WO2015178746A1 - Protéine de fusion pd-l1 et son utilisation - Google Patents

Protéine de fusion pd-l1 et son utilisation Download PDF

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Publication number
WO2015178746A1
WO2015178746A1 PCT/KR2015/005256 KR2015005256W WO2015178746A1 WO 2015178746 A1 WO2015178746 A1 WO 2015178746A1 KR 2015005256 W KR2015005256 W KR 2015005256W WO 2015178746 A1 WO2015178746 A1 WO 2015178746A1
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Prior art keywords
seq
fusion protein
amino acid
hpd
domain
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PCT/KR2015/005256
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English (en)
Korean (ko)
Inventor
성영철
이지영
송미영
임혜성
이병하
Original Assignee
주식회사 제넥신
포항공과대학교 산학협력단
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Priority claimed from KR1020150071820A external-priority patent/KR20150135148A/ko
Application filed by 주식회사 제넥신, 포항공과대학교 산학협력단 filed Critical 주식회사 제넥신
Priority to EP15795999.0A priority Critical patent/EP3147298A4/fr
Priority to US15/313,220 priority patent/US20170189476A1/en
Priority to JP2017514241A priority patent/JP2017519520A/ja
Priority to CN201580026730.1A priority patent/CN106573987A/zh
Publication of WO2015178746A1 publication Critical patent/WO2015178746A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C12N15/11—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 present invention relates to a PD-L1 fusion protein having improved stability and activity by combining the Fc region of PD-L1 with an immunoglobulin.
  • the present invention relates to a pharmaceutical composition comprising PD-L1 or a specific fragment thereof, and to a pharmaceutical composition for treating immunological diseases including PD-L1 or a specific fragment thereof.
  • hPD-Ll Human Programmed Cell Death-Ligand 1
  • PD-1 programmed death-1
  • PD-1 a hematopoietic cell
  • ⁇ ⁇ lamps dendritic cells
  • macrophages a type 1 transmembrane protein expressed in non-hematopoietic cells
  • non-hematopoietic cells such as keratinocytes, pancreatic islets, and hepatocytes.
  • PD-1 which binds to PD-L1
  • PD-L1 is expressed mainly in activated T cells, B cells, macrophages, and dendritic cells, and binds PD-L1 to cytokines of T cells. Is known to inhibit production and cell proliferation.
  • PD-L1 has been reported to bind B1 as well as PD-1 (I ⁇ unity. 2007 Jul; 27 (1): 111-22), and B7-1: CD28 binding by binding to B7-1. Blocking not only inhibits the activity of ⁇ cells but also transmits inhibitory signals through B7-1 within T cells.
  • a second signal stimulus (co-st imulat ion) is required at the same time in addition to the first signal stimulus of the T cell receptor and antigen.
  • PD-1 is a secondary signal stimulating factor (an immune check point or immune modulator) that modulates the secondary signaling activity of T cells, either activated T cells (CD8 and / or CD4) or dendritic cells.
  • programmed cel l death l igand l (PD-Ll), which is expressed on the surface of the cell, such as dendrit ic cel l, black ⁇ 7 ⁇ (CD80), etc. By binding, it is possible to inhibit T cell proliferation and to decrease cytokine expression, thereby inhibiting T cell function.
  • PD-Ll The binding between PD-l: PD-Ll is known to induce the activity of regulatory T cells (Immunol Rev. 2010 Jul; 236: 219-42), which uses IgG- and Fc to induce immunotolerance of PD-L1.
  • regulatory T cells Immunol Rev. 2010 Jul; 236: 219-42
  • IgG- and Fc IgG- and Fc to induce immunotolerance of PD-L1.
  • PD-L1 protein PD-Ll-Ig
  • CIA col lagen-induced arthrit is
  • arthritis was alleviated (Rheumatol Int. 2011 Apr; 31 Apr. 4) : 513-9).
  • PD-1 is expressed in activated T cells
  • PD-L1 protein is expected to be useful as a therapeutic agent that specifically targets active immune cells not only for autoimmune diseases but also for immune tolerance in organ transplantation. .
  • T-cell immunotolerance induction-based immunotherapeutics using agonists have not been developed until now. This can easily be developed using antibody fusion techniques for PD-1 / PD-L1 inhibitors, but PD-1 / PD-L1 signal agonists that must be developed as soluble forms of protein. This is because technical development is not easy.
  • Fc fusion of immunoglobulins is one of the techniques for increasing the half-life of protein therapeutics.
  • IgGl used in the conventional Ig fusion technique
  • ADCC antibody-dependent cellular toxicity in vivo
  • ADCC antibody-dependent eel 1 -mediated cytotoxicity
  • CDC complement dependent cytotoxicity CDC
  • autoimmune diseases Ig fusion proteins as therapeutic or immunotolerant inducers in organ transplantation do not play a role in inhibiting the inflammatory response, but rather have a problem of exacerbating inflammation.
  • the present invention provides a fusion protein comprising an Fc region of an extracellular domain or fragment thereof and a modified immunoglobulin in order to enhance the therapeutic efficacy of PD-L1.
  • a fusion protein comprising an Fc region of an extracellular domain or fragment thereof and a modified immunoglobulin in order to enhance the therapeutic efficacy of PD-L1.
  • a fragment of the extracellular domain of PD-L1 protein having a high therapeutic effect and productivity to provide a pharmaceutical composition and use comprising such fragment.
  • the present invention provides a fusion protein comprising an extracellular domain or fragment thereof of PD-L1 protein and an Fc region of modified immunoglobulin.
  • the present invention provides a nucleic acid encoding the fusion protein, a vector comprising the same and a host cell comprising the same.
  • the present invention comprises the steps of introducing a DNA molecule encoding the fusion protein to a mammalian host cell; Expressing the fusion protein in the growth medium; And it provides a method for producing the fusion protein, comprising the step of obtaining the expressed protein.
  • the present invention provides a pharmaceutical composition for preventing or treating an immune disease, and a composition for inducing immune tolerance, including the fusion protein.
  • the present invention provides a method for preventing or treating an immune disease comprising the fusion protein, and a method for inducing immune tolerance.
  • Fusion proteins comprising regions of the extracellular domain or fragments thereof and modified immunoglobulins of the PD-L1 protein according to the present invention are conventional . Compared with Ig fusion protein, it has higher stability and can be mass produced.
  • immunoregulatory T cells which have the effect of inhibiting cytokine production and cell proliferation of T cells and inhibit the function of abnormally activated immune cells and control inflammatory reaction, The effect is to adjust. Therefore, the fusion protein of the present invention is a pharmaceutical composition capable of preventing and treating immune diseases such as autoimmune diseases, inflammatory diseases and transplant rejection diseases caused by abnormal regulation of immune response, or i ⁇ une tolerance inducer There is an effect that can be usefully used as.
  • the fusion protein comprising the extracellular domain or fragment thereof of PD-L1 and the Fc region of the modified immunoglobulin according to the present invention demonstrates excellent effects in inflammatory bowel disease, colitis, psoriasis, asthma and arthritis disease models. Therefore, it can be used very effectively in the treatment of the disease.
  • FIG. 2 shows cell line screening and cell line productivity.
  • Productivity comparison of suspension cell lines for cell line screening is shown in FIG. 2 (a) and SE-HPLC results for cell line culture productivity and target protein identification are shown in FIG. 2 (b).
  • FIG. 3 shows the results of SDS-PAGE (FIG. 3 (a)) analysis and SE-HPLC (FIG. 3 (b)) analysis after purification of the recombinant PD-Ll-hyFc protein.
  • FIG. 6 is a graph illustrating the weight loss inhibitory effect (FIG. 6 (a)) and colon length reduction inhibitory effect (FIG. 6 (b)) of mPD-Ll-mFc fusion protein in a DSS-induced inflammatory bowel disease animal model. The result is.
  • FIG. 7 is a result of performing tissue staining (FIG. 7 (a)) and histological scoring analysis (FIG. 7 (b)) to analyze the therapeutic effect of mPD-Ll-mFc fusion protein in an animal model of DSS-induced inflammatory bowel disease. to be.
  • FIG. 8 is a result showing the therapeutic effect of mPD-Ll-mFc fusion protein in T cel l induced inflammatory bowel disease animal model.
  • Weight of mPD-Ll-raFc after Administration Changes were measured (FIG. 8A) and scored according to lesions (FIG. 8B) to compare.
  • FIG. 9 performs histostaining (FIG. 9 (a)) and histological scoring analysis (FIG. 9 (b)) to analyze the therapeutic effect of mPD-Ll-mFc fusion protein in T cel l induced inflammatory bowel disease animal model.
  • FIG. 9 performs histostaining (FIG. 9 (a)) and histological scoring analysis (FIG. 9 (b)) to analyze the therapeutic effect of mPD-Ll-mFc fusion protein in T cel l induced inflammatory bowel disease animal model.
  • Figure 10 is the result of observing the weight loss inhibitory effect of mPD-Ll—mFc fusion protein in T cel l induced inflammatory bowel disease animal model.
  • 11 is a result of measuring the amount of inflammatory cytokines secreted by colorectal LP cells after administration of mPD-Ll-mFc fusion protein in a Tcel 1 induced inflammatory bowel disease animal model.
  • FIG. 12 is a result of measuring tissue staining (FIG. 12 (a)) and epidermal thickness (FIG. 12 (b)) to confirm the efficacy of mPD-Ll-mFc fusion protein in an acute psoriasis mouse model.
  • Figure 13 is a result of confirming the effect of mPD Ll ⁇ mFc in the acute psoriasis mouse animal model by measuring the thickness of the ear.
  • Figure 14 is the result of confirming the effect of mPD-Ll-mFc in the acute psoriasis mouse animal model through histological staining.
  • Figure 15 shows the results of measuring the epithelial tissue thickness change according to mPD-Ll-mFc treatment in acute psoriasis mouse animal model.
  • FIG. 16 shows the results confirming the immune rejection response inhibition (immuno tolerance) of mPD-Ll-mFc in the pancreas transplantation model.
  • FIG. 16 (a) shows changes in blood glucose and weight in an untreated transplant model
  • FIG. 16 (b) shows changes in blood sugar and weight in an mPD-Ll-mFc fusion protein treated group.
  • Figure 17 is a schematic diagram showing an example of the variant structure of the hPD-Ll and hyFc fusion protein.
  • FIG. 18 is a result of comparing the productivity according to the variation of the hPD-Ll and hyFc fusion protein.
  • FIG. 18 (a) shows SE-HPLC results of the N-terminal sequence starting at 19 and
  • FIG. 18 (b) shows SE-HPLC results of the N-terminal sequence starting at 21.
  • FIG. 18 (a) shows SE-HPLC results of the N-terminal sequence starting at 19 and
  • FIG. 18 (b) shows SE-HPLC results of the N-terminal sequence starting at 21.
  • Figure 19 shows the gene constructor of the hPD-Ll-hyFc recombinant fusion protein and its manufacturing process.
  • 20 is a graph showing the productivity of the recombinant proteins (hPD-Ll ⁇ hyFc) fused to the hPD-Ll extracellular domain and fragments thereof and hyFc ( ⁇ : fusion protein produced from CH0 cell line).
  • Figure 23 is the result of confirming the CD4 + T cell inhibitory ability of hPD-Ll (VC, 19-239) -hyFcMl and hPD-Ll (V, 19-133) -hyFcMl by Tcel proli ferat ion assay.
  • Figure 24 shows the results of the CD8 + T cell inhibition of hPD-Ll (VC :, 19-239)-hyFcMl and hPD-Ll (V, 19-133) -hyFcMl was confirmed by the Tcel l prol i ferat ion assay.
  • FIG. 25 shows the results of testing PD-1 Binding aff inity of hPD-Ll-hyFc fusion protein obtained from several cell lines through ELISA assay (FIG. 24 (a)) and SPR assay (FIG. 24 (b)).
  • Figure 26 shows hPD-Ll (VC, 19-239), hPD-Ll (VC, 19-239) -IgGl (wild-type IgGl Fc), hPD-Ll (VC, 19-239) -hyFcMl and hPD in normal rat models.
  • Figure 30 shows the results confirming the ability of hPD-Ll (VC, 19-239) -hyFcMl inhibits IL-2 production and IFN-gamma production in mouse cells.
  • Figure 31 shows ⁇ cell activity against CD4 + T cells of hPD-Ll (VC, 19-239) -hyFcMl, hPD-Ll (V, 19-133) -hyFcMl and hPD-Ll (V, 19—127) -hyFcMl This is the result of confirming the inhibitory effect.
  • Figure 33 is the result of confirming the proliferation inhibitory ability according to the concentration of hPD-Ll (VC, 19-239)-hyFcMl and hPD-Ll (V, 19-133) -hyFcMl in T cells through MTT analysis.
  • FIG. 35 is a result showing the therapeutic effect of hPD-Ll-hyFc in T cel l induced inflammatory bowel disease animal model. Symptoms were scored after administration of hPD—Ll (V, 19-133) -hyFcMl (FIG. 35 (a)) and the change in weight was measured (FIG. 35 (b)).
  • 39 shows the results of measuring epithelial tissue thickness change according to hPD-Ll (VC, 19-239) -hyFcMl treatment in acute psoriasis mouse animal model.
  • 40 shows the results of scoring the phenotypic observations in the rheumatoid arthritis model to confirm the efficacy of the mPD-Ll-mFc fusion protein.
  • a fusion protein comprising an Fc region of a modified immunoglobulin and an extracellular domain or fragment thereof of a Programmed Cel l Death-Ligand 1 (PD-Ll) protein Fusion protein "," PD-Ll-Fc protein ",” PD-L1 fusion protein “or” fusion protein ").
  • the extracellular domain of PD-L1 is the Ig V-like domain of PD-Ll and the Ig C analog of PD—L1. It may be a polypeptide comprising a domain (Immunoglobul in C l ike domain).
  • the extracellular domain of PD—L1 is a protein site exposed outside the cell membrane, and may be a polypeptide consisting of amino acids 19 to 238 of SEQ ID NO: 3 or a polypeptide consisting of amino acids 19 to 239 of SEQ ID NO: 3.
  • the extracellular domain of PD-L1 includes an Ig V-like (Ig V, Ig V l ike) sequence, which is a conserved sequence similar to an immunoglobulin (Ig, immunoglobul in) and an amino acid sequence, and is conserved.
  • Ig V-like sequence is the amino acid sequence of SEQ ID NOs: 68-114 of SEQ ID NO: 3 and also contains an Ig C-like (Ig C, Ig C ike) sequence and is highly conserved
  • the sequence region is the amino acid sequence 153 to 210 of SEQ ID NO.
  • the fragment of the PD-L1 extracellular domain may include all or part of the Ig V-like domain including the Ig V-like sequence of PD-L1.
  • extracellular domain of PD-L1 or a fragment thereof may be derived from human or mouse.
  • the extracellular domain of PD-L1 may include a polypeptide consisting of the 19th to 239th amino acid sequence of SEQ ID NO: 3 (SEQ ID NO: 41) or a polythird consisting of the 19 to 239th amino acid sequence of SEQ ID NO: 1 Can be.
  • the extracellular domain of PD-L1 is about 70%, 75%, 80%, 85%, 90%, 91%, 92 and polypeptide sequence consisting of SEQ ID NO: 41 or SEQ ID NO: 1 to 239 amino acid sequence %, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more may be the same.
  • the Ig V-like domain of the PD-L1 extracellular domain is a site capable of interacting with PD-1, may be a polypeptide having amino acids 21 to 114 of SEQ ID NO: 3, 19 to The polypeptide having amino acid 114.
  • it may be a polypeptide having amino acids 21 to 120 of SEQ ID NO: 3, and may be a polypeptide having amino acids 19 to 120 of SEQ ID NO: 3.
  • It may also be a polypeptide having the amino acids 21-127 of SEQ ID NO: 3 (SEQ ID NO: 48), or a polypeptide having the amino acids 19-127 of SEQ ID NO: 3 (SEQ ID NO: 39).
  • polypeptide having amino acids 21 to 130 of SEQ ID NO: 3 19 to 130 of SEQ ID NO: 3 KR2015 / 005256 may be a polypeptide having an amino acid.
  • it may be a polypeptide having amino acids 21 to 131 of SEQ ID NO: 3, and may be a polypeptide having amino acids 19 to 131 of SEQ ID NO: 3.
  • It may also be a polypeptide having amino acids 21 to 133 of SEQ ID NO: 3, and may be a polypeptide having amino acids 19 to 133 of SEQ ID NO: 3 (SEQ ID NO: 40).
  • the Ig V-like domain of the PD-L1 extracellular domain may be a polypeptide having amino acids 21 to 114 of SEQ ID NO: 1, or may be a polypeptide having amino acids 19 to 114 of SEQ ID NO: 1.
  • it may be a polypeptide having amino acids 21 to 120 of SEQ ID NO: 1, may be a polypeptide having amino acids 19 to 120 of SEQ ID NO: 1.
  • the fragment of the PD-L1 extracellular domain when the fragment of the PD-L1 extracellular domain includes an Ig V-like domain or a fragment thereof, the fragment may further include an Ig C-like domain of the PD-L1 extracellular domain.
  • the Ig C-like domain may be a polypeptide having amino acids 133 to 225 of SEQ ID NO: 3, or may be a polypeptide having amino acids 134 to 225 of SEQ ID NO: 3.
  • the fragment of the PD-L1 extracellular domain is an Ig V-like domain or When including a fragment, the polypeptide may further comprise a polypeptide or fragment thereof comprising an Ig C-like domain of PD-L1 extracellular domain.
  • the polypeptide including the Ig C-like domain refers to an extracellular domain of PD-L1 excluding the Ig V domain, and has a polypeptide of SEQ ID NO: 3 to amino acids 134 to 239 (SEQ ID NO: 47) or SEQ ID NO: Polypeptides having the amino acids 134-238 (SEQ ID NO: 49).
  • the human PD-L1 protein has 290 amino acid residues and has an amino acid sequence represented by SEQ ID NO: 3 (Accession Number: Q9NZQ7).
  • amino acid residues 1 to 18 of the N terminus are signal sequences.
  • Mature human PD-L1 is a protein consisting of amino acids 19-290 of SEQ ID NO: 3.
  • the extracel lular domain of human PE) -L1 comprises amino acids 19 to 238 of SEQ ID NO: 3 or 19 to 239 of SEQ ID NO: 3.
  • Human PD-L1 protein comprises an Ig V-like domain of amino acids 19-127 of SEQ ID NO: 3 and an Ig C-like domain of amino acids 134-226 of SEQ ID NO: 3.
  • Mouse PD-L1 has been reported to contain 290 amino acids, which has an amino acid sequence represented by SEQ ID NO: 1 (Access ion Number: Q9EP73).
  • the amino acid residues 1 to 18 of SEQ ID NO: 1 are signal sequences, and the mature mouse PD-L1 protein consists of amino acids 19 to 290 of SEQ ID NO: 1.
  • the amino acids of 19 to 239 of SEQ ID NO: 1 are extracellular domains.
  • the mouse PD-L1 protein consists of an Ig V-like protein, amino acids 19 to 127 of SEQ ID NO: 1, and an Ig C-like dome, amino acids 224 through 133 of SEQ ID NO: 1.
  • the extracellular domain is Ig
  • the fragment of the PD-L1 extracellular domain may further include a polypeptide including an Ig C-like domain or an Ig C-like domain (an extracellular domain of PD-L1 excluding an Ig V-like domain).
  • the PD-L1 extracellular domain or fragment thereof may comprise various modified proteins or peptides. The modification may be carried out by replacing, deleting or adding one or more proteinols to wild-type PD-L1, unless the function of PEI-L1 is modified.
  • proteins or peptides are 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with wild type proteins. It may have a homology of%.
  • substitution of wild-type amino acid residues can be performed by alanine or conservative amino acid, which does not affect or weaken the charge, polarity or hydrophobicity of the entire protein.
  • extracellular domain of PD-L1 is also used as a concept including “extracellular domain of PD-L1 and fragments thereof".
  • protein polypeptide
  • peptide 1 Unless otherwise stated, may be used as interchangeable concepts.
  • PD-L1 fusion protein and "Fc region fusion protein of PD-L1-modified immunoglobulin '" refer to a PD-L1 protein, an extracellular domain of PD-L1, or a fragment thereof, wherein Fusion protein associated with an immunoglobulin Fc region
  • extracellular domain of PD-L1 may be SEQ ID NO: 41.
  • two PD-Ll-Fc complexes may form one dimer, wherein each Fc domain may bind to form a dimer.
  • PD-L1 may be directly linked to the Fc domain or may be linked via a linker.
  • the extracellular domain of PD-L1 comprises a polypeptide having 96 to 115 contiguous amino acid residues in the C-terminal direction from the N-terminus of amino acid residues at positions 19 to 133 of SEQ ID NO: 3. You can do
  • the extracellular domain of PD-L1 is preferably a polypeptide having an amino acid sequence of positions 19 to 133 of SEQ ID NO: 3, a polypeptide having an amino acid sequence of positions 19 to 131 of SEQ ID NO: 3, and a position of 19 to 130 of SEQ ID NO: 3
  • a polypeptide having an amino acid sequence, a polypeptide having an amino acid sequence of positions 19 to 127 of SEQ ID NO: 3, a polypeptide having an amino acid sequence of positions 19 to 120 of SEQ ID NO: 3, and a amino acid sequence of positions 19 to 114 of SEQ ID NO: 3 It may be characterized by comprising a polypeptide selected from the group consisting of polypeptides.
  • the extracellular domain of PD-L1 may be characterized in that it comprises a polypeptide consisting of the 19 to 239th amino acid sequence of SEQ ID NO: 1.
  • the extracellular domain of PD-L1 may be characterized in that it comprises a polypeptide consisting of the 19 th to 133 th amino acid sequence of SEQ ID NO: 1.
  • the PD-L1 protein is about 70%, 75% of the sequence of SEQ ID NO: 1 or SEQ ID NO: 3,
  • polypeptide comprising the extracellular domain or fragment thereof of the PD-L1 and the Ig C-like domain or the IgC-like domain of the PD-L1 may be linked through a linker.
  • the linker may be a polypeptide consisting of 1 to 10 amino acids, and may consist of 3 to 6 amino acids.
  • the amino acids constituting the linker are leucine (Leu, L), isoleucine (l ie, 1), alanine (Ala, A), valine (Val, V), plin (Pro, P), lysine (Lys, ), Arginine (Arg, R), asparagine (Asn, N), and glutamine (Gin, Q).
  • the linker may include amino acids of VKV, KVN, VNA and NAP, preferably SEQ ID NO: 9, SEQ ID NO: 45, or SEQ ID NO: 46.
  • the linker may be a polypeptide consisting of 3 to 15 amino acids consisting of glycine (Gly, G) and serine (Ser, S) residues, it may be composed of 6 to 11.
  • the linker may comprise a GSGGGS or GSGGGGSGGGS amino acid sequence.
  • the fusion protein of the present invention may further comprise Lingze.
  • the extracellular domain of PD-L1 and the Fc of the modified immunoglobulin may be linked through a linker.
  • the linker may be linked to the N-terminus, C-terminus or free radical of the Fc fragment and may also be linked to the N-terminus, C-terminus or free radical of PD-L1.
  • linker is a peptide linker
  • the linkage may occur at any linking site.
  • linker and Fc are separately expressed and then bound to each other, coupling is known in the art. It can be made using any of several known crosslinking agents. Examples of crosslinkers
  • imidoesters including disuccinimidyl esters such as succinimidylpropionate, and bi functional maleimides such as bis-N-maleimido-1,8-octane Including but not limited to.
  • the linker may be an albumin linker or a peptide linker.
  • the peptide linker may be a peptide of 10-20 amino acid residues consisting of Gly and Ser residues.
  • the peptide linker is leucine (Leu, L), isoleucine (lie, 1), alanine (Ala, A), valine (Val, V), plin (Pro, P), lysine (LysJO, arginine ( Arg, R), asparagine (Asn, N), serine (Ser, S) and glutamine (Gin, Q) may be a peptide consisting of 1 to 10 amino acids selected from the group consisting of.
  • the linker may include the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 45 or SEQ ID NO: 46.
  • the fusion protein may be a fusion protein, represented by the following formula (I) or ( ⁇ ):
  • FT is a multipeptide consisting of phenylalanine and threonine
  • wl, w2, w3, and w4 are each 0 or 1;
  • XI is an Ig V-like domain of PI) -L1 comprising a polypeptide having the amino acid sequence of SEQ ID NO: 48 or SEQ ID NO: 50;
  • L1 and L2 are linkers
  • X2 contains the Igunoglobulin C like domain of PD-L1 Polypeptide or fragment thereof;
  • IgFc is the Fc region of modified immunoglobulins.
  • polypeptide including the Ig C-like domain of PD-L1 may have an amino acid sequence of SEQ ID NO: 47 or SEQ ID NO: 49.
  • L1 may be composed of 1 to 10 amino acids, may be composed of 3 to 6 amino acids.
  • the amino acids are leucine (Leu, L), isoleucine (lie, 1), alanine (Ala, A), valine (Val, V), plin (Pro, P), lysine (LysJO, arginine (Arg, R) ) and asparagine (may be selected from Asn, N), the group consisting of glutamine (Gin, Q).
  • the L1 may be "having the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 45, or SEQ ID NO: 46.
  • the L2 is a polypeptide consisting of 10 to 20 amino acids consisting of glycine (Gly, G) and serine (Ser, S) residues, or leucine (Leu, L), isoleucine (lie, 1), alanine (Ala, A), valine (Val, V), plin (Pro, P), lysine (Lys, K), arginine (Arg, R) : group consisting of asparagine (Asn, N), glutamine (Gin, Q) It may be a polypeptide consisting of 1 to 10 amino acids selected from.
  • the L2 may have an amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 45 or SEQ ID NO: 46.
  • wl, w2, w3 or w4 may be 0 or 1, respectively.
  • the peptide including the Ig V-like domain and Ig C-like domain of PD—L1 may be in the form of direct binding.
  • the fusion protein may have a structural formula of Formula (Ia) or ( ⁇ — a):
  • FT-X1-L1-X2 may have an amino acid sequence of SEQ ID NO: 41.
  • the fusion protein is represented by the formula (I-b) or
  • FT-X1-L1 may have an amino acid sequence of SEQ ID NO: 40.
  • the fusion protein may have a structure with formula (Ic) or ( ⁇ -c):
  • FT-X1 may have an amino acid sequence of SEQ ID NO: 39.
  • Fc region of the modified immunoglobulin (IgFc" in Formula (I), ( ⁇ ), (I ⁇ a), ( ⁇ -a), (Ib) and ( ⁇ -b)) is IgGl, IgG2 , Fc regions of IgG3, IgD and IgG4, or a combination thereof.
  • the Fc region is modified such that no binding and / or complement binding with the Fc receptor occurs.
  • the Fc region of the modified immunoglobulin comprises a hinge region, a CH2 domain and a CH3 domain from the N-terminal to the C-terminal direction, wherein the hinge permanent includes a human IgD Hanji region, A portion of the amino acid residues of the CH2 domain of human IgD and human IgG4, wherein the CH3 VII domain may comprise a portion of the amino acid residues of the CH3 domain of human Ig G4.
  • Fc region As used herein, the terms “Fc region”, “Fc fragment” or “Fc” include reinforcement constant region 2 (CH2) and heavy chain constant region 3 (CH3) of immunoglobulin and include variable chains and light chains of immunoglobulin. Region and light chain constant region KCL1) refer to proteins that do not comprise. It may further comprise a hinge region of the heavy chain constant region. Hybrid Fc or hybrid Fc fragments are also referred to herein as "hFc” or “hyFc”.
  • Fc region variant as used herein means that some amino acids in the Fc region are substituted or prepared by combining different Fc regions. The Fc region variant may be modified to prevent cleavage at the hinge site.
  • the 144th and / or 145th amino acid of SEQ ID NO: 4 may be modified.
  • the 144th amino acid K of SEQ ID NO: 4 may be substituted with G or S
  • the 145th amino acid E may be a variant substituted with G or S.
  • the Fc region or Fc region variant of the modified immunoglobulin can be represented by the following formula:
  • N ' is the N-terminus of the polypeptide and C' is the C-terminus of the polypeptide;
  • p is an integer of 0 or 1;
  • Z 1 is an amino acid sequence having 5 to 9 consecutive amino acid residues in the N-terminal direction from position 98 of amino acid residues at positions 90 to 98 of SEQ ID NO: 4,
  • Y is from position 162 of the amino acid residue at position 99-162 of SEQ ID NO:
  • Z2 is an amino acid sequence having 4 to 37 consecutive amino acid residues in the C-terminal direction from position 163 among amino acid residues at positions 163 to 199 of SEQ ID NO: 4,
  • Z3 is an amino acid sequence having 71 to 106 contiguous amino acid residues in the N-terminal direction from position 220 of amino acid residues at positions 115 to 220 of SEQ ID NO: 'and
  • Z 4 is an amino acid sequence having 80 to 107 amino acid sequences in the C-terminal direction from the 221 position among the amino acid residues at the positions 221 to 327 of SEQ ID NO: 5.
  • the modified Ig Fc domain may be as described in US Pat. No. 7,867,491, and the production of the modified Ig Fc domain may be performed with reference to what is described in US Pat. No. 7, 867, 491.
  • the Fc fragment of the present invention may be a natural sugar chain, an increased sugar chain compared to the natural form, a reduced sugar chain compared to the natural form, or a form in which the sugar chain is removed.
  • immunoglobulin Fc sugar chains can be carried out by conventional methods known in the art such as chemical methods, enzymatic methods and genetic engineering methods using microorganisms. Removal of sugar chains from the Fc fragment was performed by the primary complement component C1. It dramatically reduces binding affinity to Clq and results in a decrease or loss of ant ibody-dependent eel 1 -mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), thereby not inducing unnecessary immune responses in vivo.
  • ADCC ant ibody-dependent eel 1 -mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • immunoglobulin Fc fragments in deglycosylated or aglycosylated forms may be more suitable for the purposes of the present invention as carriers of drugs.
  • the term "deglycosylat ion” means that the sugar is enzymatically removed from the Fc fragment
  • the term "aglycosylat ion” means that the Fc fragment is a prokaryote, preferably. It means that it is produced in an unglycosylated form by co / /.
  • the Fc region of the modified immunoglobulin is SEQ ID NO: 6 (hyFc), SEQ ID NO:
  • the Fc region of the modified immunoglobulin may include the amino acid sequence of SEQ ID NO: 2 (nonlytic mouse Fc).
  • the extracellular domain of PD-L1 may bind to the N-terminus or C-terminus of the Fc region of the modified immunoglobulin, a fusion protein comprising the Fc region of the PD-L1 extracellular domain-modified immunoglobulin May have an amino acid sequence of SEQ ID NOs: 10-23.
  • the fusion protein comprising the Fc region of the PD-L1 extracellular domain-modified immunoglobulin may have an amino acid sequence of SEQ ID NO: 12, 13, 18 or 19, and in another embodiment, It may have an amino acid sequence of the number 14, 15, 16, 17, 20, 21, 22 or 23.
  • the fusion protein comprising the Fc region of the PD-L1 extracellular domain—modified immunoglobulin is 70%, 75%, 80%, 85%, 90%, with amino acids of SEQ ID NOs: 10-23, It may have a sequence having a homology of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • nucleic acid molecule may be a glycopeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10 to 23.
  • the nucleic acid molecule may include a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 24 to 37.
  • the nucleic acid molecule may further comprise a signal sequence or a leader sequence.
  • signal sequence refers to a biologically active molecular drug; Refers to a fragment that directs the secretion of the fusion protein and is cleaved after being translated in the host cell.
  • the signal sequence of the present invention is a polynucleotide encoding an amino acid sequence that initiates the movement of a protein across an endoplasmic ret iculum (ER) membrane.
  • Signal sequences useful in the present invention include antibody light chain signal sequences, eg, antibody 14.18 (Gi ll ies et al., J. Immunol. Meth 1989.
  • antibody heavy chain signal sequences eg, M0PC141 antibody. Heavy chain .
  • Signal sequences Sakano et al., Nature 1980. 286: 676-683
  • other signal sequences see, eg, Watson et al., Nucleic Acid Research 1984. 12: 5145-5164). .
  • signal peptides include a basic N-terminal region, a central hydrophobic region, and a more polar C. It consists of three zones: the terminal zone.
  • the deep hydrophobic region contains 4 to 12 hydrophobic residues that immobilize the signal sequence through the membrane lipid bilayer during migration of the immature polypeptide.
  • eu preferred ⁇ sequence is cut in a common eu eu JINHO eu peptidase (signal pept idases) of the ER lumen (lumen) by cellular enzymes known as
  • the signal sequence tPa t issue Plasminogen Act ivat ion
  • HSV gDs or growth may be a secretion signal sequence of the hormone.
  • i can be a secretion signal sequence used in higher eukaryotes, including mammals such as, and more preferably tPa sequence or amino acid sequence 1 to 18 of SEQ ID NO: 1 or SEQ ID NO: 3. Most preferably, it may include the DNA sequence of SEQ ID NO: 38.
  • the secretion signal sequence of the present invention is It is used to substitute codon with high frequency of expression in host cell. Can be.
  • Another aspect of the invention is to provide an expression vector comprising an isolated nucleic acid molecule encoding a fusion protein consisting of the extracellular domain of PD-L1 and the Fc region of a modified immunoglobulin.
  • vector is understood to be a nucleic acid means comprising a nucleotide sequence that can be introduced into a host cell, recombined and inserted into the host cell genome, or spontaneously replicated as an episome.
  • vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and analogs thereof.
  • viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.
  • host cell refers to prokaryotic and eukaryotic cells into which recombinant expression vectors can be introduced.
  • transformed and “transfected” refer to the introduction of nucleic acids (eg, vectors) into cells by many techniques known in the art.
  • gene expression or “expression” of a protein of interest is understood to mean transcription of DNA sequences, translation of mRNA transcripts, and secretion of Fc fusion protein products or antibodies or antibody fragments.
  • Useful expression vectors can be RcCMV lnvitrogen, Carlsbad) or variants thereof.
  • Useful expression vectors include human CMV (cytomegalovirus) promoters to facilitate the continuous transcription of genes of interest in mammalian cells, and bovine growth hormone polyadenylation signal sequences to increase the steady-state levels of RNA after transcription. can do.
  • the expression vector is pAD15, which is a modified vector of RcCMV.
  • Another aspect of the invention provides a host cell comprising said expression vector. To provide. Suitable host cells can be transformed or transfected with the DNA sequences of the present invention and used for the expression and / or secretion of the protein of interest.
  • Currently preferred host cells that can be used in the present invention include immortal hybridom3 ⁇ 4 eel Is, NS / 0 myeloma eel Is, 293 cells, Chinese hamster ovary cells (CHO cel l) , HeLa cells, CapT cells (human amniotic fluid derived cells), and COS cells.
  • One expression system used to express high levels of fusion proteins, antibodies, or fragments thereof in mammalian cells comprises a DNA construct encoding a secretion cassette comprising a signal sequence and an immunoglobulin Fc region in the 5 'to 3' direction. to be.
  • Another aspect of the present invention to provide a pharmaceutical composition for the prevention or treatment of immune diseases comprising a fusion protein consisting of the extracellular domain or fragment thereof of PD-L1 and the Fc region of the modified immunoglobulin.
  • the immune disease may be a disease selected from the group consisting of autoimmune diseases, inflammatory diseases, and disease of rejection of cells, tissues, or organs.
  • the autoimmune diseases include arthritis (acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, spondylitis)
  • Arthritis juveni le-onset rhematoid arthrit is, osteoarthritis, arthritis arteritis, chronicar progrediente, deformans arthritis, polyarthritis polyarthritis, polyarthrit is chronica primaria anti-male ', such as rheumatoid arthritis and osteoarthritis (react ive arthrit is), and spastic myelitis (ankylosing spondyl it is)], inflammatory hyperproliferative skin disorders.
  • Psoriasis contact dermatitis, chronic contact dermatitis, allergic dermatitis, such as plaque psoriasis, gutatte psoriasis, purulent psoriasis, and psoriasis of the nai Is , Allergic contact dermatitis, Chronic idiopathic urticaria, including herpetiformis dermatitis, and dermatitis, including atopic dermatitis, X-linked and IgM syndrome, chronic allergic urticaria, and chronic autoimmune urticaria urticaria such as (urticaria), polymyositis / dermatomyositis (polymyositis / dermatomyositis), juvenile dermatomyositis (juveni le dermatomyositis), addictive, skin peeling (toxic epidermal necrolysis), scleroderma (scleroderma) (including systemic scleroderma), systemic
  • encephalomyelitis such as stiff-person syndrome, allergic encephalomyelitis or encephalomyelitis al lergica, and experimental allergic encephalomyelitis (EAE), thymic cell-associated myasthenia gradiosis Myasthenia gravis, cerebellar degeneration (cerebel lar de) generation, neuroromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, time syndrome Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytic interstitial pneumonia (lymphoid interstitial pneumonitis), bronchiolitis obliteran (non-transplantation) vs NSIP, Guil Iain-Bar re syndrome,
  • heungban erythema elevatum et diutinum
  • fetus Erythroblastosis fetalis
  • eosinophilic faciitis Shulman's syndrome
  • Felty's syndrome flariasis
  • chronic ciliary inflammation heterochromatous ciliary inflammation
  • hemorrhoids Cyclitis, such as iridocyclitis or Fuch's cyclitis, Henoch-Schonlein purpura
  • human immunity Virus human immunodeficiency virus; HIV infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndromes , Congenital Lubola infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmunity Autoimmune gonadal failure, Sydenham 's chorea, poststreptococcal nephritis thromboangitis ubiterans, thyrotoxico
  • Aut 0 immune polyglandular syndrome type I adult-initiated idiopathic hypoparathyroidism (AOIH), alopecia totalis, di lated cardiomyopathy , Epidermolis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or non-pyogenic sinusitis Acute or chronic sinusitis, ethmoid, frontal, maxilla, maxi 1 lary, or sphenoid sinusitis, eosinophil-disorders such as eosinophilia, pulmonary infiltration eosinophi 1 ia , eosinophilia-Mia glial increase ⁇ group (ia eosinophi 1 myalgia syndrome), multiple ropeul syndrome (Lof ler f 's syndrome),' chronic eosinophilia pneumonia ( chronic eosinoph
  • the inflammatory diseases include, but are not limited to, rheumatic diseases (rheumatic arthritis, osteoarthritis, psoriatic arthritis), spondyloarthropathies (ankylosing spondylitis, reactive arthritis), Reiter's syndrome Including but not limited to, crystal arthropathies (including but not limited to gout, pseudogout, calcium pyrophosphate deposition disease), Lyme disease Lyme disease, polymyalgia rheumatica, connective tissue diseases [systemic lupus erythematosus, systemic sclerosis, polymyositis skin .
  • rheumatic diseases rheumatic arthritis, osteoarthritis, psoriatic arthritis
  • spondyloarthropathies ankylosing spondylitis, reactive arthritis
  • Reiter's syndrome Including but not limited to, crystal arthropathies (including but not limited to gout, pseudogout, calcium pyrophosphate deposition disease), Ly
  • Myocarditis (dermatomyositis), Sjogren's syndrome]; Vasculitides (including but not limited to polyarteritis nodosa, Wegener's granulomatosis, Church-Strauss syndrome); Inflammatory diseases, including the consequences of trauma or ischaemia; Sako Id increases (sarcoidosis); atherosclerotic vascular disease (atherosclerotic vascular disease), atheromatous "arteriosclerosis (atherosclerosis) and vascular barrier disease (vascular occlusive disease) [Artemia reumseong arteriosclerosis, ischemic heart disease (ischaemic heart disease), myocardial Vascular diseases, including but not limited to myocardial infarction, stroke, peripheral vascular disease; and vascular stent restenosis; It may be selected from the group consisting of uveitis, corneal disease, corneal disease, ulceris, iridocyclitis, and ocular diseases including cataracts.
  • the immune disease may preferably be selected from the group consisting of autoimmune diseases, inflammatory diseases, transplant rejection diseases, colitis, psoriasis, asthma, autoimmune diabetes, inflammatory bowel disease and arthritis.
  • another aspect of the present invention to provide a composition for induction of immune tolerance comprising a fusion protein consisting of the Fc region of the extracellular domain of PD-L1 and the modified immunoglobulin.
  • immune tolerance refers to a condition in which the immune system does not show tissue destruction responsiveness to a specific antigen without being in a sustained immunosuppressive state. Proteins can be used for immune tolerance involving regulatory T cells.
  • the fusion protein may be used to suppress the immune response generated during tissue transplantation.
  • the fusion protein comprising the extracellular domain of PD-L1 or a fragment thereof may comprise a pharmacological carrier.
  • the pharmacological carrier can be any carrier as long as it is a non-toxic material suitable for delivering the antibody to a patient. Distilled water, alcohols, fats, waxes and inert solids may be included as carriers. Pharmaceutically acceptable adjuvants (solvents, dispersants) may also be included in the pharmaceutical composition.
  • fusion proteins comprising the PD-L1 extracellular domain or fragments thereof can be administered to a subject in a variety of ways.
  • the pharmaceutical composition can be administered parenterally, examples being subcutaneous, intramuscular or intravenous. Such compositions can typically be sterilized according to well known sterilization techniques.
  • the composition may include pharmacologically acceptable auxiliaries and adjuvants, toxic modulators and analogs thereof required to control physiological conditions such as pH adjustment, for example, sodium acetate, sodium chloride ( sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like.
  • the fusion protein concentration can vary widely, for example up to about 0.5% by weight, in general or at least about 13 ⁇ 4 to 15% or 20% and depending on the particular method of administration selected It may be selected based on preference on volume, viscosity ies and the like.
  • Another aspect of the invention provides a method of treating a disease by administering a composition comprising a PD-L1 fusion protein of the invention as a pharmacologically active ingredient.
  • Such methods include administering an effective amount of a PD-L1 fusion protein of the invention to a mammal having a medical condition that is or is not directly associated with the disease of interest.
  • nucleic acids such as DNA or RNA encoding a preferred PD-L1 fusion protein can be administered to a subject, preferably a mammal, as a therapeutic agent.
  • cells comprising a nucleic acid encoding a PD-Ll fusion protein can be administered to a subject, preferably a mammal, as a therapeutic agent.
  • the PD-L1 fusion protein may also be administered in a therapeutically effective amount to a subject, preferably a mammal, including a human.
  • the chimeric polypeptide may be administered by intravenous, subcutaneous, oral, buccal, sublingual, nasal, parenteral, rectal, vaginal or pulmonary route.
  • compositions of the invention can be administered by any route.
  • the compositions of the present invention can be provided to an animal directly (e.g., by injection, implantation or topical administration to a tissue site, topically) or by any suitable means systemically (e.g., parenterally or orally).
  • the compositions of the invention are intravenous, subcutaneous, ophthalmic, intraperitoneal, intramuscular, oral, rectal, intraorbital, intratracerebral, intracranial, intraspinal, ventricular
  • parenterally such as intravenous, intrathecal, intracistenal, intracapsular, intranasal or aerosol administration
  • the composition is preferably aqueous or aqueous.
  • the carrier or vehicle is physiologically acceptable and therefore can be added to the composition and delivered to the patient, which does not adversely affect the electrolyte and / or volume balance of the patient.
  • the body fluid formulation for the formulation may generally include physiologic saline.
  • DNA constructs comprising nucleic acids encoding PD-L1 fusion proteins of the invention can be used as part of a gene therapy protocol that carries nucleic acids encoding PD-L1 fusion protein constructs.
  • the present invention can be administered with any biologically effective carrier an expression vector that infects and expresses the PD-L1 fusion protein in vivo in a particular cell type to reconstruct or complement the function of the desired PD-L1 fusion protein.
  • any formulation or composition capable of efficiently carrying a gene encoding a desired PD-L1 fusion protein or a fusion protein thereof in a cell in vivo.
  • nucleic acids encoding PD-L1 fusion proteins For gene therapy with nucleic acids encoding PD-L1 fusion proteins,
  • the gene of interest can be inserted into a viral vector, or recombinant bacterial plasmid or recombinant eukaryotic plasmid, including recombinant retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex virus-Kherpes simplex virus-1).
  • Dosages of nucleic acids encoding fusion proteins with the present invention range from 0.1 mg to 100 mg in humans.
  • the preferred dosage of nucleic acid encoding a fusion protein of the invention is 1 mg to 10 mg in humans.
  • the preferred dosage of nucleic acid encoding a fusion protein of the invention is 2 mg to 10 mg in humans.
  • Optimal amounts and dosage forms can be determined by routine experimentation within the skill of the art.
  • Unit doses of the fusion protein of the invention range from 0.01 mg / kg to humans
  • the unit dose of the fusion protein is 1 mg / kg to 100 mg / kg in humans. In another embodiment, the unit dose of the fusion protein is from 5 mg / kg to 20 mg / kg in humans.
  • the unit dosage may vary depending on the disease to be treated and the presence of side effects. However, the optimal dosage can be determined using ordinary people experiments.
  • Administration of the fusion protein may be by periodic rapid infusion (per iodi c bolus injections), by external reservoirs (e.g. intravenous bags) or by medial (e.g. bioerodable implants). continuous intravenous, subcutaneous or intraperitoneal administration.
  • compositions of the present invention may be administered in combination with other drugs or physiologically active substances having a prophylactic or therapeutic effect on the disease to be prevented or treated, or in combination with such other drugs (combinat ion formul t ion). It may be formulated in a form.
  • the method for preventing or treating a disease using the fusion protein or composition of the present invention may include administering another drug or physiologically active substance having a prophylactic or therapeutic effect in combination with the fusion protein or composition of the present invention.
  • the route of administration, timing of administration, and dosage may be determined according to the type of disease, the disease state of the patient, the purpose of treatment or prevention, and the other drug or combination of physiologically active substances.
  • mFc non-bacterial mouse Fc
  • the mouse PD-Ll (Programmed Cel l Death-Li gand l, mPD-Ll) gene is a known amino acid sequence (Access i on number: Q9EP73, In SEQ ID NO: 1, an extracellular domain (extracel hil ar domain) was used.
  • the fusion partner Fc portion is an ant ibody dependent cel l cytotoxici ty) ⁇ -Variant sequences were used to prevent CDC (Complement Dependent Cytotoxici ty), and the Fc portion (SEQ ID NO: 2) of the mutated mouse IgG2a was fused to mPD-Ll to express recombinant protein. A vector was secured.
  • the pAD15-mPD-Ll-mFc plasmid containing the raPD-Ll-mFc structural gene (SEQ ID NO: 24) was constructed as shown in FIG. 1 so that the gene could be expressed in an animal cell line.
  • the produced expression vector was transformed into the CHO Chinese Hamster Ovary Cel l) -DG44 cell line by electroporation (Electroporat ion). Cells with high expression level were selected by ELISA quantitative analysis of the transformed cell lines, and 'MTX ampl if icat ion-monoclonal selection-productivity while increasing the amount of methotrexate. High expression cell lines were selected by repeating the 'measurement' process (FIG. 2 (a)).
  • Example 2 Securing the mPD-Ll-mFc Protein
  • the mPD obtained in Example 1 above was obtained. Proteins of interest were isolated and purified from cell cultures produced from -Ll-mFc suspension cell lines.
  • the protein purification process was monitored over time at UV 280nm. As a result, it was confirmed that the target protein was eluted from the peak appearing as the elution buffer (Elut ion buffer: 0.1M glycine, pH 3.0) passed through the Protein A resin column (FIG. 3).
  • the purified mPD-Ll-mFc protein was confirmed by SDS-PAGE analysis, which showed a size of about 150 KDa under non-reducing condit ion and about 75 KDa under reducing condit ion. By confirming, it was confirmed that mPD-Ll-mFc had the form of a homodimer (FIG. 3A).
  • Example 3 In vitro Activity Evaluation Using mPD-Ll-mFc Protein
  • mouse spleen cells were used for immunosuppressive efficacy. It was confirmed in vitro.
  • the ratio of the anti_0) 3 and mPD-Ll-mFc protein was coated on the microbead 1: 1 or 1: 4.
  • the ratio of beads to mouse splenocytes was 10: 1 to stimulate splenocytes (5 ⁇ 10 6 beads: 5 ⁇ 10 5 splenocytes).
  • DSS-induced enteritis model To determine the efficacy of mPD-Ll ⁇ mFc in DSS-induced enteritis model. To determine the efficacy of mPD-Ll ⁇ mFc in DSS-induced enteritis model. To determine the efficacy of mPD-Ll-mFc protein in vivo, DSS similar to the human acute inflammatory growth disease model in IBD (inflammatory bowel disease) Dextran Sodium Sulfate) -induced mouse enteritis model was used.
  • IBD inflammatory bowel disease
  • Dextran Sodium Sulfate Dextran Sodium Sulfate
  • CD4 + CD25-CD45RB high T cells were isolated from splenocytes of C57BL / 6 mice using a flow cytometry (Fluorescent act ivat cel l sorting, FACS).
  • FACS Fluorescent act ivat cel l sorting
  • mPD-Ll-mFc protein obtained in Example 2 was injected at 20 week intervals from 3 weeks after T cell injection.
  • CTLA4-IgGl fusion protein Orencia
  • For the dosage and usage of the control group see Internal (I ial Journal of inf lammat ion, 2012: 412178). Changes in the weight of the mice were observed during the entire experiment and the cl inical scores were recorded in the mice.
  • the mouse PD-Ll-mFc fusion protein has an inhibitory effect on T cell proliferation, and is effective in acute and chronic inflammatory growth diseases, and expression of inflammatory cytokines accompanying lesions Confirmed to be inhibited.
  • the PD-Ll-Fc protein is highly applicable to the treatment of inflammatory reactions associated with IBD disease and IBD.
  • Example 5 Confirmation of psoriasis treatment efficacy using mPD-Ll-mFc protein 40 mg of ImiquimodUMQ in both ears of experimental mice; ALDARA CREAM (3M) was applied for 6 days to induce acute psoriasis.
  • mPD-Ll_raFc The therapeutic effect of mPD-Ll_raFc was examined after dividing the mouse group into the normal mouse group, the group not treated after psoriasis induction, and the group treated with mPD-Ll-mFc protein simultaneously with psoriasis induction.
  • mPD-Ll-mFc was intraperitoneally administered at 200 ug per mouse on days 1, 2, 4 and 6.
  • the histopathology of the ear was examined 7 days after the initial administration (FIG. 12 (a)) of the psoriasis such as epidermal thickness in the psoriasis induced group by IMQ.
  • the pathological tissues were observed and compared (FIG. 12 (b)).
  • the group treated with mPD-Ll-mFc had a statistically significant decrease in epidermal thickness compared to the control group.
  • IMQ Imiquimod
  • Mice group, normal mouse group, psoriasis induction group, anti-P40 antibody (mouse ant i-p40 ant ibody) group, mPD-Ll-mFc protein and anti-P40 antibody group And mPD-Ll-mFc were divided into groups treated together, and the efficacy of treatment was examined.
  • Anti-P40 antibodies were intraperitoneally administered at 100 ug per mouse on Days 1 and 4.
  • mPD-Ll-mFc was intraperitoneally administered at 200 ug per mouse on days 1, 2, 4 and 6.
  • ear thickness was observed daily to confirm the phenotype of psoriasis onset.
  • the mPD-Ll-mFc and anti-P40 antibody administration group showed a statistically significant delayed onset effect compared to the untreated group (FIG. 13).
  • the histopathology of the ear was examined and compared to the psoriasis-derived tissues such as epidermal thickness in the psoriasis-induced group by IMQ, and the mPD-Ll-mFc-treated group was compared to other groups. It was statistically significant that the epidermal thickness was reduced (FIG. 14).
  • epidermal thickness was also found to be statistically significantly reduced in the group treated with mPD-Ll-mFc compared to the other group not administered (FIG. 15).
  • RA rheumatoid arthritis
  • Cl col lagen induced arthrit is a rat model of rheumatoid arthritis (RA)
  • the treatment efficacy of mPD-Ll-mFc was examined using mouse hair 3 ⁇ 4.
  • CIA mice were induced to RA by administering the same amount of collagen at regular intervals of C57BL / 6 (The Jackson Laboratory, US) to complete Freund's adjuvant (CFA) at two week intervals.
  • the CIA mouse model consisted of the no drug group, the group receiving 100 ug and 300 ug of mPD—Ll-mFc (obtained in Example 2) or the group receiving 300 ug of anti-TNF-alpha antibody, respectively. It was.
  • Each drug was intraperitoneally administered three times a week for four weeks, after which the efficacy of mPD-Ll-mFc was observed.
  • a healthy islet transplantat ion of another mouse species (C57 / BL6) was transplanted into a mouse (BALB / c (H-2d)) whose pancreas was destroyed by streptozosin (STZ).
  • the mouse model was divided into groups that were not drug-administered and groups that received 100 ug of mPD-Ll-mFc (obtained in Example 2).
  • MPD-Ll-mFc was then administered on the day of transplanting the islets (day 0), 7 and 14 days after transplantation, and the blood glucose and weight of each individual were measured from 2 weeks before the end of the transplantation to the end of the experiment.
  • the reference blood sugar is indicated by a dotted line
  • the reference weight is indicated by a gray area.
  • an expression vector containing a human PD-L1 structural gene a human PD-L1 gene having a known amino acid sequence (Accession number: Q9NZQ7) was used.
  • a construct containing only an extracellular domain extracel lular domain was prepared and fused with a modified Fc domain to obtain a recombinant protein expression vector.
  • hyFc hybrid Fc
  • the hyFc protein is a hybrid type of the Fc of human IgD and the Fc of human lgG4, and may exhibit an excellent increase in body support when bound to a bioactive protein compared to the conventionally modified immunoglobulin and Fc region.
  • recombinant protein expression vectors were prepared using the amino acid sequence of the SEQ ID NO: 6 (hyFc) or the amino acid sequence of the SEQ ID NO: 7 (hyFcMl).
  • hPD-Ll-hyFc fusion proteins and hPD-Ll-hyFcMl fusion proteins have the amino acid sequences shown in SEQ ID NOs: 12-23.
  • the nucleic acid sequences encoding the hPD-Ll-hyFc fusion protein and the hPD—Ll-hyFcMl fusion protein are as shown in SEQ ID NOs: 26 to 37.
  • hPD-Ll-l inker-hyFc fusion protein was prepared, and in this example, GS6 or GS11 was used as a linker.
  • GS6 means peptide consisting of the amino acid sequence GSGGGS and GS11 means peptide consisting of the amino acid sequence GSGGGGSGGGS.
  • Example 9 Securing hPD-Ll-hyFc (human PD-Ll-hyFc) Protein The expression level of various PD-Ll-hyFc produced using the CAP-T and CH0 production systems of Example 8 was determined by ELISA assay.
  • Fig. 20 In order to separate and purify various types of hPD-Ll-hyFc from the culture solution, the protein purification process was monitored over time under UV280nm conditions using Protein A resin. As shown in FIG. 22, the target protein was eluted by analyzing the peak appearing as the elution buffer (Elut ion buffer: 0.1M glycine, pH 3.0) passed through the purification column through SE-HPLC analysis. In addition, the size was confirmed by SDS-PAGE analysis (FIG. 21).
  • Elut ion buffer 0.1M glycine, pH 3.0
  • hPD-Ll-hyFc fusion proteins including the V-like domain of PD-L1 and the V- and C-like domains of PD-L1, were evaluated.
  • a mixture was prepared using 2 ug / ml anti-CD3 antibody and each fusion protein (hPD—Ll-hyFc) in a ratio of 1: 1 or 1: 4, and then mixed into 5 ⁇ 10 5 / wel l mouse splenocytes. Treated.
  • PD-L1 the extracellular domain of PD-L1 (hPD-Ll (V, 19-239) -hyFcMl) and fragments thereof (hPD—Ll (V, 19-133) -hyFcMl) resulted in CD4 + T cells of mouse spleen cells and It was confirmed to inhibit CD8 + T cell proliferation (decreased Ki-67 expression) (FIGS. 23 and 24). In particular, the CD8 + T cells showed a more significant concentration-dependent cell proliferation reduction effect than CD4 + T cells.
  • Example 10-2. Binding Affinity Measurement Test PD-L1 is known to bind to PD-1 to inhibit the proliferation of T cells or inhibit the secretion of inflammatory cytokines. Thus, pharmacological activity was predicted by measuring the binding affinity between the various types of PD-Ll-hyFc fusion proteins described in FIG. 16 and PD-1.
  • each hPD-Ll-hyFc fusion protein was treated by concentration, and then binding affinity was evaluated by ELISA analysis.
  • the fusion protein OiPD-Ll (V, 19-133) -hyFcMl containing only the V domain of PD-L1, as well as the fusion protein containing the V and C domains of PD-L1 (hPD-Ll (VC, 19-239) ) -hyFcMl) was also confirmed to have excellent binding force with PE L (Fig. 25 (a)).
  • binding affinity was tested through SPR analysis.
  • a protein GLC sensor chip Bio-Rad, Cat #. 176-5011) coated with hPD-1 was prepared. After the coating was completed, treated with hPD-LlCVC, 19-239), hPD-LKVC, 19-239) -hyFcMl and hPD-LKV, 19-133) -hyFcMl at concentrations of 1000, 500, 250 and 100 50 nM, respectively.
  • the test was conducted. Each fusion protein was flowed on the hPD-Ll coated chip at a rate of 40 or 50 ul / min per minute for 240 seconds or 300 seconds. Then, the base value was confirmed using the regeneration buffer 10 mM NaOH and the above steps were repeated. Then, the binding curve was confirmed using a protein binding analyzer (Proteon XPR36, BI0-RAD, USA).
  • Example 11 Measurement of half-life of PD-L1 fusion protein (see KR10-2008-0094781A) In order to confirm the pharmacokinetics of the fusion protein according to the present invention, half-life of the body was measured and the contents disclosed in KR10-2008-0094781A Reference was made.
  • Example 11-1 Preparation of Fusion Proteins
  • PD-L1 fusion protein was prepared by fusion of the PD-L1 extracellular domain or fragment thereof and immunoglobulin Fc.
  • Fc wild-type IgG Fc derived from humans and hyFc and hyFcMl prepared in Example 9 were used.
  • the extracellular domain of PD-L1 is full-length peptide (VC, 19-239), hPD-LKVC comprising 21 to 239 amino acid residues of SEQ ID NO: 3, 21-239), 19 to 133 amino acid residues of SEQ ID NO: 3 HPD-Ll (V ′ 19-133) comprising: hPD-Ll (V, 19-130) comprising 19 to 130 amino acid residues of SEQ ID NO: 3, hPD comprising 19 to 127 amino acid residues of SEQ ID NO: 3 -Ll (V, 19-127), hPD—19,120 comprising amino acid residues of SEQ ID NO: 3—LKV, 19-120) and hPD-Ll, comprising 19—114 amino acid residues of SEQ ID NO: 3 -114) fragments were used and performed in the same manner as in Examples 5-6.
  • hPD-Ll (VC, 19-239), hPD-Ll (VC, 21-239), hPD-Ll (V, 19-133), hPD-LKV, 19-130), hPD-Ll (V , 19-127), hPD-LKV, 19-120), hPD-Ll (V, 19-114), hPD-LKVC, 19-239) -hFc (IgGl), hPD-Ll (VC, 21-239) — HFc, hPD-Ll (V, 19-133) -hFc, hPD-LKV, 19-130) -hFc, hPD-Ll (V, 19-127) -hFc, hPD-Ll (V, 19-120) -hFc, hPD-LKV, 19-114) -hFc, hPD-LKVC, 19-239) -hyFc, hPD-Ll (VC, 21-239) -
  • GS6 in the present example means a peptide consisting of the amino acid sequence GSGGGS and GS11 means a peptide consisting of the amino acid sequence GSGGGGSGGGS.
  • Example 11-2 Pharmacokinetic Test of Fusion Proteins To compare the half-life of the extracellular domain fragment of PD-L1 obtained and its recombinant fusion protein, PD- without Fc binding as a control via the intravenous route to male SpragueDawleyRats Charles River Laboratories, Wilmington) 2 mg / kg of L1 protein (Sino Biological Inc., Cat # 10084-H08H) was administered.
  • Blood was obtained before and after infusion 5 minutes, 30 minutes, 2, 8, 24, 48, 72, 96, 120, 144, 168, 216, 264, 336 hours. Blood samples were incubated for 30 minutes at room temperature for pooling. After centrifugation at 3000 rpm for 10 minutes, serum of each sample was obtained and stored in a cryo freezer. Samples were quantified at several dilution ratios using assays capable of specifically detecting PD-L1.
  • HPD-Ll (VC, 19-239), hPD-LKVC, 21-239), hPD-Ll (V, 19 133), hPD-Ll (V, 19-130), via subcutaneous injection or intravenous injection route, hPD-LKV, 19-127), hPD-LKV, 19-120), hPD-LKV, 19-114), hPD-LKVC, 19-239) -hFc (I gGl), hPD-Ll (VC, 21- 239) -hFc, hPD-LKV, 19-133) -hFc, hPD-LKV, 19-130) -hFc, hPD-LKV, 19-127) -hFc, hPD-LKV, 19-120) -hFc, hPD -LKV, 19-114) -hFc, hPD-LKVC, 19-239) -hyFc, hPD-Ll (VC, 21-239) -hyF
  • Example 12 Comparison of in vitro activity using PD-L1 or fragments or fusion proteins thereof -T cell proliferation inhibition and inflammatory cytokine expression comparison
  • the ratio of anti-CD3 to each hPD-Ll-hyFc fusion protein was coated on the microbead at 1: 1 or 1: 4.
  • the ratio of beads to mouse splenocytes was 10: 1 to stimulate splenocytes (5 ⁇ 10 6 beads: 5 ⁇ 10 5 splenocytes).
  • the expression level of Ki 67, a cytotoxic factor was analyzed by flow cytometry (FACS) on mouse spleen cells. The effect of Ll-hyFc was measured.
  • hPD-Ll (VC, 19-239) -hyFcMl was treated at concentrations of 0, 10 and 50 ug / ml, respectively. 48 hours after treatment, the level of IL-2 was confirmed by ELISA assay.
  • hPD-Ll VC, 19-239) -hyFcMl or CTLA-4-Ig (0rencia) (control) were added to PBMCs obtained from RA patients to confirm human-derived T-cell proliferation inhibitory ability. After treatment, 72 hours later, CFSE staining confirmed prol i ferat ion.
  • hPD-Ll (VC) -hyFc protein exhibits not only an inhibitory effect on human T-cells but also an inhibitory effect of cytokines associated with T-cell activation. It was found that the T-cells can be effectively suppressed.
  • DCs Dendritic cells
  • VC hPD-Ll
  • CTLA-4-Ig proteins purified hPD-Ll (VC) -hyFcMl and CTLA-4-Ig proteins.
  • IL-6 is known to be secreted from the activated DC as a representative proinf lammatory cytokine, and IL-6 expression was used as an evaluation index.
  • GM-CSF and IL-4 were treated with 5 ug / ml and cultured for 6 days under conditions in which dendritic cells were stimulated with LPS.
  • hPD-Ll VC, 19-239) -hyFcMl or CTLA-4-Ig (0rencia) was treated with 2, 10 and 50 ug / ml, respectively.
  • human IL-6 mRNA expression was compared by quantitative real-t ime PCR. As a result, it was found that hPD—Ll (VC, 19-239) -hyFcMl reduced IL-6 expression level in DC cells (FIG. 29).
  • Example 15-1 Inhibition of IL-2 Production by Concentration of hPD-Ll-hyFc Recombinant Protein IL-2 Inhibition of T Cell Activation of hPD-Ll (VC) -hyFcMl and CTLA-4-Ig Protein Using Cells Isolated from Mouse Spleen And IFN-gamma measurements.
  • the isolated mouse splenocytes were cultured in anti-CD3 (2 ug / ml) and ant i—CD28 coated plates and treated with hPD-Ll (VC, 19-239 HiyFcMl 0, 10, 50 ug / ml, respectively). The cells were cultured for 48 hours, and then the amount of IL-2 and IFN-gamma, which are indicators of the level of T cell activity, was measured by ELISA.
  • IL-2 and splenocytes were treated by treatment of hPD—Ll (VC, 19-239) -hyFcMl.
  • Example 15-2 Confirmation of IFN-gamma Inhibitory Effect of hPD-Ll-hyFc Recombinant Protein IFNs in Pancreatic Cells Using hPD-Ll (19-239) -hyFcMl, hPD-Ll (19-133) -hyFcMl, or hPD-Ll (19-127) -hyFcMl Recombinant Protein in T Cells Isolated from Mouse Pancreas Inhibition of total T cells and CD4 + T cells was evaluated by measuring -gamma secretion.
  • hPD-Ll (19-114) -hyFc and hPD-Ll (19-120) -hyFc which are fragments of the V domain.
  • isolated mouse splenocytes were cultured in anti-CD3 (2 ug / ml) and ant i-CD28 coated plates, hPD-Ll (VC, 19-239) -hyFc, hPD-Ll (V, 19-133) -hyFc, hPD-Ll (V, 19-127) were incubated for 48 hours with 2 ug of treatment. Thereafter, a culture was obtained and IFN-gamma, a T cell active cytokine present in the culture, was measured by ELISA assay.
  • 19 to 127 fragments of the amino acid sequence of SEQ ID NO: 3 can be expected to be an important site in showing the immunosuppressive effect in the extracellular domain of PD-L1, as well as productivity of the fusion protein for shorter fragments.
  • Some results were found to be somewhat inefficient in Figure 20. Therefore, it was predicted that it would be desirable to use a polypeptide having a length of 19 to 127 fragments or more of the amino acid sequence of SEQ ID NO: 3 in the development of an Fc fusion protein therapeutic using the domain of PD-L1.
  • Example 15-3 Example 15-3.
  • the isolated mouse splenocytes were cultured in anti-CD3 (2 ug / ml) coated plate and hPD-Ll (VC, 19-239) -hyFcMl, hPD-Ll (V) at concentrations of 250, 500 or 1000 nM. ; 19-133) was treated with hyFcMl or CTLA-4-Ig (0rencia) . After culturing for 72 hours, fflT analysis was performed to measure the extent of T cell proliferation.
  • hPD-Ll (VC, 19-239) -hyFcMl, hPD-Ll (V, 19-133) -hyFcMl or CTLA-4-Ig (0rencia) all inhibited the proliferation of T cells in a concentration-dependent manner.
  • all types of hPD-Ll-hyFc fusion proteins were found to have superior T-cell inhibitory ability than CTLA-4-Ig (0rencia) as a control (FIG. 33).
  • Example 15-5 Example 15-5.
  • 100 ng / ml PMA was pre-treated in 2.5 X 10 4 cel ls / 15 ul / wel l to activate the cells for 24 hours, and then the respective concentrations of 0, 1, 1.9, 3.9, 7.8, 15.5, 31, 62, 124 uM.
  • the fluorescence signal intensity was measured after incubation for 4 to 5 hours in a luminometer.
  • hPD-Ll V (:, 19-239) -hyFc, hPD-Ll (V, 19-133) -hyFcMl and CTLA-4-Ig (0renci a) were all concentration dependent. Confirming the inhibition of IL-2 secretion of T cells demonstrated the T cell inhibitory effect in human T cell lines (FIG. 34).
  • CD4 + CD25XD45RB high T cells were intraperitoneally injected.
  • Example 16-2 Evaluation of efficacy of hPD-Ll-hyFc recombinant protein by confirming viability of chronic inflammatory bowel disease model In vivo activity of hPD-LKVC, 19-239) -hyFcMl and hPD-Ll (V, 19-133) -hyFcMl fusion proteins To assess the efficacy of palliation and inhibition of enteritis following PD-L1 protein administration in a chronic mouse enteritis model induced by T cells.
  • CD4 + CD25XD45RB high T cells were isolated from spleen cells of C57BL / 6 mice using flow cytometry.
  • mice deficient in T and B cells were intraperitoneally injected with 5 ⁇ 10 5 CD4 + CD25XD45RB high T cells isolated. From 3 weeks after T cell injection, hPD-Ll (VC :, 19-239) -hyFcMl and hPD-Ll (V, 19-133) -hyFcMl fusion proteins were intraperitoneally 3 times at 20 weeks or 200 ug intervals. The number of mice injected intra- and surviving for the entire experimental period was recorded.
  • Example 17 Evaluation of psoriasis treatment effect of hPD-Ll-hyFc recombinant protein To confirm the efficacy of hPD-Ll-hyFc in acute psoriasis mouse model, 40 mg of Imiquimod (IMQ) was added to the ears of experimental mice. Acute psoriasis was induced by applying to both ears daily.
  • IMQ Imiquimod
  • mice were divided into normal mice, psoriasis induction and untreated groups, and psoriasis induction, and anti-P40 antibody and hPD-LKVC (19-239) -hyFcMl fusion protein.
  • Anti-P40 antibody was intraperitoneally administered 25 ug per mouse on day 1 and day 4.
  • hPD-Ll (F) -hyFc was intraperitoneally administered at 200 ug per mouse on days 1, 2, 4 and 6. After initial administration, ear thickness was observed daily to confirm the phenotype of psoriasis onset.
  • the hPD-Ll (VC, 19-239) -hyFcMl and anti—P40 antibody administration groups There was a statistically significant delay in onset effect compared to the group that did not receive (FIG. 37).
  • the histopathology of the ear was examined and compared with the psoriasis tissues such as epidermal thickness in the psoriasis-induced group by IMQ.
  • the group administered hPD-Ll (VC, 19-239) -hyFcMl showed statistically significant epidermal thickness reduction compared to the other groups not administered (FIG. 38).
  • the hPD-Ll fusion protein has a therapeutic effect in psoriasis and showed the efficacy of Dongdong compared to the anti-P40 antibody which is a conventional therapeutic agent.

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Abstract

Cette invention concerne une protéine de fusion constituée d'un domaine extracellulaire de PD-L1 et d'une région Fc d'immunoglobuline modifiée. Le domaine extracellulaire de PD-L1 et un fragment de celui-ci ont une excellente fonction immunomodulatrice, et peuvent être utilisés en tant que médicament pour l'immunomodulation quand ils sont couplés à une région Fc d'immunoglobuline modifiée. En conséquence, la protéine de fusion PD-L1 selon l'invention s'est avérée avoir un excellent effet dans des modèles de maladies inflammatoires de l'intestin, de rectocolite, psoriasis, asthme, et arthrite, et peut donc être utilisée de manière très efficace dans le traitement desdites maladies.
PCT/KR2015/005256 2014-05-23 2015-05-26 Protéine de fusion pd-l1 et son utilisation WO2015178746A1 (fr)

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JP2017514241A JP2017519520A (ja) 2014-05-23 2015-05-26 Ｐｄ−ｌ１融合タンパク質およびその使用
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RU2620552C1 (ru) * 2016-05-23 2017-05-26 Федеральное государственное бюджетное образовательное учреждение дополнительного профессионального образования "Российская медицинская академия непрерывного профессионального образования" Министерства здравоохранения Российской Федерации (ФГБОУ ДПО РМАНПО Минздрава России) Способ лечения тяжелых форм псориаза
US9920123B2 (en) 2008-12-09 2018-03-20 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
CN110520534A (zh) * 2016-11-09 2019-11-29 恩根尼公司 程序性死亡配体1的肠表达

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9920123B2 (en) 2008-12-09 2018-03-20 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
RU2620552C1 (ru) * 2016-05-23 2017-05-26 Федеральное государственное бюджетное образовательное учреждение дополнительного профессионального образования "Российская медицинская академия непрерывного профессионального образования" Министерства здравоохранения Российской Федерации (ФГБОУ ДПО РМАНПО Минздрава России) Способ лечения тяжелых форм псориаза
CN110520534A (zh) * 2016-11-09 2019-11-29 恩根尼公司 程序性死亡配体1的肠表达

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