任何启动子/增强子元件控制。可用于控制嵌合多肽分子表达的启动子包括但不限于长末端重复序列(Squinto等，1991，Cell，65：1-20)；SV40早期启动子区、CMV、M-MuLV、胸苷激酶启动子、金属硫蛋白(metallothionine)基因的调控序列；原核表达载体如b-内酰胺酶启动子或tac启动子(见Scientific American(1980)，242：74-94)；来自酵母或其它真菌的启动子元件如Gal 4启动子、ADH、PGK、碱性磷酸酶和源于基因如弹性蛋白酶I基因的组织特异性转录控制区。
作为宿主用于重组蛋白的细胞系是本领域周知的，包括多种可从美国典型培养物保藏中心(ATCC)获得的永生化细胞系。这些细胞系中包括中国仓鼠卵巢(CHO)细胞、NSO、SP2细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝癌细胞(例如Hep G2)、A549细胞及多种其它细胞系。在一个优选的技术方案中，融合蛋白抗体在CHO细胞(dhfr-CHO细胞，以DHFR为筛选标记)表达。表达***的另一个实施例是GS(谷氨酸合成酶)基因表达***，具体可参考WO87/04462、 WO89/01036和EP338841等。当编码诸如IL10-Fc融合蛋白的核酸(或含核酸的载体)导入到哺乳动物宿主细胞中时，可通过将宿主细胞培养足以在宿主细胞中表达IL10-Fc融合蛋白或将融合蛋白分泌到宿主细胞生长的培养基中。
可用任何本领域已知标准蛋白纯化方法从培养基中回收IL10-Fc融合蛋白，例如免疫亲和柱纯化、硫酸盐沉淀、离子交换色谱、疏水相互作用色谱、反向色谱或凝胶过滤等纯化技术或其任意的组合。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。对于本发明的融合蛋白的亲和层析纯化，可以使用具有蛋白A或蛋白G的基质。
药物组合物
另一方面，本发明提供包含本文中提供的任何IL10-Fc融合蛋白的药物组合物，本发明的药物组合物包含治疗有效量的IL10-Fc融合蛋白和药学可接受的载体。药学可接受的载体是指在所采用的剂量和浓度一般对接受者无毒性，即在适当时对动物(例如人)施用时不产生不利、变应性或其它不当反应的分子实体和组合物。药学可接受的载体包括任何和所有的溶剂、缓冲剂、分散介质、涂料材料、表面活性剂、抗氧化剂、防腐剂(例如抗细菌剂、抗真菌剂)、等张剂、吸收延缓剂、盐、防腐剂、抗氧化剂、蛋白质、药物、药物稳定剂、聚合物、凝胶、粘合剂、赋形剂、崩解剂、润滑剂、甜味剂、芳香剂、染料、此类类似的材料及其组合。
本发明所述的药物组合物，可以静脉内、皮内、动脉内、腹膜内、 颅内、关节内等方式给药。本发明的融合蛋白尤其适合于胃肠外给药，特别是通过注射施用，例如皮下、皮内、静脉内、动脉内、肌内、鞘内或腹膜内注射施用。对于注射，可以将本发明的融合蛋白在水性溶液，优选地在生理学相容的缓冲液中配制。或者，融合蛋白可以为粉末形式，用于在使用前用合适的媒介物例如无菌水溶解。
治疗方法
可以将本文中提供的任一种IL10-Fc融合蛋白用在治疗方法中。
对于在治疗方法中的使用，将以与临床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。
在某些实施方案中，提供本发明的IL10-Fc融合蛋白或药物组合物用作药物及用于治疗或预防有此需要的个体中的疾病，所述的疾病包括病毒性疾病、炎性疾病、免疫相关病症、纤维化病症和增殖性病状等。
IL-10为在免疫调节和炎症中具有多效性作用的细胞因子。其由肥大细胞产生，消解这些细胞在过敏反应的部位的炎性效应。虽然其能够抑制促炎性细胞因子诸如IFN-γ、IL-2、IL-3、TNFα和GM-CSF的合成，但IL-10对某些T细胞和肥大细胞也具有刺激性并且刺激B细胞成熟、增殖和抗体产生。IL-10可阻断NF-κB活性并且参与JAK-STAT信号转导途径的调控。其还诱导CD8+T细胞的细胞毒性活性和B细胞的抗体产 生，并且其抑制巨噬细胞活性和促肿瘤炎症。CD8+T细胞的调节是剂量依赖性的，其中较高剂量诱导较强的细胞毒性反应。
IL-10在CD8+T细胞的活化中起着多个作用。例如，IL-10在记忆CD8+T细胞中诱导效应分子(IFNγ、穿孔蛋白和颗粒酶B)。此类记忆CD8+T细胞是负责提供受试者的长期抗病毒保护作用的细胞。虽然当IL-10不存在时记忆CD8+T细胞的产生和扩增可发生(Vicari，A.和Trinchieri，G.,(2004)Immuno.Rev.202:223-236)，但IL-10直接活化此类细胞的事实提供独特的和备选的治疗方法。
IL10-Fc融合蛋白具有IL-10相似的生物学活性。鉴于上述，本公开的实施方案基于CD8+T细胞与癌症和病毒感染之间的联系。因此，治疗和/或预防癌症相关疾病、病症和病状的某些方法，诸如维持例如大于0.5ng/mL、大于1ng/mL或大于0.1ng/mL的平均IL10-Fc融合蛋白血清浓度应当也适用于此类疾病的治疗。本公开涵盖本文所述的IL10-Fc融合蛋白在治疗或预防广泛的疾病、病症或病状和/或其症状中的用途。根据本公开，IL10-Fc融合蛋白用于治疗或预防增殖性病状或病症，包括癌症，例如子宫、宫颈、乳腺、***、睾丸、胃肠道、肾、膀胱、骨、骨髓、皮肤、头或颈、皮肤、肝脏、胆囊、心脏、肺、胰腺、唾液腺、肾上腺、甲状腺、脑、神经节、中枢神经***(CNS)和周围神经***(PNS)的癌症，以及造血***和免疫***的癌症。在特定实施方案中，肿瘤或癌症是结肠癌、卵巢癌、乳腺癌、黑色素瘤、肺癌、胰腺癌、胶质母细胞瘤或白血病等。
在一个实施方案中，公开了所述IL10-Fc融合蛋白药物用于治疗疾病 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，所述的疾病包括炎性病状、免疫相关病症、纤维化病症和癌症等。所述的个体是哺乳动物，优选是人。根据疾病的类型和严重程度，大于约0.1ng/mL(例如，0.1-2ng/mL、0.1-1ng/mL、0.5-1.5ng/mL或1.1-2.1ng/mL)的血清谷浓度的IL10-Fc融合蛋白可以是用于对患者施用的起始候选剂量，不管是例如通过一次或多次分开的施用，还是通过连续输注进行。
优选的，当受试者为人时，可以以大于2.0μg/kg/日、大于2.5μg/kg/日、大于3.0μg/kg/日、大于5μg/kg/日、大于8μg/kg/日、大于10μg/kg/日、大于12μg/kg/日、15μg/kg/日、大于18μg/kg/日、大于20μg/kg/日、大于21μg/kg/日、大于22μg/kg/日、大于23μg/kg/日、大于24μg/kg/日或大于25μg/kg/日的剂量施用IL10-Fc融合蛋白。使用本领域中公知的技术，能从体外数据例如动物模型估算出初始剂量。本领域中的普通技术人员能容易地基于动物数据优化对人的施用。
实施例
以下是本发明的方法和组合物的实施例。应理解鉴于上文提供的一般性描述，可以实践各种其他实施方案。
实施例1：IL10-IgG4 Fc融合蛋白基因构建
将SEQ ID NO：3翻译成DNA序列，并根据CHO细胞的密码子偏好性优化，获得IL10-Fc融合蛋白的表达序列SEQ ID NO：7。在该优化好的序列5’端添加NheI酶切位点和kozac序列(SEQ ID No.8)，在3’ 端添加终止密码子和XhoI酶切位点(SEQ ID No.9)，得到融合蛋白的完整表达框。人工合成完整表达框序列，***到pIRES质粒的NheI和XhoI酶切位点之间，得到pIRES-IL10-Fc表达质粒。该质粒经线性化后，电转染至CHO-s细胞，加入MSX筛选出阳性克隆。
实施例2：IL10-IgG4 Fc融合蛋白的表达及纯化
实施例1中获得的阳性克隆，经过两轮有限稀释，筛选出3个表达量较优的克隆，经扩大培养，接种到7L发酵罐进行补料培养(图2)，表达目的蛋白(图3)。发酵结束后4500rpm离心6min，收集上清调整pH至4.0后4℃保存。
上清先用10KDa的超滤膜包超滤浓缩；然后用Mabselect Sure进行初步亲和层析，收集融合蛋白。亲和层析流动相为：A1:25mM PB+50mM Nacl,pH7.0，B1:20mM Gly,pH3.0，B2:20mM柠檬酸缓冲液,pH3.0。层析柱先用流动相A1平衡，上样后先用流动相B1洗脱杂质，再用流动相B2洗脱融合蛋白，收集到的洗脱也用1M Tris-His pH8.0调节至pH中性。此步收集到的粗纯样品，在通过Capto adhere层析柱精纯。样品经pH7.0上样结合后，用pH4.0洗脱得到较纯的样品(95％以上)。亲和层析纯化后电泳图如图4所示，测定IL10-IgG4 Fc融合蛋白分子量为90kD左右。
实施例3：体内半衰期测定
通过尾静脉注射，将rhIL-10(Rochy Hill公司)和由实施例2获得的IL10-Fc融合蛋白按200ng/Kg体重剂量分别注入平均体重约200g的SD大鼠)。注射后，在不同时间点(0、1、2、4、6、8、12、24、36、48、60、72、96小时)通过剪尾取血法收集血样，肝素钠抗凝，将采集的血 样12000g离心5min，收集血清。
将血样用人IL-10 ELISA试剂盒(购自Bender Medsystem公司)并参照说明书检测血清中融合蛋白的含量，结果取平均值。结果显示，本发明制备IL10-Fc融合蛋白的体内消除半衰期大于22.6小时，而用人rhIL-10经尾静脉注射后消除半衰期为2-4小时，表明本发明制备得到IL10-Fc融合蛋白在体内的半衰期相对于rhIL-10对照品延长了5-6倍以上。
实施例4：抗肿瘤活性实验
研究证明IL-10不仅可以通过活化NK细胞发挥抗肿瘤效应，还可以通过活化T细胞抑制肿瘤的发展。研究表明IL-10治疗的荷瘤小鼠可以诱导IFN-γ和颗粒酶的表达。该作用可能是由对肿瘤内CD8+T细胞有特异性的IL-10信号通路介导的：IL-10激活CD8+T细胞中磷酸化的STAT1和STAT3，从而诱导CD8+T细胞的增殖和IFN-γ、细胞毒性蛋白穿孔素及颗粒蛋白酶的表达；IFN-γ可以诱导肿瘤细胞和单核巨噬细胞中MHC-Ⅰ类抗原提呈分子的表达，协助CD8+T细胞杀死大部分抗原特异性肿瘤细胞；激活CD8+T细胞中的TCR可以有效地诱导抗细胞凋亡信号和细胞增殖信号。总之，IL-10不仅可以通过提高NK细胞的细胞毒性活性增强肿瘤杀伤效应，还可以通过介导肿瘤内特异性细胞毒性CD8+T细胞的浸润和激活、IFN-γ和颗粒蛋白酶的表达和增强肿瘤抗原提呈等而提高瘤内CD8+T细胞的肿瘤杀伤能力和IFN-γ诱导的抗原提呈能力，从而增强了抗肿瘤免疫逃逸的功能。
为了检测IL10-Fc刺激CD8+T的细胞毒作用,从小鼠脾脏分离出 CD8+细胞，在体外培养并激活后，加入不同浓度的IL10-Fc(以IL-10为对照)作用后，可刺激细胞产生细胞毒作用(颗粒酶/穿孔素表达提升)并刺激IFNγ表达(如图5)。尽管，体外活性方面，IL10-Fc只有IL10的30％-40％左右，但是考虑到IL10-Fc体内半衰期显著延长，其体内生物学活性并未显著下降。
实施例5：IL10-Fc体内药效测试实验
首先构建SPC免疫组库耐受的动物模型，将SPC-PDL1肺癌细胞株和人外周血提取的PBMC混合接种于小鼠右侧腋下，每处接种3x10 6，约7-9天后，待多数瘤体增殖至90-250mm 3时(平均值在120-140mm 3)。接着排除瘤体小于90mm 3或大于250mm 3的小鼠或其它异常情况，剩余小鼠按随机分配方法，将各小鼠均等地分配到人IgG4抗体(HuIgG4)空白组、PD-1抗体阳性对照组和IL10-Fc组(每组各10只)，然后按不同组别要求进行6次抗体治疗或IL10-Fc。
小鼠给药方案为：根据组别给予不同抗体，腹腔注射，首次给药按250ug/只，之后按200ug/只/次(10mg/kg)，q3dx6次；IL10-Fc采用在瘤体周围皮下给药，每个瘤体每次给药5ug(100ul溶液)，q3dx6次；观察给药治疗后瘤体增殖变化情况和小鼠状态，在给药结束后继续观察和测量1-2周瘤体增殖情况。按实验方案设计，对各组进行6次给药，给药期间，PD-1抗体和IL10-Fc组整体上瘤体体积和增殖速度差不多，且明显慢与对照HuIgG4组，统计学上具有显著性差异(p﹤0.05)，如图6所示。初步的动物试验证明，IL10-Fc在小鼠体内抑制肿瘤细胞生长的效果与PD-1抗体接近，显示该药物在肿瘤治疗中的潜在医疗价值。
Figure PCTCN2018116074-appb-000002
Figure PCTCN2018116074-appb-000003
Figure PCTCN2018116074-appb-000004
Figure PCTCN2018116074-appb-000005
Figure PCTCN2018116074-appb-000006
Figure PCTCN2018116074-appb-000007
Figure PCTCN2018116074-appb-000008

Claims (31)

  1. 一种IL10-Fc融合蛋白，其中IL-10的C末端直接或通过连接肽与人IgG Fc蛋白的N端相连；其中，IL-10序列与Seq ID No：1所示一致；连接肽序列通式为(G 4S) n、(SG 4) n或G 4(SG 4) n，n是介于1和10之间的数字；人IgG Fc蛋白选自人类IgG2、IgG4的天然序列Fc区或变体Fc区。
  2. 根据权利要求1所述的IL10-Fc融合蛋白，其特征在于：所述的人类IgG2 Fc序列如SEQ ID NO：10所示。
  3. 根据权利要求1所述的IL10-Fc融合蛋白，其特征在于：所述的人类IgG4 Fc序列如SEQ ID NO：11所示。
  4. 根据权利要求1所述的IL10-Fc融合蛋白，其特征在于：所述的连接肽序列通式为[GlyGlyGlyGlySer] n，n为1-4的整数。
  5. 根据权利要求1所述的IL10-Fc融合蛋白，其特征在于：所述的IL10-Fc融合蛋白为IL-10的C末端通过连接肽[GlyGlyGlyGlySer] 3与人IgG Fc蛋白的N端相连；其中，IL-10序列与Seq ID No：1所示一致，人IgG Fc蛋白选自人类IgG2、IgG4的天然序列Fc区或变体Fc区。
  6. 根据权利要求5所述的IL10-Fc融合蛋白，其特征在于：所述的IL10-Fc融合蛋白为IL10-人IgG2 Fc融合蛋白，其序列如SEQ ID NO：12所示。
  7. 根据权利要求5所述的IL10-Fc融合蛋白，其特征在于：所述的IL10-Fc融合蛋白为IL10-人IgG4 Fc融合蛋白，其序列如SEQ ID NO:13所示。
  8. 根据权利要求1所述的IL10-Fc融合蛋白，其特征在于：所述的IL10-Fc融合蛋白为IL-10的C末端直接或通过连接肽与人IgG4 Fc变体蛋白的N端相连；其中，IL-10序列与Seq ID No：1所示一致；连接肽序列通式为(G 4S) n、(SG 4) n或G 4(SG 4) n，n是介于1和10之间的数字；人IgG4 Fc变体蛋白包含SEQ ID NO：2的序列,其中:
    16位的X1为Pro或Glu；
    17位的X2为Phe、Val、或Ala；
    18位的X3为Leu、Glu、或Ala；
    80位的X4为Asn或Ala；以及
    230位的X5为Lys或不存在。
  9. 根据权利要求1所述的IL10-Fc融合蛋白，其特征在于：所述的IL10-IgG4 Fc融合蛋白中连接肽序列通式为[GlyGlyGlyGlySer] n，n为1-4的整数。
  10. 根据权利要求9所述的IL10-Fc融合蛋白，其特征在于：所述的IL10-IgG4 Fc融合蛋白中连接肽序列通式为[GlyGlyGlyGlySer] 3。
  11. 根据权利要求1所述的IL10-Fc融合蛋白，其特征在于：所述的IL10-IgG4 Fc融合蛋白包括下列蛋白质：
    IL10-[GlyGlyGlyGlySer] 3-IgG4 Fc，其中Fc第16位的X1为Glu，17位的X2为Ala，18位的X3为Ala，80位的X4为Asn，以及230 位的X5不存在，其序列如SEQ ID NO：3所示；
    IL10-[GlyGlyGlyGlySer] 3-IgG4 Fc，其中Fc第16位的X1为Glu，17位的X2为Ala，18位的X3为Ala，80位的X4为Ala，以及230位的X5不存在，其序列SEQ ID NO：4所示；
    IL10-GlyGlyGlyGlySer-IgG4 Fc，其中Fc第16位的X1为Glu，17位的X2为Ala，18位的X3为Ala，80位的X4为Asn，以及230位的X5为Lys，其序列SEQ ID NO：5所示；
    IL10-IgG4 Fc，其中Fc第16位的X1为Glu，17位的X2为Ala，18位的X3为Ala，80位的X4为Ala，以及230位的X5不存在，其序列SEQ ID NO：6所示；
    IL10-[GlyGlyGlyGlySer] 3-IgG4 Fc，其中Fc第16位的X1为Glu，17位的X2为Ala，18位的X3为Ala，80位的X4为Asn，以及230位的X5为Lys；
    IL10-GlyGlyGlyGlySer-IgG4 Fc，其中Fc第16位的X1为Glu，17位的X2为Ala，18位的X3为Ala，80位的X4为Asn，以及230位的X5不存在；
    IL10-[GlyGlyGlyGlySer] 3-IgG4 Fc，其中Fc第16位的X1为Pro，17位的X2为Phe，18位的X3为Ala，80位的X4为Ala，以及230位的X5不存在；
    IL10-[GlyGlyGlyGlySer] 3-IgG4 Fc，其中Fc第16位的X1为Pro，17位的X2为Val，18位的X3为Ala，80位的X4为Ala，以及230位的X5不存在；
    IL10-IgG4 Fc，其中Fc第16位的X1为Glu，17位的X2为Ala，18位的X3为Ala，80位的X4为Ala，以及230位的X5为Lys；IL10-[GlyGlyGlyGlySer]2-IgG4 Fc，其中Fc第16位的X1为Pro，17位的X2为Ala，18位的X3为Ala，80位的X4为Asn，以及230位的X5为Lys。
  12. 一种多核苷酸，其编码权利要求1-11任一项所述的IL10-Fc融合蛋白。
  13. 一种多核苷酸，其与编码权利要求1-11任一项所述的IL10-Fc融合蛋白的多核苷酸序列具有至少约80％、85％、90％、95％、96％、97％、98％、99％或100％的同源性。
  14. 根据权利要求12所述的多核苷酸，其基因序列如SEQ ID NO：7所示。
  15. 一种包含权利要求12-14任一项的多核苷酸的载体，特别是表达载体。
  16. 根据权利要求15所述的载体，其特征在于：所述的载体选自原核表达载体如pET系列载体，pQE系列载体；酵母表达载体pPICZ-α-A，pHIL-D2，pPIC9或pHIL-S1；动物细胞表达载体pIRES质粒、pSVK3或pMSG；
  17. 一种宿主细胞，其包含权利要求12的多核苷酸或权利要求15的载体。
  18. 根据权利要求17所述的宿主细胞，其特征在于：所述的宿主细胞选自细菌、酵母、昆虫或哺乳动物细胞。
  19. 根据权利要求18所述的宿主细胞，其特征在于：所述的宿主细胞选自中国仓鼠卵巢(CHO)细胞、NSO、SP2细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝癌细胞Hep G2或A549细胞。
  20. 一种用于生产权利要求1-11任一项所述的IL10-Fc融合蛋白的方法，包括以下步骤：
    (i)在适合于表达IL10-Fc融合蛋白的条件下培养权利要求17的宿主细胞，并
    (ii)回收IL10-Fc融合蛋白。
  21. 根据权利要求20所述的方法，其特征在于：步骤(ii)回收IL10-Fc融合蛋白方法包括免疫亲和柱纯化、硫酸盐沉淀、离子交换色谱、疏水相互作用色谱、反向色谱或凝胶过滤等纯化技术或其任意的组合。
  22. 一种药物组合物，其包含治疗有效量的权利要求1-11任一项所述IL10-Fc融合蛋白和药学可接受的载体。
  23. 权利要求1-11任一项所述的IL10-Fc融合蛋白或权利要求22所述的药物组合物，其用作药物。
  24. 权利要求1-11任一项所述的IL10-Fc融合蛋白或权利要求22所述的药物组合物，其用于治疗或预防有此需要的个体中的疾病，所述的疾病包括病毒性疾病、炎性疾病、免疫相关病症、纤维化病症或增殖性病状等。
  25. 权利要求1-11任一项所述的IL10-Fc融合蛋白或权利要求22所述的药物组合物用于治疗或预防增殖性病状或病症，包括癌症。
  26. 权利要求1-11任一项所述的IL10-Fc融合蛋白或权利要求22所述的药物组合物用于治疗或预防癌症，包括子宫、宫颈、乳腺、***、睾丸、胃肠道、肾、膀胱、骨、骨髓、皮肤、头或颈、皮肤、肝脏、胆囊、心脏、肺、胰腺、唾液腺、肾上腺、甲状腺、脑、神经节、中枢神经***(CNS)和周围神经***(PNS)的癌症，以及造血***和免疫***的癌症。
  27. 权利要求1-11任一项所述的IL10-Fc融合蛋白或权利要求22所述的药物组合物用于治疗或预防癌症，所述的肿瘤或癌症是结肠癌、卵巢癌、乳腺癌、黑色素瘤、肺癌、胰腺癌、胶质母细胞瘤或白血病等。
  28. 权利要求1-11任一项所述的IL10-Fc融合蛋白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，所述的疾病包括炎性病状、免疫相关病症、纤维化病症和癌症等。
  29. 根据权利要求28所述的方法，所述的个体是哺乳动物。
  30. 根据权利要求29所述的方法，所述的个体是人。
  31. 根据权利要求30所述的方法，当所述的个体是人时，以大于2.0μg/kg/日、大于2.5μg/kg/日、大于3.0μg/kg/日、大于5μg/kg/日、大于8μg/kg/日、大于10μg/kg/日、大于12μg/kg/日、15μg/kg/日、大于18μg/kg/日、大于20μg/kg/日、大于21μg/kg/日、大于22μg/kg/日、大于23μg/kg/日、大于24μg/kg/日或大于25μg/kg/日的剂量施用IL10-Fc融合蛋白。
PCT/CN2018/116074 2018-11-18 2018-11-18 一种重组人白细胞介素10融合蛋白及其应用 WO2020097946A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201880092332.3A CN111989340A (zh) 2018-11-18 2018-11-18 一种重组人白细胞介素10融合蛋白及其应用
PCT/CN2018/116074 WO2020097946A1 (zh) 2018-11-18 2018-11-18 一种重组人白细胞介素10融合蛋白及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/116074 WO2020097946A1 (zh) 2018-11-18 2018-11-18 一种重组人白细胞介素10融合蛋白及其应用

Publications (1)

Publication Number Publication Date
WO2020097946A1 true WO2020097946A1 (zh) 2020-05-22

Family

ID=70730381

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/116074 WO2020097946A1 (zh) 2018-11-18 2018-11-18 一种重组人白细胞介素10融合蛋白及其应用

Country Status (2)

Country Link
CN (1) CN111989340A (zh)
WO (1) WO2020097946A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021231447A1 (en) * 2020-05-12 2021-11-18 Regeneron Pharmaceuticals, Inc. Novel il10 agonists and methods of use thereof
WO2022149881A1 (ko) * 2021-01-07 2022-07-14 주식회사 프로젠 신규 il-10 변이체 융합단백질 생산용 세포주 및 그의 용도

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112625137B (zh) * 2019-10-08 2021-10-08 北京东方百泰生物科技股份有限公司 一种人白细胞介素10-Fc融合蛋白及其医药用途
CN114621353B (zh) * 2020-12-08 2024-04-02 广东菲鹏制药股份有限公司 一种重组融合蛋白及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2379388A1 (en) * 1999-07-13 2001-01-18 George N. Cox, Iii Immunoglobulin fusion proteins
US20090111146A1 (en) * 2003-09-02 2009-04-30 National Institute Of Advanced Industrial Science Antibody Drug
EP2258841A1 (en) * 2003-06-23 2010-12-08 The Regents of the University of Colorado Methods for treating pain
CN101948543A (zh) * 2010-08-30 2011-01-19 中国科学技术大学 一种融合蛋白及其编码基因与应用
CN106061997A (zh) * 2014-02-06 2016-10-26 豪夫迈·罗氏有限公司 白介素‑10免疫缀合物
WO2018073185A1 (en) * 2016-10-17 2018-04-26 Vetoquinol Sa Modified antibody constant region

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MX2017005467A (es) * 2014-10-27 2017-11-30 Inhibrx Lp Polipéptidos de fusión de serpina y métodos para utilizar los mismos.
CN108623692B (zh) * 2017-03-20 2020-11-17 徐寒梅 一种融合蛋白及其制备方法和其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2379388A1 (en) * 1999-07-13 2001-01-18 George N. Cox, Iii Immunoglobulin fusion proteins
EP2258841A1 (en) * 2003-06-23 2010-12-08 The Regents of the University of Colorado Methods for treating pain
US20090111146A1 (en) * 2003-09-02 2009-04-30 National Institute Of Advanced Industrial Science Antibody Drug
CN101948543A (zh) * 2010-08-30 2011-01-19 中国科学技术大学 一种融合蛋白及其编码基因与应用
CN106061997A (zh) * 2014-02-06 2016-10-26 豪夫迈·罗氏有限公司 白介素‑10免疫缀合物
WO2018073185A1 (en) * 2016-10-17 2018-04-26 Vetoquinol Sa Modified antibody constant region

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021231447A1 (en) * 2020-05-12 2021-11-18 Regeneron Pharmaceuticals, Inc. Novel il10 agonists and methods of use thereof
US11673930B2 (en) 2020-05-12 2023-06-13 Regeneran Pharmaceuticals, Inc. IL10 agonists and methods of use thereof
WO2022149881A1 (ko) * 2021-01-07 2022-07-14 주식회사 프로젠 신규 il-10 변이체 융합단백질 생산용 세포주 및 그의 용도

Also Published As

Publication number Publication date
CN111989340A (zh) 2020-11-24

Similar Documents

Publication Publication Date Title
JP6689329B2 (ja) 免疫グロブリンＦｃ変異体
KR102609197B1 (ko) 인터류킨 15 단백질 복합체 및 그의 용도
CN108948207B (zh) 一种人白细胞介素10-Fc融合蛋白及其编码基因与应用
EP2409991B1 (en) Antibody constant region variant
AU2004309050B2 (en) IL-7 fusion proteins
WO2020097946A1 (zh) 一种重组人白细胞介素10融合蛋白及其应用
JP2019033755A (ja) インターロイキン−１０融合タンパク質及びその使用
CA2520138C (en) Improved fc fusion proteins
EP3113858B1 (en) Il-15/il-15ralpha based conjugates purification method
JP2016105714A (ja) 代替軽鎖の発現
WO2018166468A1 (zh) IgG样长效免疫融合蛋白及其应用
US11512122B2 (en) IL-7-FC-fusion proteins
JP2023509952A (ja) 新規４－１ｂｂｌ三量体含有抗原結合分子
CN105073977B (zh) 重组酵母转化体和用其制备免疫球蛋白Fc片段的方法
JP2022538139A (ja) 変異体インターロイキン－２及び抗ｃｄ８抗体を含む免疫複合体
EP4253423A1 (en) Bispecific recombinant protein and use thereof
US20230340054A1 (en) Interleukin-2 muteins and uses thereof
JP2022514187A (ja) ＩＶＩＧの代替のための多量体ハイブリッドＦｃ蛋白質
CN115916831A (zh) 包含抗lag-3抗体和il-2的融合蛋白及其用途
WO2021063313A1 (zh) 改变效应功能的Fc变体及其融合蛋白
WO2022007885A1 (zh) 融合多肽和多肽二聚体及其用途
US20240132562A1 (en) Heterodimeric fc cytokines and uses thereof
WO2023060165A2 (en) Interleukin-10 muteins and fusion proteins thereof
JP2024520569A (ja) ｓＰＤ－１及びＩＬ－１５を有する二重特異性Ｆｃ融合タンパク質

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18940012

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18940012

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 18940012

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 20.12.2021)