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WO2021153979A1 - Protéine de fusion comprenant un anticorps anti-taa, un anticorps anti-pd-l1 et il-2 et les utilisations correspondantes - Google Patents

Protéine de fusion comprenant un anticorps anti-taa, un anticorps anti-pd-l1 et il-2 et les utilisations correspondantes Download PDF

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WO2021153979A1
WO2021153979A1 PCT/KR2021/001054 KR2021001054W WO2021153979A1 WO 2021153979 A1 WO2021153979 A1 WO 2021153979A1 KR 2021001054 W KR2021001054 W KR 2021001054W WO 2021153979 A1 WO2021153979 A1 WO 2021153979A1
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Prior art keywords
fusion protein
cancer
amino acid
antibody
cells
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Korean (ko)
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성영철
윤진원
신은주
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Genexine Inc
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Genexine Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • the present invention relates to a fusion protein comprising an anti-TAA antibody, an anti-PD-L1 antibody and IL-2, and uses thereof. More specifically, the present invention relates to a fusion protein comprising an anti-CEA antibody, an anti-PD-L1 antibody and IL-2, and uses thereof.
  • the anticancer drug market occupies the largest portion of the overall drug market and has the highest growth potential (CACR) of 11.6%, which is expected to expand to 153 trillion won in 2020.
  • CACR growth potential
  • immuno-oncology drugs have a mechanism in which the immune system attacks cancer cells by stimulating the body's immune system.
  • tumor cells expressing tumor-specific antigens can be eliminated by an individual's immune system at an early stage of development.
  • actively proliferating tumor cells express immune checkpoint proteins such as PD-L1 to evade immune surveillance.
  • immune checkpoint proteins are known as a mechanism to evade the immune response of tumor cells by interfering with the anticancer immune surveillance system.
  • Immune checkpoint inhibitors released with the mechanism of inhibiting the binding of PD-1 and PD-L1 include pembrolizumab (Keytruda, pembrolizumab, anti-PD-1: Merck), nivolumab (Opdivo, nivolumab, anti-PD- 1: BMS), and atezolizumab (Tecentriq, atezolizumab, anti-PD-L1: Roche).
  • Ipilimumab (Yervoy, Ipilimumab, anti-CTLA4: BMS), the world's first FDA-approved immune checkpoint inhibitor prior to the immune checkpoint inhibitor for PD-1 and PD-L1, binds to T-cell surface CTLA-4 It was approved for the first time as a treatment for melanoma patients as a mechanism to prevent cells from being incapacitated and to help the proliferation of T cells.
  • the number of T cells in the tumor tissue was markedly insufficient.
  • the patient's immune status is one of the important factors that determine the response to the immune checkpoint inhibitor.
  • the immune system is disturbed by chemotherapy and radiation therapy, resulting in 'immune cold or immune desert', which is speculated to be the cause of low reactivity to immune checkpoint inhibitors. Therefore, there is a need to develop a substance that activates the patient's immune system and research on a new treatment method that uses it in combination with an existing immune checkpoint inhibitor.
  • cytokines are substances that regulate various immune responses, and are important factors that determine the development, proliferation, maintenance, activity, and death of immune cells according to their types and characteristics.
  • Various cytokine therapeutics are being developed for the purpose of activating the immune system of patients.
  • IL-2 cytokines IL-2, IL-7, IL-15, IL-21
  • cytokine therapeutics have a limitation in that they cannot confirm distinct therapeutic efficacy due to their short half-life.
  • the present inventors have studied to develop an effective immuno-oncology agent capable of inhibiting the immune checkpoint protein while activating the immune system.
  • the fusion protein containing the anti-CEA antibody, anti-PD-L1 antibody and IL-2 is excellent.
  • the present invention was completed by confirming that it exhibits an anticancer effect.
  • one aspect of the present invention provides a fusion protein of the following structural formula 1.
  • Another aspect of the present invention provides a fusion protein dimer in which the two fusion proteins are bound.
  • Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the fusion protein or the fusion protein dimer as an active ingredient.
  • the fusion protein of the present invention has excellent binding ability between CEA, a tumor-associated antigen, and PD-L1, an immune checkpoint protein, and can specifically bind to tumor cells expressing CEA and/or PD-L1.
  • the fusion protein of the present invention can induce proliferation of T cells and NK cells, activate immune cells, and exhibit excellent anticancer effects when administered to cancer-inducing mice. Therefore, the fusion protein of the present invention can be usefully used to treat cancer diseases.
  • FIG. 1 is a view showing the results of analysis by SDS-PAGE after purifying the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer according to an embodiment of the present invention.
  • FIG. 2 is a view showing the results of SE-HPLC analysis after formulating the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer, which is an embodiment of the present invention.
  • Figure 3 is a diagram measuring the binding force between the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer according to an embodiment of the present invention and a cell expressing CEA.
  • FIG. 4 is a diagram measuring the binding force between the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein of an embodiment of the present invention and a cell expressing PD-L1.
  • FIG. 5 is a diagram measuring the binding force between the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer according to an embodiment of the present invention and cells expressing CEA and PD-L1.
  • FIG. 6 shows PD-1/PD-L1 binding inhibition after treatment of cells expressing PD-1 and cells expressing PD-L1 with ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer according to an embodiment of the present invention It is a view confirming the change of the NFAT-Luc signal by
  • FIG. 7 is a view confirming the proliferation rate of immune cells such as T cells and NK cells after human-derived peripheral blood mononuclear cells were treated with the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer according to an embodiment of the present invention.
  • FIG. 9 is a diagram illustrating the half-life of the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer according to an embodiment of the present invention.
  • FIG. 10 is a diagram showing the amount of accumulation in the body of each tissue of the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer according to an embodiment of the present invention.
  • FIG. 11 is a diagram showing the distribution of CD8+ T cells, NK cells and Treg cells according to time after administration of the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer according to an embodiment of the present invention to solid cancer-induced mice.
  • FIG. 12 is a diagram illustrating the measurement of tumor size and absolute lymphocyte count after administration of the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer, which is an embodiment of the present invention, to solid cancer-inducing mice.
  • Fusion protein comprising anti-CEA antibody, anti-PD-L1 antibody and IL-2
  • the N ' is the N-terminus of the fusion protein
  • the C ' is the C-terminus of the fusion protein
  • A is a single-chain variable fragment (scFv) of an antibody that specifically binds to a tumor-associated antigen
  • B is an scFv of an antibody that specifically binds to PD-L1 (Programmed death-ligand 1)
  • C is an Fc domain of an immunoglobulin, a fragment thereof, or a variant thereof
  • D is IL-2, a fragment thereof or a variant thereof
  • the L 1 , L 2 and L 3 provide a fusion protein that is a peptide linker, respectively.
  • A is a tumor-associated antigen, wherein the tumor-associated antigen is not cancer-specific among antigens expressed according to cell carcinoma, and refers to an antigen present in a trace amount even in the corresponding normal cells.
  • A may be an scFv of an antibody that specifically binds to any one tumor-associated antigen selected from the group consisting of CEA (Carcinoembryonic antigen), AFP ( ⁇ -fetoprotein), and MUC1 (Mucin 1).
  • A may be an scFv of an antibody that specifically binds to CEA.
  • the CEA is a tumor-related antigen present in the blood found in cancers such as colon cancer, breast cancer, lung cancer, liver cancer, noncancerous diseases, or smokers, and can be used to check the progress of cancer treatment and whether or not it recurs.
  • AFP is a tumor-associated antigen detected in blood found in patients with carcinomas such as liver cancer, gastric cancer, ovarian fetal cancer, hepatitis, and cirrhosis.
  • carcinomas such as liver cancer, gastric cancer, ovarian fetal cancer, hepatitis, and cirrhosis.
  • AFP is a fetal serum protein that is produced during gestation and begins to decline after birth to levels observed in adults after 18 months.
  • the MUC1 is a protein found in specific epithelial cells, and is measured higher in breast cancer, ovarian cancer, lung cancer, and prostate cancer patients than in normal subjects. By measuring the expression level of MUC1 in the blood, treatment and recurrence of cancer can be predicted.
  • A may be composed of the amino acid sequence represented by SEQ ID NO: 1.
  • the B may consist of the amino acid sequence represented by SEQ ID NO: 2.
  • the C may be an Fc domain of an immunoglobulin, a fragment thereof, or a variant thereof.
  • the variant of the Fc domain may be an Fc domain of a modified immunoglobulin or a fragment thereof.
  • the Fc domain of the modified immunoglobulin has a modified binding ability with an Fc receptor and/or complement, thereby causing antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). This may be weakened.
  • the modified immunoglobulin may be selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and combinations thereof.
  • the term "Fc domain”, “Fc fragment” or “Fc” includes the heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of an immunoglobulin, but includes the variable regions of its heavy and light chains. and light chain constant region 1 (CL1). It may further comprise a hinge region of the heavy chain constant region.
  • the immunoglobulin Fc domain fragment may include the CH2 and CH3 domains of an antibody.
  • the CH2 and CH3 domains may be derived from IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD or IgE. Modified Fc or modified Fc fragments are also referred to herein as “hFc” or “hyFc”.
  • the term “Fc domain variant” refers to one in which some amino acids in the Fc domain are substituted or prepared by combining different types of Fc domains.
  • the Fc domain variant can prevent cleavage at the hinge region.
  • the 144th amino acid and/or the 145th amino acid of SEQ ID NO: 9 may be modified.
  • it may be a variant in which K, which is the 144th amino acid of SEQ ID NO: 9, is substituted with G or S, and E, which is the 145th amino acid, is substituted with G or S.
  • the Fc fragment of the present invention may be in the form of a native sugar chain, an increased sugar chain compared to the native type, a decreased sugar chain compared to the native type, or a form in which the sugar chain is removed.
  • Immunoglobulin Fc sugar chains can be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Removal of sugar chains from the Fc fragment sharply reduces the binding affinity of the primary complement component C1 to C1q and results in a decrease or loss of ADCC or CDC, thereby not inducing an unnecessary immune response in vivo.
  • the immunoglobulin Fc fragment in a deglycosylated or aglycosylated form may be more suitable for the purpose of the present invention as a drug carrier.
  • deglycosylation refers to enzymatic removal of sugars from an Fc fragment.
  • aglycosylation means that the Fc fragment is produced in an unglycosylated form by prokaryotes, preferably E. coli.
  • the Fc domain of the modified immunoglobulin comprises the amino acid sequence of SEQ ID NO: 9 (hyFc), SEQ ID NO: 5 (hyFcM1), SEQ ID NO: 12 (hyFcM2), SEQ ID NO: 13 (hyFcM3) or SEQ ID NO: 14 (hyFcM4) may be doing
  • the Fc domain of the modified immunoglobulin may include the amino acid sequence of SEQ ID NO: 5 (non-lytic mouse Fc).
  • C used is a modified immunoglobulin Fc domain (hyFcM1), which is a variant of the immunoglobulin Fc domain, and C may consist of the amino acid sequence shown in SEQ ID NO: 5.
  • D may be IL-2 or a variant thereof.
  • IL-2 is from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). Any wild-type IL-2 obtained.
  • the term includes untreated IL-2, and any form of IL-2 obtained from treatment in cells.
  • the term also includes wild-type variants of IL-2, eg, splice variants or allelic variants.
  • human IL-2 may consist of the amino acid sequence shown in SEQ ID NO: 7.
  • mutant of IL-2 includes a truncated form of IL-2, and a form in which IL-2 is linked to another molecule by fusion or chemical conjugation, and IL-2 It may include any mutant form of various forms of
  • the mutant of IL-2 may be a mutant obtained by substituting some amino acids of wild-type IL-2.
  • the IL-2 variant may be modified to have low toxicity in vivo.
  • toxicity in vivo may mean a side effect caused by binding of IL-2 to the alpha chain of the IL-2 receptor.
  • various IL-2 variants have been developed, and these IL-2 variants are as disclosed in US Patent 5,229,109 and Korean Patent 1667096.
  • the 38th or 42nd amino acid of SEQ ID NO: 7 may be substituted.
  • one embodiment of the IL-2 variant may be one in which wild-type 38th arginine is substituted with alanine (R38A).
  • one embodiment of the variant of IL-2 may be a form in which the wild-type 42 th phenylalanine is substituted with another amino acid, specifically, the IL-2 variant has the 42 th amino acid of the sequence of SEQ ID NO: 7 F42A, F42G , F42S, F42T, F42Q, F42E, F42N, F42D, F42R and F42K may be substituted.
  • the variant of IL-2 may be a form in which the 38th and 42nd amino acids are substituted in wild-type IL-2.
  • the IL-2 variant may include any one amino acid substitution selected from R38A, F42A, and combinations thereof in the amino acid sequence shown in SEQ ID NO: 7.
  • the variant of IL-2 may have the amino acid sequence of SEQ ID NO: 8.
  • the A and B may be coupled through 1 to 50 peptide linkers (first linker, L 1 ).
  • the peptide linker comprises 1 to 50 contiguous amino acids, 5 to 45 contiguous amino acids, 10 to 40 contiguous amino acids, or 15 to 35 contiguous amino acids, or 20 to 30 contiguous amino acids. It may consist of amino acids. In one embodiment, L 1 may consist of 20 amino acids.
  • the A and B may be coupled through a peptide linker (first linker, L 1 ) consisting of the amino acid sequence shown in SEQ ID NO: 3.
  • the B and C may be coupled through 1 to 50 peptide linkers (second linker, L 2 ).
  • L 1 is 1 to 50 consecutive amino acids, 5 to 45 consecutive amino acids, 10 to 40 consecutive amino acids, or 15 to 35 consecutive amino acids, or 20 to 30 amino acids can
  • L 2 may consist of 30 amino acids.
  • L 2 may include at least one cysteine.
  • L 2 may include one, two, or three cysteines.
  • the L 2 may be derived from the hinge of the immunoglobulin.
  • L 2 may be composed of the amino acid sequence represented by SEQ ID NO: 16.
  • the C and D may be coupled through 1 to 50 peptide linkers (third linker, L 3 ).
  • the peptide linker may consist of 1 to 50 consecutive amino acids.
  • L 3 may consist of 4 amino acids.
  • the C and D may be coupled through a peptide linker (third linker, L 3 ) consisting of the amino acid sequence shown in SEQ ID NO: 6.
  • the fusion protein may consist of the amino acid sequence represented by SEQ ID NO: 15.
  • Another aspect of the present invention provides a fusion protein dimer in which two fusion proteins comprising the anti-CEA antibody, the anti-PD-L1 antibody and IL-2 are bound.
  • the fusion protein comprising the anti-CEA antibody, the anti-PD-L1 antibody and IL-2 is as described above.
  • the bond between the fusion proteins constituting the dimer may be formed by a disulfide bond by a cysteine present in the linker, but is not limited thereto.
  • the fusion proteins constituting the dimer may be the same, but may be different fusion proteins.
  • the dimer may be a homodimer.
  • polynucleotide encoding a fusion protein comprising an anti-CEA antibody, an anti-PD-L1 antibody and IL-2.
  • the polynucleotide may be a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 15.
  • the fusion protein comprising the anti-CEA antibody, the anti-PD-L1 antibody and IL-2 is as described above. As long as the polynucleotides encode the same amino acid sequence, one or more bases may be substituted.
  • the polynucleotide may further include a nucleic acid encoding a signal sequence.
  • signal sequence refers to a signal peptide that directs secretion of a target protein.
  • the signal peptide is cleaved after translation in the host cell.
  • the signal sequence is an amino acid sequence that initiates the movement of the protein through the ER (endoplasmic reticulum) membrane.
  • the signal sequence is well known in the art, and typically contains 16 to 30 amino acid residues, but may include more or fewer amino acid residues.
  • a typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region.
  • the central hydrophobic region contains 4 to 12 hydrophobic residues that anchor the signal sequence through the membrane lipid bilayer during migration of the immature polypeptide.
  • the signal sequence is cleaved in the lumen of the ER by cellular enzymes commonly known as signal peptidases.
  • the signal sequence may be a secretion signal sequence of tissue Plasminogen Activation (tPa), a signal sequence of Herpes simplex virus glycoprotein D (HSV gDs), or a growth hormone.
  • tPa tissue Plasminogen Activation
  • HSV gDs Herpes simplex virus glycoprotein D
  • a growth hormone a secretion signal sequence used in higher eukaryotic cells including mammals and the like may be used.
  • Another aspect of the present invention provides a vector comprising the polynucleotide.
  • the vector can be introduced into a host cell, recombination and insertion into the host cell genome. or said vector is understood as a nucleic acid means comprising a polynucleotide sequence capable of spontaneous replication as an episome.
  • Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and analogs thereof.
  • examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.
  • the vector may be plasmid DNA, phage DNA, etc., commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), E. coli-derived plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis plasmids (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, ⁇ gt10, ⁇ gt11, ⁇ ZAP, etc.), animal virus vectors (retroviruses) ), adenovirus, vaccinia virus, etc.), insect virus vectors (baculovirus, etc.). Since the vector exhibits different protein expression levels and modifications depending on the host cell, it is preferable to select and use a host cell most suitable
  • the term "gene expression” or “expression” of a target protein is understood to mean transcription of a DNA sequence, translation of an mRNA transcript, and secretion of a fusion protein product or fragment thereof.
  • a useful expression vector may be RcCMV (Invitrogen, Carlsbad) or a variant thereof.
  • the expression vector includes a human cytomegalovirus (CMV) promoter to promote continuous transcription of a target gene in mammalian cells, and a bovine growth hormone polyadenylation signal sequence to increase the stable-state level of RNA after transcription. can do.
  • CMV human cytomegalovirus
  • Another aspect of the present invention provides a transformed cell into which the vector is introduced.
  • the host cell of the transformed cell may include, but is not limited to, a cell of prokaryotic, eukaryotic, mammalian, plant, insect, fungal or cellular origin.
  • a cell of prokaryotic, eukaryotic, mammalian, plant, insect, fungal or cellular origin As an example of the prokaryotic cell, E. coli may be used. In addition, yeast may be used as an example of eukaryotic cells.
  • CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK 293 cells or HEK293T cells may be used as the mammalian cells. , but is not limited thereto, and any cell that can be used as a mammalian host cell known to those skilled in the art is available.
  • the CaCl 2 precipitation method when introducing an expression vector into a host cell, the CaCl 2 precipitation method, the CaCl 2 precipitation method using a reducing substance called DMSO (dimethyl sulfoxide), which increases the efficiency by using the Hanahan method, electroporation method, calcium phosphate precipitation method , protoplast fusion method, agitation method using silicon carbide fiber, agrobacterium mediated transformation method, transformation method using PEG, dextran sulfate, lipofectamine and drying/inhibition mediated transformation method can be used.
  • DMSO dimethyl sulfoxide
  • the glycosylation-related gene possessed by the host cell is manipulated through a method known to those skilled in the art, and the sugar chain pattern of the fusion protein (e.g., sialic acid, fucosylation, glycation) can be adjusted.
  • the sugar chain pattern of the fusion protein e.g., sialic acid, fucosylation, glycation
  • Another aspect of the present invention provides a method for producing a fusion protein comprising an anti-CEA antibody, an anti-PD-L1 antibody, and an IL-2 comprising culturing the transformed cell.
  • the production method comprises the steps of i) culturing the transformed cells to obtain a culture; and ii) recovering the fusion protein from the culture.
  • the method of culturing the transformed cells may be performed using a method well known in the art. Specifically, the culture may be continuously cultured in a batch process or injection batch or repeated fed batch process (fed batch or repeated fed batch process).
  • Another aspect of the present invention is a fusion protein comprising an anti-CEA antibody, an anti-PD-L1 antibody and IL-2 or a fusion protein dimer to which the two fusion proteins are bound as an active ingredient for the prevention or treatment of cancer
  • a pharmaceutical composition is provided.
  • the fusion protein comprising the anti-CEA antibody, the anti-PD-L1 antibody and IL-2 or the fusion protein dimer to which the two fusion proteins are bound is as described above.
  • the fusion protein or dimer thereof of the present invention may specifically bind to CEA, a tumor-associated antigen expressed in tumor cells, by including an scFv of an antibody that specifically binds to CEA.
  • the fusion protein of the present invention or a dimer thereof includes an antibody scFv that specifically binds to PD-L1, thereby binding to PD-L1 expressed in tumor cells and inhibiting PD-L1/PD-1 binding.
  • the fusion protein or dimer thereof of the present invention can activate immune cells by including IL-2 or a fragment or variant thereof. That is, the fusion protein of the present invention or a dimer thereof specifically binds to tumor cells, can block the immune evasion mechanism of tumor cells, and can activate immune cells around tumor cells, thereby preventing or treating cancer diseases. can be usefully used for
  • the cancer is gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer and lymphoma. may be selected from the group.
  • the preferred dosage of the pharmaceutical composition varies depending on the condition and weight of the patient, the degree of disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art.
  • the active ingredient exhibits anticancer activity or an arbitrary amount (effective amount) depending on the use, formulation, purpose of formulation, etc. as long as it can exhibit a therapeutic effect on infectious disease may be included, and a typical effective amount will be determined within the range of 0.001 wt % to 20.0 wt % based on the total weight of the composition.
  • the term “effective amount” refers to an amount of an active ingredient capable of inducing an anticancer effect or an infectious disease treatment effect. Such effective amounts can be determined empirically within the ordinary ability of one of ordinary skill in the art.
  • the term “treatment” may be used to include both therapeutic treatment and prophylactic treatment. In this case, prevention may be used in the sense of alleviating or reducing a pathological condition or disease of an individual.
  • the term “treatment” includes any form of administration or application for treating a disease in a mammal, including a human. The term also includes inhibiting or slowing the disease or its progression; restoring or repairing damaged or missing function, partially or completely relieving the disease; or stimulate inefficient processes; It includes the meaning of alleviating serious diseases.
  • the term “efficacy” refers to one or more parameters, such as survival or disease-free survival over a period of time, such as 1 year, 5 years, or 10 years. can be determined by the parameter may include suppressing the size of at least one tumor in the subject.
  • “enhanced efficacy” e.g., improvement in efficacy
  • terapéuticaally effective amount refers to an amount of a compound or composition effective to prevent or treat a target disease, which is sufficient to treat the disease at a reasonable benefit/risk ratio applicable to medical treatment, and It means an amount that does not cause side effects.
  • the level of the effective amount may be determined by the patient's health status, disease type, severity, drug activity, drug sensitivity, administration method, administration time, administration route and excretion rate, treatment period, combination or factors including drugs used concurrently and other factors well known in the medical field.
  • a therapeutically effective amount refers to an amount of a drug effective to treat cancer.
  • the pharmaceutical composition may further include a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be any non-toxic material suitable for delivery to a patient. Distilled water, alcohol, fats, waxes and inert solids may be included as carriers. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.
  • the pharmaceutical composition may be prepared as a parenteral formulation according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient.
  • pharmaceutically acceptable means that it does not inhibit the activity of the active ingredient and does not have toxicity beyond which the application (prescription) target is adaptable.
  • the pharmaceutical composition When the pharmaceutical composition is prepared for parenteral use, it may be formulated in the form of injections, transdermal administrations, nasal inhalants and suppositories together with suitable carriers according to methods known in the art.
  • a suitable carrier may be sterile water, ethanol, polyol such as glycerol or propylene glycol, or a mixture thereof, preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, or sterilized for injection. Water, an isotonic solution such as 5% dextrose, etc. may be used.
  • Formulation of pharmaceutical compositions is known in the art, and specifically, reference may be made to the literature [Remington's Pharmaceutical Sciences (19th ed., 1995)] and the like. This document is considered a part of this specification.
  • a preferred dosage of the pharmaceutical composition is in the range of 0.01 ug/kg to 10 g/kg per day, or 0.01 mg/kg to 1 g/kg, depending on the patient's condition, weight, sex, age, patient's severity, and administration route. can be Administration may be performed once or divided into several times a day. Such dosages should not be construed as limiting the scope of the present invention in any respect.
  • compositions of the present application are mammals and humans, particularly preferably humans.
  • the pharmaceutical composition of the present application may further include any compound or natural extract known to have an anticancer activity or therapeutic effect on an infectious disease, and safety has already been verified for the increase/reinforcement of anticancer activity.
  • the administration route, dosage, and frequency of administration of the fusion protein or fusion protein dimer may be administered to a subject in various ways and amounts depending on the patient's condition and presence or absence of side effects, and the optimal administration method, dosage and frequency of administration are A person skilled in the art can select within an appropriate range.
  • the fusion protein or fusion protein dimer may be administered in combination with other drugs or physiologically active substances with known therapeutic effects for the disease to be treated, or formulated in the form of a combination preparation with other drugs.
  • Another aspect of the present invention is a fusion protein comprising an anti-CEA antibody, an anti-PD-L1 antibody and IL-2 or a fusion protein dimer to which the two fusion proteins are bound as an active ingredient for the prevention or treatment of cancer provide a way
  • the therapeutically effective amount includes the specific composition including the type and extent of the response to be achieved, whether or not other agents are used if necessary, the individual's age, weight, general health, sex and diet, administration time, administration route, and composition. It is preferable to apply differently depending on various factors including the secretion rate, treatment period, and drugs used together or concurrently with a specific composition and similar factors well known in the pharmaceutical field. Therefore, it is preferable to determine the effective amount of the composition suitable for the purpose of the present invention in consideration of the foregoing.
  • the subject is applicable to any mammal, and the mammal includes not only humans and primates, but also domestic animals such as cattle, pigs, sheep, horses, dogs and cats.
  • ⁇ CEA anti-CEA antibody
  • ⁇ PDL1 anti-PD-L1 antibody
  • IL2v variant of IL-2
  • an anti-CEA antibody scFv having the amino acid sequence shown in SEQ ID NO: 1 was prepared.
  • an anti-PD-L1 antibody scFv having the amino acid sequence shown in SEQ ID NO: 2 was prepared using the amino acid sequence of the anti-PD-L1 antibody.
  • the prepared scFv of the anti-CEA antibody and the scFv of the anti-PD-L1 antibody were combined with a peptide linker (first linker) consisting of the amino acid sequence shown in SEQ ID NO: 3, which was combined with SEQ ID NO: on the N-terminal side of the FC domain. It was bound with a peptide linker (second linker) consisting of the amino acid sequence represented by 16.
  • the mutant of IL-2 was manufactured to include two mutants, which are expected to increase the therapeutic efficacy through selective binding to NK cells while decreasing the binding ability to IL-2 receptor ⁇ .
  • the IL-2 variant has amino acid substitutions of R38A and F42A in human IL-2 (SEQ ID NO: 7), and the one consisting of the amino acid sequence shown in SEQ ID NO: 8 was used.
  • the variant of IL-2 was coupled to the C-terminal side of the FC domain with a peptide linker (third linker) consisting of the amino acid sequence shown in SEQ ID NO: 6.
  • the ⁇ CEA-hyFc gene construct in which ⁇ CEA, the second linker and hyFc are combined in the order from the N-terminus to the C-terminus An expression vector containing Furthermore, an expression vector containing the ⁇ CEA-hyFc-IL2v gene construct in the form of ⁇ CEA, the second linker, hyFc, the third linker and IL2v combined in the order from the N-terminus to the C-terminus was prepared.
  • an expression vector containing the ⁇ PDL1- ⁇ CEA-hyFc gene construct in the form of ⁇ PDL1, the first linker, ⁇ CEA, the second linker and hyFc joined in the order from the N-terminus to the C-terminus was prepared.
  • an expression vector containing a hyFc-IL2v gene construct in which hyFc, a third linker, and IL2v are combined in the order from the N-terminus to the C-terminus was prepared.
  • ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer In order to produce the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer, suspension-adapted CHO cells were inoculated at 6 ⁇ 10 6 cells/ml (seeding), and then the ⁇ CEA- ⁇ PDL1-hyFc-IL2v prepared in Example 1 was performed. Transformation was achieved by treatment with 200 ml of an expression vector containing the gene construct and ExpiFectamine TM CHO reagent. To obtain a large amount of ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer, ExpiFectamine TM CHO enhancer and ExpiCHO TM feed were added the day after transformation, and the culture temperature was shifted to 32°C ( shift). After 7 days, the culture medium was collected and centrifuged at 3,600 rpm for 15 minutes to obtain a supernatant. Thereafter, the superna
  • ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer containing an Fc domain using AKTA Prime (GE Healthcare Life sciences) and Protein A resin was purified.
  • AKTA Prime GE Healthcare Life sciences
  • Protein A resin Protein A resin was purified.
  • As the elution buffer 100 mM Glycine-HCL, 50 mM L-Arginine and 5% Glycerol (pH 3.0) were used.
  • a fusion protein dimer with a purity of 90% or more was obtained only by purification using Protein A resin. Only a fraction with high purity was isolated and formulated in 50 mM hepes, 250 mM sucrose, pH 5.0. As a result of confirming the formulated ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer through SDS-PAGE, it was confirmed at about 250 kDa under non-reducing conditions, and about 100 kDa under reducing conditions. (FIG. 1, non-reducing: fusion protein dimer, reduced: fusion protein monomer). In addition, as a result of confirming the purity through SE-HPLC analysis, a purity of 90.22% was confirmed ( FIG. 2 ).
  • Example 1 Expression vector containing ⁇ PDL1- ⁇ CEA-hyFc-IL2v gene construct, expression vector containing ⁇ CEA-hyFc gene construct, expression vector containing ⁇ CEA-hyFc-IL2v gene construct, ⁇ PDL1- ⁇ CEA-hyFc Using the expression vector containing the gene construct and the expression vector containing the hyFc-IL2v gene construct, the ⁇ PDL1- ⁇ CEA-hyFc-IL2v fusion protein was produced in the same manner as for the production of the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein, respectively.
  • a dimer, ⁇ CEA-hyFc fusion protein dimer, ⁇ CEA-hyFc-IL2v fusion protein dimer, ⁇ PDL1- ⁇ CEA-hyFc fusion protein dimer and hyFc-IL2v fusion protein dimer were produced.
  • CEA-expressing MKN45 cells Korea Cell Line Bank (KCLB), Cat. No. 80103 was used to confirm the degree of binding.
  • each fusion protein dimer was diluted to a concentration of 300 ⁇ g to 0 ⁇ g by concentration and incubated at 4° C. for 30 minutes.
  • a PBS (Welgene, Cat.No. LB004-02-500ml) buffer containing 1% FBS (Hyclone, Cat.No. SH30084.03) was prepared for use as a FACS assay buffer. Thereafter, after washing with FACS assay buffer, 1.5 ⁇ l of a secondary antibody, PE-anti-human IgG antibody (Biolegend, Cat. incubated for 30 min. After washing with FACS assay buffer, it was transferred to a FACS tube for FACS analysis.
  • the EC50 value of the MKN45 cell line and the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer was about 15.8 nM.
  • the EC50 value of the MKN45 cell line and the ⁇ PDL1- ⁇ CEA-hyFc-IL2v fusion protein dimer was about 104.7 nM ( FIG. 3 ).
  • ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer and ⁇ PDL1- ⁇ CEA-hyFc-IL2v fusion protein dimer produced in Example 2 bind to the immune checkpoint protein
  • PD-L1, PD-L1 expressing MDA - The degree of binding was confirmed using MB-231 cell line (Korea Cell Line Bank (KCLB), Cat. No. 30026).
  • each fusion protein dimer was diluted by concentration while diluting 2 times at 12,000 ng/ml, and treated at 4° C. for 30 minutes. incubated during Thereafter, after washing with FACS assay buffer, 1.5 ⁇ l of a secondary antibody, PE-anti-human IgG antibody (Biolegend, Cat. incubated for 30 min. After washing with FACS assay buffer, it was transferred to a FACS tube for FACS analysis.
  • a secondary antibody PE-anti-human IgG antibody (Biolegend, Cat. incubated for 30 min.
  • FACS assay buffer After washing with FACS assay buffer, it was transferred to a FACS tube for FACS analysis.
  • the EC50 value of the MDA-MB-231 cell line and the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer was about 4.98 nM.
  • Example 5 Confirmation of binding of ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer with cells expressing PD-L1 and CEA: in vitro
  • mice The degree of binding was confirmed using the derived MC38 cell line (Kerafast, Cat.No. ENH204-FP).
  • a mouse-derived MC38 cell line expressing human-derived CEA and PD-L1 was prepared, and this cell line was named "C4 cell line”.
  • the C4 cell line was prepared by inserting the gene encoding the amino acid sequence of SEQ ID NO: 17 using XhoI and XbaI restriction enzymes into Lenti-XTM vector (Clontech, Cat. No. 632164) to construct a lentivirus. . Thereafter, the lentivirus was transfected into the MC-38-CEA cell line (Kerafast, Cat. No. ENH202).
  • the transfection mixture (PLUSTM Reagent, Invitrogen, Cat. No. 11514015) with 1.5 ml of plasmid DNA complex
  • Lenti-XTM 293T cell line (Clontech, Cat. No. 632180).
  • the plasmid DNA complex is a plasmid and Lenti- X Packaging Single Shots plasmid (Clontech, Cat.No. 631275).
  • the supernatant of the Lenti-XTM 293T cell line that produced the lentivirus was obtained and filtered through a 0.45 ⁇ m filter.
  • the filtered supernatant was treated with MC-38-CEA and transfected. After culturing for 48 hours, to check whether hPD-L1 was expressed, the expression level was checked using APC-anti human PD-L1 antibody (BD Bioscience, Cat. No. 563741). In addition, cell lines well expressing hPD-L1 were selected and stored through single cell selection.
  • ⁇ CEA-hyFc fusion protein dimer ⁇ CEA-hyFc-IL2v fusion protein dimer, and ⁇ PDL1-hyFc-IL2v fusion protein dimer were used as comparison groups.
  • each fusion protein dimer was diluted by concentration while diluting 2 times at 12,000 ng/ml, and incubated at 4° C. for 30 minutes. Thereafter, after washing with FACS assay buffer, 1.5 ⁇ l of a secondary antibody, PE-anti-human IgG antibody (Biolegend, Cat. incubated for 30 min. After washing with FACS assay buffer, it was transferred to a FACS tube for FACS analysis.
  • the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer had a high EC50 value of about 2.6 nM despite the protein molecular weight of about 250 kDa ( FIG. 5 ).
  • ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer inhibits PD-1/PD-L1 binding.
  • ⁇ CEA-hyFc fusion protein dimer, ⁇ CEA-hyFc-IL2v fusion protein dimer, and ⁇ PDL1-hyFc-IL2v fusion protein dimer were used as comparison groups.
  • PD-1 Effector Cells Promega, Cat.No. J115A
  • PD-L1-aAPC/CHO- K1 cells Promega, Cat. No. J109A
  • the TCR signal is blocked by the binding of PD-1/PD-L1 expressed by both cells.
  • the NFAT-Luc fluorescence signal was detected using a fluorescence microscope.
  • the two cells are treated with the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer, the PD-1/PD-L1 binding is inhibited and the TCR signal is transmitted, thereby activating the lower NFAT-Luc signal.
  • Example 7 ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer Confirmation of immune cell proliferation effect: in vitro
  • the immune cells were treated with the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer, and then the proliferation rate of each immune cell was compared.
  • a group treated with recombinant IL-2 (rhIL-2, Proleukin® Novartis, Cat. No. 653601261) was set as a control group.
  • PBMC peripheral blood mononuclear cells
  • the FACS antibody (PerCP/Cyanine5.5 anti-human CD3 Antibody ( Biolegend, Cat.No. 300430), Brilliant Violet 650TM anti-human CD4 Antibody (Biolegend, Cat.No. 300536), Brilliant Violet 605TM anti-human CD8a Antibody (Biolegend, Cat.No. 301040), BV786 Mouse Anti -Human CD56 (BD Biosciences, Cat. No. 564058), (1) PE anti-mouse/rat/human FOXP3 Antibody (Biolegend, Cat. No. 320008)) after reacting at 4° C.
  • the FACS antibody PerCP/Cyanine5.5 anti-human CD3 Antibody ( Biolegend, Cat.No. 300430), Brilliant Violet 650TM anti-human CD4 Antibody (Biolegend, Cat.No. 300536), Brilliant Violet 605TM anti-human CD8a Antibody (Biolegend, Cat.No. 301040), BV786 Mouse Anti -Human CD56 (BD Biosciences, Cat. No. 5
  • PE-anti-human IgG antibody Biolegend, Cat. No. 490304
  • the primary antibody, PE-anti-human IgG antibody was treated with 1.5 ⁇ l of each sample in 100 ⁇ l of FACS assay buffer and reacted at 4° C. for 30 minutes. Thereafter, after washing with FACS assay buffer, it was transferred to a FACS tube for analysis.
  • the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer increases the proliferation of NK cells and CD8+ T cells, which are important for tumor killing, to a level similar to that of recombinant IL-2, and in the case of Tregs, by inducing significantly lower proliferation, It was confirmed that the anticancer efficacy was superior to that of 2.
  • Example 8 ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer Confirmation of immune cell activation effect: in vitro
  • the amount of tumor cells killed by immune cells after treatment with the ⁇ CEA- ⁇ PDL1-hyFcRn-IL2v fusion protein dimer was measured. confirmed through.
  • the killed tumor cells were measured by checking the 7AAD+ cells that were stained by intervening in the DNA of the dead cells and measuring the amount of LDH from the dead cells.
  • Human-derived PBMCs were treated with the ⁇ CEA- ⁇ PDL1-hyFcRn-IL2v fusion protein dimer for each concentration and activated, and then cultured with tumor cells at a ratio of 20:1. At this time, the group treated with recombinant IL-2 (rhIL-2) was set as a comparison group.
  • human PBMC Effector cell
  • tumor cells Tuget cell
  • E:T tumor cell
  • a 7-AAD (1000X stock) solution was diluted 1:1000 to 30 minutes before FACS analysis, and the cells were treated and FACS analysis was performed.
  • human PBMC Effector cell
  • tumor cells Tuget cell
  • E:T tumor cell
  • the supernatant was obtained from the cell culture solution, and 100 ⁇ l of the LDH measure reaction mixture (250 ⁇ l catalyst, 11.25 mL dye) was treated in each well, and incubated at room temperature for 30 minutes while blocking light. Then, the absorbance was measured at wavelengths of 490 nm to 492 nm and 620 nm using a microplate reader.
  • PK analysis was performed.
  • ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer 1 mg/kg was subcutaneously administered to 8-week-old male SD rats, and blood was collected every hour. confirmed through.
  • Anti-human IgG Fc antibody was coated on an ELISA plate at 4°C overnight or at 25°C for 2 hours, then treated with a blocking buffer (2.5% skim milk, 1% BSA in PBS) and at 25°C for 1 hour. During the blocking process was carried out. Thereafter, a calibration standard diluted to an appropriate concentration, QC, and rat serum samples were loaded into each well and incubated at 37° C. for 1 hour.
  • Anti-human IgG Fc-HRP antibody (Abcam, ab97225) was treated and reacted at 25° C. for 30 minutes. After that, a TMB substrate was added to cause a color reaction, and the reaction was stopped by treatment with a 0.2 NH 2 SO 4 solution, and absorbance was measured at a wavelength of 450 nm. At this time, the absorbance for the concentration of the calibration standard was obtained by obtaining a graph with a 4-parameter logistics regression equation to calculate the concentration of each sample.
  • the reason for preparing a triple-specific fusion protein compared to a single- or bi-specific antibody is to secure and safely use a drug with the disadvantages of a single- or bi-specific antibody.
  • each fusion protein dimer is prepared in a buffer containing no amine-containing chemical, treated with CFTM750 dye dissolved in DMSO, and then at room temperature for about 1 hour. It was reacted by shaking slowly. Thereafter, unbound dye was removed by centrifugation, and each dye-labeled fusion protein was diluted in PBS to a desired concentration and used. At this time, the yield of the reaction was confirmed by measuring the absorbance at a wavelength of 280 nm and a wavelength of 755 nm, which is the maximum absorption wavelength of CFTM750.
  • solid cancer-induced mice were prepared by transplanting the C4 cell line into 8-week-old male SD rats. Solidity-induced mice were stained with hyFc-IL2v fusion protein dimer, hyFc-IL2wt fusion protein dimer, or ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer, and then subcutaneously administered once at 1 mg/kg. Two days later, solid cancer-induced mice were sacrificed and the amount of fusion protein dimers accumulated in each tissue was compared.
  • the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer was accumulated in the tumor tissue (C4), and the degree of accumulation in the lung, liver, kidney and spleen was lower than that of the control group. (Fig. 10). Through this, it was confirmed that the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer can lower the toxicity that may be caused by accumulation in the lung, liver, and spleen.
  • C57BL/6 mice were transplanted with the C4 cell line expressing human-derived CEA and PD-L1 prepared in Example 5 to prepare solid cancer-inducing mice.
  • the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer was subcutaneously administered to the solid cancer-induced mice at 5 mg/kg.
  • NK cells showed the greatest increase and decrease in blood and spleen about 5 days after injection of ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer. then decreased.
  • CD8+ T cells showed the greatest increase on the 5th day in the blood and spleen, and then decreased slowly thereafter, and it was confirmed that the increased state continued until the 9th day in the tumor tissue.
  • Treg also showed an increase on the 3rd day, but it was not a significant result (FIG. 11).
  • the anticancer effect of the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer was confirmed using solid cancer-induced mice. Specifically, after transplanting the C4 cell line expressing human-derived CEA and PD-L1 prepared in Example 5 with a number of 5x10 5 cells into the right flank of a mouse, 5 mg of ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer The tumor size and absolute lymphocyte count over time were measured in the group subcutaneously injected once at /kg and in the group repeated twice a week. At this time, the group administered with PBS was set as a negative control.
  • the tumor size of the group administered with the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer was reduced, and in particular, the tumor size of the group administered repeatedly was significantly reduced.
  • the absolute lymphocyte count of the group administered with the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer was increased compared to the negative control group ( FIG. 12 ).
  • the ⁇ CEA- ⁇ PDL1-hyFc-IL2v fusion protein dimer exhibited excellent anticancer effect.

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Abstract

La présente invention concerne une protéine de fusion comprenant un anticorps anti-TAA, un anticorps anti-PD-L1 et IL-2 et les utilisations correspondantes, la protéine de fusion : présentant une excellente affinité de liaison à l'antigène carcino-embryonnaire (ACE), qui est un antigène associé à une tumeur et à PD-L1, qui est une passerelle immunitaire ; pouvant se lier plus particulièrement à des cellules tumorales exprimant ACE et/ou PD-L1 ; pouvant induire la prolifération de lymphocytes T et de cellules NK et activer des cellules immunitaires ; et pouvant présenter d'excellents effets anticancéreux lorsqu'elle est administrée à des souris induites par le cancer et pouvant ainsi être avantageusement utilisée dans le traitement de maladies cancéreuses.
PCT/KR2021/001054 2020-01-31 2021-01-27 Protéine de fusion comprenant un anticorps anti-taa, un anticorps anti-pd-l1 et il-2 et les utilisations correspondantes Ceased WO2021153979A1 (fr)

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KR20180119135A (ko) * 2017-04-24 2018-11-01 주식회사 제넥신 4-1bbl 변이체 및 이를 포함하는 융합 단백질
WO2019147837A2 (fr) * 2018-01-24 2019-08-01 Beijing Percans Oncology Co. Ltd. Protéines de fusion de cytokines

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