WO2020175886A1 - 항-ang2 항체 및 이의 용도 - Google Patents

항-ang2 항체 및 이의 용도 Download PDF

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WO2020175886A1
WO2020175886A1 PCT/KR2020/002687 KR2020002687W WO2020175886A1 WO 2020175886 A1 WO2020175886 A1 WO 2020175886A1 KR 2020002687 W KR2020002687 W KR 2020002687W WO 2020175886 A1 WO2020175886 A1 WO 2020175886A1
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seq
antibody
light chain
heavy chain
variable region
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PCT/KR2020/002687
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English (en)
French (fr)
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남주령
변상순
고종일
김도윤
이주형
하정민
박천호
이은아
이원섭
유진산
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주식회사 파멥신
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Priority to CA3131250A priority Critical patent/CA3131250A1/en
Priority to SG11202108832XA priority patent/SG11202108832XA/en
Priority to US17/429,208 priority patent/US20220204603A1/en
Priority to JP2021550123A priority patent/JP7177284B2/ja
Priority to EP20762642.5A priority patent/EP3932946A4/en
Priority to CN202080028107.0A priority patent/CN113728004A/zh
Priority to AU2020227580A priority patent/AU2020227580A1/en
Publication of WO2020175886A1 publication Critical patent/WO2020175886A1/ko

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    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors

Definitions

  • the present invention relates to an antibody that specifically binds to and inhibits function by angiopoietin-2 (Angiopoietin-2; Ang-2).
  • Angiogenesis refers to the mechanism by which new blood vessels are created from existing blood vessels, and is known to play an important role in organ formation, normal physiological growth, and wound healing.
  • tumor growth and metastasis It is known to play an important role, and abnormal angiogenesis is known to play a crucial role in diseases such as tumor growth and metastasis, age-related macular degeneration, diabetic retinopathy, psoriasis, rheumatoid arthritis, and chronic inflammation.
  • ASD Age-related Macular Degeneration
  • DR diabetic retinopathy
  • Diabetic Macular Edema DME
  • macular degeneration and diabetic retinopathy are major causes of blindness worldwide.
  • VEGF Vascular Endothelial Growth Factor
  • ANG2 Engiopoietin 2
  • ANG2 binds to the Tie2 receptor in the endothelial cells of the blood vessel wall. It is known as a cytokine that promotes neovascularization.
  • ANG2 binds to the Tie2 receptor in the endothelial cells of the blood vessel wall. It is known as a cytokine that promotes neovascularization.
  • ANG2 binds to the Tie2 receptor in the endothelial cells of the blood vessel wall. It is known as a cytokine that promotes neovascularization.
  • suppression of ANG2 signal suppresses intratumoral angiogenesis, thereby exerting anticancer effects.
  • the expression of ANG is known to exhibit anticancer effects. It is known that it is highly present in the waterproofing fluid of the sexual patient's eye.
  • Angiopoietin-2 is an antagonistic ligand of Tie2, a receptor present in vascular endothelial cells.
  • Angiopoietin-l (Angiopoietin-2) is an agonist of Tie2.
  • Angiopoietin-l; Angl) competes for Tie2 binding, thereby inhibiting signal transduction by Tie2.
  • Angiopoietin-l; Angl a ligand that activates Tie2 receptors, stabilizes blood vessels by maintaining the barrier function of vascular endothelial cells ( It acts as a key regulator that maintains stabilization.
  • vascular endothelial cells In the state of overexpression or inflammation of VEGF, vascular endothelial cells
  • Angl promotes the junctional integrity of vascular endothelial cells, thereby inducing stabilization of vascular endothelial cells and reducing vascular permeability, while Ang2 increased in activated vascular endothelial cells binds to Tie-2. By doing so, it is involved in the movement of vascular endothelial cells and tip formation, which in turn promotes the formation of new blood vessels.
  • Ang-2 contributes to the formation of angiogenesis in cancer tissues.
  • cancer tissues cooption occurs in which cancer cells select existing blood vessels for the formation of angiogenesis. Then, in the Ang-2 pathway, angiogenesis occurs. Due to the deterioration of the blood vessels, which destroys the function of the existing blood vessels, the environment within the cancer tissues is
  • VEGF vascular endothelial growth factor
  • the present inventors developed an anti-Show 13 ⁇ 42 antibody that exhibits a binding force for the purpose of Show 13 ⁇ 42, and these anti-Show 13 ⁇ 42 antibodies have been developed.
  • the purpose of the present invention is to provide a novel antibody or antigen-binding fragment thereof against 1 ⁇ 2 2 of the show.
  • Another object of the present invention is to provide a nucleic acid encoding the antibody or antigen-binding fragment thereof.
  • Another object of the present invention is to provide a vector containing the nucleic acid, a cell transformed with the vector, and a manufacturing method thereof.
  • Another object of the present invention is to provide an angiogenesis inhibitor comprising the antibody or antigen-binding fragment thereof and a composition for the treatment of diseases associated with angiopoietin-2 activation and overproduction.
  • Another object of the present invention is to provide an angiogenesis inhibitor comprising the antibody or an antigen-binding fragment thereof and a composition for diagnosis of diseases associated with angiopoietin-2 activation and overproduction.
  • Another object of the present invention is to provide a composition for the prevention or treatment of ocular diseases comprising the antibody or its antigen-binding fragment.
  • Another object of the present invention is to provide a composition for the prevention or treatment of tumors or cancers comprising the antibody or antigen-binding fragment thereof.
  • Another object of the present invention is to provide a composition for co-administration with the above antibody or the show 13 ⁇ 42 antibody or the show13 ⁇ 42 antibody containing the antibody or its antigen-binding fragment.
  • the present invention is a heavy chain selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19 and 25
  • Heavy chain 0 is 12 selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20 and 26, and
  • a heavy chain variable region comprising a heavy chain 00113 selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 51, 52 and 53, and
  • Light chain 0 is 11 selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22, and 28,
  • Light chain 00112 selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23 and 29, and
  • the present invention also contains the nucleic acid encoding the antibody or antigen-binding fragment thereof.
  • the present invention also provides a vector containing the nucleic acid.
  • the present invention also provides a cell transformed with the vector.
  • the present invention also provides a method for preparing the antibody or antigen-binding fragment thereof, comprising the following steps: (a) culturing the cell; and (b) the antibody or its antibody in the cultured cell. The step of recovering the antigen-binding fragment.
  • the present invention also provides an angiogenesis inhibitor comprising the antibody or an antigen-binding fragment thereof, and a composition for treatment of diseases associated with angiopoietin-2 activation and or overproduction.
  • An angiogenesis inhibitor comprising an antibody or an antigen-binding fragment thereof and a composition for diagnosis of a disease associated with angiopoietin-2 activation and or overproduction is provided.
  • the present invention also provides a tumor or a tumor comprising the antibody or an antigen-binding fragment thereof. It provides a composition for preventing or treating cancer.
  • the present invention also provides a composition for preventing or treating ocular diseases comprising the antibody or an antigen-binding fragment thereof.
  • the present invention also provides a composition for preventing or treating an eye disease.
  • the present invention also provides the antibody or an antigen-binding fragment thereof. It provides a composition for co-administration with Ang2 antibody or Ang2 antibody containing.
  • FIG. 1 is a result of confirming that the selected monoclonal scFv phage has the ability to inhibit Ang2/Tie2 binding.
  • FIG. 2 is an SDS-PAGE result of selected anti-Ang2 antibodies under reduction and non-reduction conditions for products after temporary expression and purification.
  • Fig. 3 is an ELISA result of evaluating the binding of Ang2 and Ang2 to human and mouse of the transient expression and purification anti-Ang2 antibody.
  • Fig. 4 is a result showing the ability of selected anti-Ang2 antibodies to neutralize human and mouse Ang2ATie2 binding.
  • Fig. 5 is a result showing the ability of the selected anti-Ang2 antibodies to neutralize the binding of Ang2/integrin.
  • Figure 6 shows that the selected anti-Ang2 antibody can inhibit Ang2/Tie2 signal transmission.
  • Figure 7 is the result of confirming the purity of each purification step by expressing the selected anti-Ang2 scFv antibody in E. coli.
  • Figure 8 is an anti-Ang2 scFv antibody expressed in Escherichia coli against human Ang2 protein
  • Figures 9 to 11 are the results of confirming the in vivo efficacy of the selected anti-Ang2 ScFv antibodies in a CNV mouse model.
  • heavy chain 00111 selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19 and 25,
  • SEQ ID NO: 2, 8, 14, 20 and 26 heavy chain selected from the group consisting of 12, and
  • a heavy chain variable region comprising a heavy chain 00113 selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 51, 52 and 53, and
  • Light chain 0 is 11 selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22, and 28,
  • Light chain 00112 selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23 and 29, and
  • An antibody that binds to a light chain variable region comprising a light chain 00113 selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24 and 30, or It is about its antigen-binding fragment.
  • antibody refers to an anti-show 1 ⁇ 2 antibody that specifically binds to Show 13 ⁇ 42. In the scope of the present invention, not only the complete antibody form that specifically binds to Show 13 ⁇ 42, but also the antibody molecule described above It also includes a pseudo antigen-binding fragment.
  • a complete antibody has two full-length light chains and two full-length heavy chains, each of which is linked by a heavy chain and a disulfide bond.
  • the heavy chain constant region is gamma, etc.), Mu (B), and Subclasses with alpha ( «), delta (6) and Msilon 4 types are gamma 1 external 1), gamma 2 external 2), gamma 3 (work), gamma 4 external 4), alpha 1 (x1) and alpha. It has 2 ((x2).
  • the constant region of the light chain has kappa ( ⁇ and lambda (X) types).
  • Antigen-binding fragments or antibody fragments of an antibody refer to fragments that have an antigen-binding function, and include M, M, M 2 and the like. Is the variable region of the light and heavy chain and the constant region of the light chain and the first of the heavy chain.
  • M ⁇ ’ is a hinge region containing one or more cysteine residues at the (:-end) of the 011 domain of the heavy chain (13 ⁇ 4
  • Cysteine residues in the domain are produced by disulfide bonds. Is the smallest antibody fragment that has only the heavy chain variable region and the light chain variable region.
  • the double chain 1 0; ⁇ 0-(;1 111 1) is a non-covalent bond to which the heavy chain variable region and the light chain variable region are connected, and the short chain 1 ( In general, 13 ⁇ 416-11 1, 1 ⁇ ) can form a dimer-like structure like a double chain because the variable region of the heavy chain and the variable region of the light chain are covalently linked through a peptide linker or (:-terminal).
  • Such antibody fragments can be obtained by using protein hydrolase (for example, by limiting the whole antibody to papain, you can obtain the protein, and by cutting with pepsin, you can obtain the 2 fragment), and genetic recombination technology. It can also be produced through [56]
  • the antibody according to the present invention is in the form of Fv (eg, scFv) or is in the form of a complete antibody.
  • the heavy chain constant region is gamma (Y), mu (B), alpha (a), Either isotype can be selected from Delta (6) or Amsilon 4, for example, the constant region is gamma l (IgGl), gamma 3 (IgG3) or gamma 4 (IgG4); the light chain constant region is kappa or Can be lambda type.
  • variable region domain VH containing an amino acid sequence with a sufficient variable region sequence and a full-length heavy chain containing the three constant region domains CH1, CH2 and CH3 and fragments thereof are all meant.
  • ⁇ Light chain'' refers to both the full-length light chain and fragments thereof, including the variable region domain VL and the constant region domain CL, containing an amino acid sequence with sufficient variable region sequences to confer specificity on the antigen.
  • Antibodies of the present invention are monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFV), single chain antibodies, Fab fragments, F(ab') fragments,
  • Disulfide-binding Fvs sdFV
  • anti-idiotype antibodies or epitope-binding fragments of these antibodies, etc. are included, but are not limited thereto.
  • the monoclonal antibody refers to an antibody obtained from a substantially homogeneous group of antibodies, that is, the same, except for possible naturally occurring mutations in which the individual antibodies occupying the group may exist in trace amounts.
  • Monoclonal antibodies are highly specific and highly specific. It is an enemy and is induced against a single antigenic site. Typically, it contains different antibodies directed against different determinants (epitopes).
  • each monoclonal antibody is directed against a single determinant on the antigen.
  • This “epitope” is a protein-determining site where antibodies can specifically bind
  • Epitopes are usually composed of a group of chemically active surface molecules, such as amino acids or sugar side chains, and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. Stereoscopic epitopes and non-stereoscopic epitopes The epitope is distinguished in that the bond to the former is lost in the presence of a denaturing solvent, but not to the latter.
  • the "humanized” form of non-human (eg murine) antibody is a non-human
  • humanized antibodies are non-human species (donor antibodies), e.g. mice, rats, rabbits or non-humans that possess the desired specificity, affinity, and ability to retain residues from the hypervariable regions of the recipient. It is a human immunoglobulin (receptor antibody) that has been replaced by a residue from the hypervariable region of primates.
  • donor antibodies e.g. mice, rats, rabbits or non-humans that possess the desired specificity, affinity, and ability to retain residues from the hypervariable regions of the recipient. It is a human immunoglobulin (receptor antibody) that has been replaced by a residue from the hypervariable region of primates.
  • the "human antibody” is a molecule derived from human immunoglobulin, and it means that all amino acid sequences constituting the antibody including the complementarity determining region and structural region are composed of human immunoglobulin. 2020/175886 1»(:1 ⁇ 1 ⁇ 2020/002687
  • heavy and/or light chains are the same or homologous to the corresponding sequence in an antibody that is derived from a particular species or belongs to a particular antibody class or subclass, while the rest of the chain(s) is derived from another species or is another antibody. Same or homologous to the corresponding sequence in an antibody belonging to the class or subclass
  • chimeric antibodies immunoglobulins
  • fragments of these antibodies that exhibit a desired biological activity.
  • an ⁇ antibody variable domain'' refers to a complementarity determining region (0011; that is, 00111, 00112, and 00113), and the light and heavy chain portions of an antibody molecule comprising the amino acid sequence of the skeletal region.
  • ⁇ 3 ⁇ 4 refers to the variable domain of the heavy chain.
  • the silver light chain refers to the variable domain.
  • the "complementarity determining region" ⁇ 011 refers to the amino acid residue of the antibody variable domain, which is an entity necessary for antigen binding. Each variable domain is typically, It has three regions identified as 00112 and 00113.
  • the present invention includes a heavy chain variable region comprising heavy chain 00113 of SEQ ID NO: 1 and a light chain variable region comprising light chain 00yo3 of SEQ ID NO: 2.
  • the antibody or antigen-binding fragment thereof that binds to the show 13 ⁇ 42
  • Heavy chain 0 of SEQ ID NO: 1 Heavy chain 0 of 11, heavy chain 0 of SEQ ID NO: 2, heavy chain variable region comprising heavy chain 00113 of SEQ ID NO: 3, of SEQ ID NO: 4
  • Heavy chain variable region comprising 00113, light chain 00111 of SEQ ID NO: 16, SEQ ID NO: 17
  • a light chain variable region comprising the light chain 00113 of 18,
  • a light chain variable region comprising light chain 00112, and light chain 00113 of SEQ ID NO: 24, the heavy chain of SEQ ID NO: 25 heavy chain 00112 of SEQ ID NO: 26, and heavy chain of SEQ ID NO: 27
  • the heavy chain variable region containing 00113, the light chain of SEQ ID NO: 28 of SEQ ID NO: 29 Light chain variable region comprising, 1] heavy chain 00111 of SEQ ID NO: 13, heavy chain 00112 of SEQ ID NO: 14 and heavy chain of SEQ ID NO: 51
  • Heavy chain variable region comprising 00113, light chain 00111 of SEQ ID NO: 16, SEQ ID NO: 17
  • the "IV' fragment is an antibody fragment containing a complete antibody recognition and binding site.
  • the “me” fragment is the variable and constant domain of the light chain and the variable and first constant domain of the heavy chain.
  • Me(')2 antibody fragments usually contain a pair of mesoporous fragments that are covalently linked near their carboxy ends by hinge cysteines between them.
  • It may further include a polypeptide linker between.
  • Show 13 ⁇ 42 antibody may contain single chain or double chain. Functionally, the binding affinity of Show13 ⁇ 42 antibody is within the range of 10 ⁇ to W- 12 M. For example, the binding affinity of Show 13 ⁇ 42 antibody is 10 6 M. To 10 12 10 7 M to 10 12 10 8 M to 10 12 10 9 M to
  • the antibody or antigen-binding fragment thereof that binds to the show 13 ⁇ 42 may include a heavy chain variable region selected from the group consisting of SEQ ID NOs: 32, 36, 40, 44, 48, 55, 57, 59 and 61.
  • the antibody or antigen-binding fragment thereof that binds to the show 13 ⁇ 42 is SEQ ID NO: 34, 38,
  • It may contain a light chain variable region selected from the group consisting of 42, 46 and 50.
  • It may include a heavy chain variable region of 61 and a light chain variable region of SEQ ID NO: 42.
  • phage display lies in the fact that it can quickly and efficiently classify sequences that bind with high affinity to the target antigen by targeting a large library of randomized protein variants.
  • Peptide and protein libraries are phage imaged. Display on has been used to screen millions of polypeptides to identify polypeptides with specific binding properties.
  • This phage display technology provided a powerful tool for the generation and selection of novel proteins that bind to specific ligands (eg, antigens).
  • a large library of protein variants is generated, and target antigens and Sequences that bind with high affinity can be quickly classified.
  • Nucleic acid encoding a variant polypeptide is fused with a nucleic acid sequence encoding a viral envelope protein, e.g. gene III protein or gene VIII protein.
  • Encoding protein or polypeptide A monophasic phage display system was developed in which a nucleic acid sequence was fused with a nucleic acid sequence encoding a part of gene III protein. In the monophage display system, the gene fusion is expressed at a low level, and the wild type gene III protein is also expressed, so that particle infectivity is maintained.
  • Libraries were prepared in a number of ways, for example, by inserting a random DNA sequence to alter a single gene or by cloning a related gene line. Targeting the library, antibodies or antigen-binding proteins carrying the desired characteristics Can be screened for the expression of
  • Phage display technology has several advantages over conventional hybridoma and recombination methods for producing antibodies with the desired characteristics. These technologies do not use animals and have a large variety of sequences in a short time. Make it possible to generate antibody libraries. The production of hybridomas or the production of humanized antibodies may require several months of production period. Also, since immunity is not required at all, phage antibody libraries are toxic or low-antigen antigens. Antibodies can also be generated for the phage antibody library. 2020/175886 1»(:1 ⁇ 1 ⁇ 2020/002687
  • New therapeutic antibodies can be generated and identified.
  • lymphoid tissues can be used to create unsensitized or non-immune antigen-binding libraries.
  • the versatility of the library may depend on the size of the library.
  • the size of the library is reduced by improper folding of the antibody or antigen-binding protein and by inefficient production due to the presence of zicodons.
  • Expression in bacterial cells is expressed in the antibody or antigen-binding domain as appropriate. If not folded, it can be inhibited. Expression can be improved by alternating residues at the surface of the variable/constant interface or at the selected residues.
  • the sequence of the skeletal region can be obtained from the antibody phage library in bacterial cells.
  • Diversity can be created by randomizing areas. The use of all 20 amino acids creates a highly diverse variant antibody sequence and increases the chances of identifying new antibodies.
  • the anti-show 1 ⁇ 2 2 antibody of the present invention may include not only the sequence of the anti-show 1 ⁇ 2 2 antibody of the present invention described in this specification, but also its biological equivalents, for example, to further improve the binding affinity of the antibody and/or other biological properties.
  • modifications may include, for example, deletions, insertions and/or substitutions of amino acid sequence residues of the antibody.
  • amino acid mutations may have the relative similarity of amino acid side chain substituents, e.g., hydrophobicity, hydrophilicity.
  • arginine, lysine and histidine are all positively charged residues; alanine, glycine and serine have similar sizes; phenylalanine, It can be seen that trimtophan and tyrosine have a similar shape; therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, trimtophan and tyrosine are biologically functional equivalents. have.
  • the above practical identity is at least 90% homology when the sequence of the present invention and any other sequence described above are aligned to the maximum possible correspondence, and the aligned sequence is analyzed using an algorithm commonly used in the industry. , Most preferably, it refers to a sequence that shows a homology of at least 95%, 96% or more, 97% or more, 98% or more, 99% or more.
  • Alignment methods for sequence comparison are well known in the art. NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NCBI, etc., and sequence analysis such as blastp, blasm, blastx, tblastn and tblastx on the Internet
  • the antibody of the present invention or its antigen-binding fragment is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% compared to the specified sequence or the whole described in the specification. , 98%, 99%, or more homology.
  • homology can be determined by sequence comparison and/or alignment by methods known in the art. For example, sequence comparison algorithms (i.e. BLAST) Alternatively, BLAST 2.0), manual alignment, and visual inspection can be used to determine the percent sequence homology of the nucleic acid or protein of the present invention.
  • the antibody of the present invention or the nucleic acid encoding its antigen-binding fragment can be separated to produce the antibody or its antigen-binding fragment recombinantly.
  • the nucleic acid is isolated, inserted into a replicable vector, and further cloned ( DNA amplification) or additional expression. Based on this, the present invention relates to a vector containing the nucleic acid from another point of view.
  • Nucleic acid'' has the meaning of comprehensively including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units in nucleic acids, are not only natural nucleotides, but also analogs with modified sugar or base sites (analogue ).
  • the sequence of nucleic acids encoding the heavy and light chain variable regions of the present invention may be modified. The modification includes addition, deletion, or non-conservative or conservative substitution of nucleotides.
  • the nucleic acid is a heavy chain variable region selected from the group consisting of SEQ ID NOs: 31, 35, 39, 43, 47, 54, 56, 58, 60 encoding the heavy chain variable region
  • the nucleic acid may include a light chain variable region selected from the group consisting of SEQ ID NOs: 33, 37, 41, 45 and 49 encoding the light chain variable region.
  • nucleic acid and light chain of SEQ ID NO: 31 encoding the heavy chain variable region 2020/175886 1»(:1 ⁇ 1 ⁇ 2020/002687
  • nucleic acid of SEQ ID NO: 35 encoding the heavy chain variable region and the nucleic acid of SEQ ID NO: 37 encoding the light chain variable region;
  • nucleic acid of SEQ ID NO: 39 encoding the heavy chain variable region and the nucleic acid of SEQ ID NO: 41 encoding the light chain variable region;
  • nucleic acid of SEQ ID NO: 43 encoding the heavy chain variable region and the nucleic acid of SEQ ID NO: 45 encoding the light chain variable region;
  • nucleic acid of SEQ ID NO: 47 encoding the heavy chain variable region and the nucleic acid of SEQ ID NO: 49 encoding the light chain variable region;
  • nucleic acid of SEQ ID NO: 54 encoding the heavy chain variable region and the nucleic acid of SEQ ID NO: 41 encoding the light chain variable region;
  • nucleic acid of SEQ ID NO: 56 encoding the heavy chain variable region and the nucleic acid of SEQ ID NO: 41 encoding the light chain variable region;
  • nucleic acid of SEQ ID NO: 58 encoding the heavy chain variable region and the nucleic acid of SEQ ID NO: 41 encoding the light chain variable region;
  • nucleic acid of SEQ ID NO: 60 encoding the heavy chain variable region and the nucleic acid of SEQ ID NO: 41 encoding the light chain variable region may be included.
  • the DNA encoding the antibody can be specifically bound to the DNA encoding the heavy and light chains of the antibody using a conventional process.
  • Vector components generally include, but are not limited to, one or more of the following: signal sequence, replication origin, one or more marker genes, enhancement factor elements, promoters, and transcription termination sequences.
  • vector used in this specification refers to the gene of interest in the host cell.
  • Means for expression include plasmid vectors; cosmid vectors; bacteriophage vectors, adenovirus vectors, retroviral vectors, and viral vectors such as adeno-associated virus vectors.
  • the nucleic acid encoding an antibody in the vector is a nucleic acid encoding an antibody.
  • “Operatively linked” refers to a sequence that regulates nucleic acid expression (eg, a promoter, a signal sequence, or
  • Promoter eg Promoter, 1 promoter, 1 11 ⁇ 5 promoter, lpp promoter, Promoter, 8-6 promoter, promoter and 17 promoter, etc.
  • ribosome binding sites for initiation of translation, and transcription/translation termination sequences are generally included.
  • eukaryotic cells as the host, Derived from the genome of mammalian cells 2020/175886 1»(:1 ⁇ 1 ⁇ 2020/002687
  • Promoter e.g. metallotionine promoter, (3-actin promoter, human heroglobin promoter and human muscle creatin promoter) or promoter derived from mammalian virus (e.g. adenovirus late promoter, vaccinia virus 7.5 promoter, promoter) , Cytomegalovirus (CMV) promoter, medical Promoter, mouse mammary tumor virus (MMTV) promoter, 1 3/4 promoter of Moloney virus, promoter of Msteinva virus ⁇ 6 ⁇ 0 and Rous Sarco virus (promoter of 1 « ⁇ 0) can be used, It generally has a polyadenylation sequence as a transcription termination sequence.
  • mammalian virus e.g. adenovirus late promoter, vaccinia virus 7.5 promoter, promoter
  • CMV Cytomegalovirus
  • medical Promoter e.g. adenovirus late promoter, vaccinia virus 7.5 promoter, promoter
  • MMTV mouse mammary tumor virus
  • vectors are used to facilitate purification of antibodies expressed therefrom.
  • sequence to be fused is, for example, glutathione-binding protein.
  • the present invention relates to cells transformed with the vectors mentioned above.
  • the cells used to generate the antibodies of the present invention may be prokaryotic, yeast or higher eukaryotic cells, limited to this. It doesn't work.
  • Bacillus strains such as Juryngensis, Streptomyces (Wa and Mygo, Pseudomonas (8 mountains 011101 8) (for example, Pseudomonas putida (8 mountains 011101 8) 31 (Urine)), Proteus Mirabilis 11 3 ⁇ 41 8) and
  • Staphylococcus (81)11) 400 ⁇ 018) (For example, Staphylococcus
  • Prokaryotic host cells such as Carnosus (81) 11) 40 to 8 (Pyo Mi Ye-ae)) can be used.
  • the present invention relates to a method for producing the antibody or antigen-binding fragment thereof, comprising the step of culturing the cells; and) recovering the antibody or antigen-binding fragment thereof from the cultured cells. It's about.
  • the cells can be cultured in various media. Among commercial media, they can be used as culture media without limitation. All other essential supplements known to the person skilled in the art may be included in an appropriate concentration. Culture conditions, for example temperature , Etc. are already being used with host cells selected for expression, which will be apparent to those skilled in the art. [124] The recovery of the antibody or its antigen-binding fragments removes impurities, for example by centrifugation or ultrafiltration, and makes the resultant, for example, friendly.
  • the present invention relates to a composition for the prevention or treatment of tumors containing the antibody as an active ingredient.
  • the antibody may be an IgG or a fragment containing a variable region, namely ScFv, Fab.
  • the heavy chain The variable region may be IgGl, IgG2, IgG3, or IgG4.
  • the present invention includes, for example, (a) a pharmaceutically effective amount of an antibody against Ang2 or an antigen-binding fragment thereof according to the present invention; and (b) the prevention or treatment of eye diseases including a pharmaceutically acceptable carrier.
  • the present invention may also be a method for preventing or treating an ocular disease comprising administering the antibody or antigen-binding fragment thereof to a tumor patient.
  • the present invention is furthermore, the antibody or its antigen-binding fragment.
  • the antigen-binding fragment may be used for interfering with the mechanism of Ang2 and for preventing or treating eye diseases through this.
  • the cornea is an avascular tissue and must always remain transparent to preserve vision.
  • angiogenesis is known to appear in the eye as well, causing angiogenesis-related diseases of the eyeball.
  • Angiogenesis in the cornea impairs the transparency of the eye, leading to a loss of vision, and angiogenesis in the retina produces abnormal blood vessels, resulting in blood bleeding, leading to blindness through degeneration of the retinal cells.
  • the present invention provides eye diseases such as retinopathy of prematurity,
  • corneal neovascularization e.g., diabetic retinopathy
  • choroidal neovascular disease e.g., macular degeneration
  • the present invention includes, for example, (a) a pharmaceutically effective amount of an antibody against Ang2 or an antigen-binding fragment thereof according to the present invention; and (b) the prevention of tumors or cancers containing a pharmaceutically acceptable carrier, or
  • the present invention may be a therapeutic pharmaceutical composition.
  • the present invention may also be a method for preventing or treating a tumor or cancer comprising administering the antibody or antigen-binding fragment thereof to a tumor or cancer patient.
  • Antibodies or antigen-binding fragments thereof may be used for inhibiting the mechanism of Ang2 and for preventing or treating tumors or cancers through them.
  • the disease applied to the above composition, tumor or cancer is typically Ang2.
  • tumors or cancers that are desirable for treatment are melanoma (e.g. metastatic malignant melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g. hormone refractory prostate carcinoma). ), Pancreatic Adenocarcinoma, Breast Cancer (In some cases, Triple Negative Breast Cancer, Colon Cancer, Lung Cancer (for example, non-small cell lung cancer), 2020/175886 1»(:1 ⁇ 1 ⁇ 2020/002687
  • Esophageal cancer head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer,
  • the present invention includes refractory or recurrent cancers that can be treated using the antibodies of the present invention.
  • the present invention relates to an angiogenesis inhibitory pharmaceutical composition
  • an angiogenesis inhibitory pharmaceutical composition comprising the above anti-show 1 ⁇ 2 antibody or its antigen-binding fragment as an active ingredient.
  • Another example is the anti-show 1 ⁇ 2 antibody or the above anti-show 1 ⁇ 2 antibody or It provides a pharmaceutical composition for the prevention and/or treatment of diseases related to angiopoietin-2 activation and/or overproduction comprising an antigen-binding fragment thereof as an active ingredient.
  • the present invention provides a method for inhibiting angiogenesis, comprising, for example, administering a therapeutically effective amount of the anti-show 1 ⁇ 2 antibody or antigen-binding fragment thereof to a patient in need of inhibition of angiogenesis.
  • the angiogenesis inhibitory method may further include the step of identifying a patient in need of angiogenesis inhibition prior to the administration step.
  • treatment of the anti-show 1 ⁇ 2 antibody or antigen-binding fragment thereof Administering an appropriate effective amount to a patient in need of prevention and/or treatment of a disease associated with angiopoietin-2 activation and/or overproduction,
  • a method for preventing and/or treating diseases associated with angiopoietin-2 activation and/or overproduction includes angiopoietin-2 activation and/or overproduction prior to the administration step. It may further include steps to identify patients in need of prevention and/or treatment of diseases associated with production.
  • the pharmaceutical composition may additionally contain a pharmaceutically acceptable carrier, and the carrier is commonly used in the formulation of drugs, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, Calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate,
  • a pharmaceutically acceptable carrier is commonly used in the formulation of drugs, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, Calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate,
  • compositions may be one or more selected from the group consisting of propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc., but is not limited thereto.
  • the above pharmaceutical compositions are also diluents, excipients, and lubricants commonly used in the manufacture of pharmaceutical compositions.
  • Wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents may further include one or more selected from the group consisting of preservatives.
  • the effective amount of the pharmaceutical composition, or the antibody or antigen-binding fragment thereof can be administered orally or parenterally.
  • parenteral administration intravenous, subcutaneous, intramuscular, intraperitoneal, endothelial, and topical administration It can be administered by intranasal administration, intrapulmonary administration, and rectal administration, etc.
  • oral compositions can be formulated to be coated with an active agent or to protect against digestion in the stomach.
  • the composition can be administered by any device capable of moving the active substance to the target cell.
  • the content of anti-show 13 ⁇ 42 antibody or antigen-binding fragment thereof in the pharmaceutical composition is 2020/175886 1»(:1 ⁇ 1 ⁇ 2020/002687
  • Formulation method, administration method, patient's age, weight, sex, morbidity, food, administration time, administration interval, route of administration, excretion rate and response sensitivities can be prescribed in various ways.
  • the daily dose of 13 ⁇ 42 antibody or antigen-binding fragment thereof is 0.001 to 100013 ⁇ 4/13 ⁇ 4, specifically 0.01 to 10013 ⁇ 4/13 ⁇ 4, more specifically 0.1 In general, it may be in the range of 0.1 to 20 !3 ⁇ 4/!3 ⁇ 4, but is not limited thereto.
  • the daily dosage is formulated as a single formulation in unit dosage form, formulated in appropriate quantities, or manufactured by putting it in a multi-dose container. Can be
  • the pharmaceutical composition can be administered in combination with other drugs, such as other angiogenesis inhibitors or agents for treating diseases associated with angiopoietin-2 activation and/or overproduction, and the dosage, method of administration, and types of other drugs. Can be appropriately prescribed according to the patient's condition.
  • other drugs such as other angiogenesis inhibitors or agents for treating diseases associated with angiopoietin-2 activation and/or overproduction, and the dosage, method of administration, and types of other drugs. Can be appropriately prescribed according to the patient's condition.
  • the pharmaceutical composition may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be formulated in the form of extracts, powders, powders, granules, tablets, or capsules.
  • a dispersant or It may additionally contain stabilizers.
  • the anti-show 1 ⁇ 2 2 antibody or the pharmaceutical composition containing the antigen-binding fragment thereof contains an antibody or an antigen-binding fragment, it can be formulated into an immune liposome.
  • Liposomes containing antibodies can be prepared according to methods well known in the art.
  • the immune liposome is phosphatidylcholine, cholesterol, and
  • lipid composition containing polyethylene glycol-derivated phosphatidylethanolamine it can be prepared by reverse phase evaporation.
  • a fragment of the antibody is disulfide-replaced through a reaction. It can be conjugated to liposomes.
  • the anti-show 13 ⁇ 42 antibody or its antigen-binding fragment specifically binds to Angiopoietin-2, it is used to check the activation and/or overproduction of Angiopoietin-2. Therefore, another example of the present invention is angiopoietin-2 activation and/or overproduction and/or angiopoietin-2 activation and / Or providing a diagnostic pharmaceutical composition for a disease related to overproduction.
  • the biological sample may be selected from the group consisting of cells, tissues, body fluids, etc. obtained from a patient.
  • the step of determining whether the antigen-antibody reaction is performed is known in the art. For example, it can be measured through conventional enzymatic reactions, fluorescence, luminescence and/or radiation detection, specifically,
  • FI A luminescence immunoassay
  • LIA luminescence immunoassay
  • Western bloting etc.
  • FI A luminescence immunoassay
  • LIA luminescence immunoassay
  • Western bloting etc. can be measured by a method selected from the group consisting of, but is not limited thereto.
  • Patients subject to administration or diagnosis of the pharmaceutical composition may be mammals including primates including humans and monkeys, and rodents including mice and rats.
  • Diseases associated with the angiopoietin-2 activation and/or overproduction include cancer; cancer metastasis; Eye diseases such as prematurity retinopathy, corneal neovascularization, diabetic retinopathy, choroidal neovascular disease, macular degeneration (eg, age-related macular degeneration); asthma; rheumatoid arthritis; Psoriasis; Inflammatory diseases such as pneumonia, chronic inflammation; Cardiovascular disease such as hypertension, arteriosclerosis, or sepsis.
  • the cancer may be an overexpression of angiopoietin-2, and may be solid or blood cancer, but is not limited thereto.
  • the preparation used its own non-human scFv (human naive ScFv) library, such as Korean Patent Application (Publication Patent No. 10-2008-0109417).
  • 96-well immunity human scFv (human naive ScFv) library
  • the grown cells are centrifuged at 7000 rpm for 10 minutes and centrifuged once more in the same way.
  • the collected supernatant is 1/5 (v/v) of the supernatant and 20% PEG/2.5m NaCl is added and iced.
  • Discard the supernatant dissolve the precipitate with 3 ml of TBS, filter it through 0.45 [xm filter, store it in 4, and use it in the next panning process. Repeat this process 3-4 times to bind to the antigen.
  • the antibody was confirmed by performing ELISA.
  • Example 2 Monoclonal ScFv phage screening that specifically binds to Ang2 and neutralizes the binding with Tie2 (binding ELIS A/competitive ELISA) [151] The over night in the panning (panning) the end of the process the last round cell stock (round cell stock) the CM agar plate (agar plate) 200 500 colonies is junhu diluted to crushing to be formed 37 O C the next day colonies (colony) Let if it over night at 96 weldip increase rate (96 well deep plate) in 2xYT medium (CM 34 g / m ⁇ + 1% glucose) was placed 200 into Single colonies on gakwel 37 O C, 3000rpm. The next day, 2xYT medium (CM 34/ho/M + 1% glucose) in a new 96-well deep plate
  • the cells grown the next day are centrifuged at 3000 rpm for 10 minutes and stored in 4 O C.
  • the laid Ag is washed 3 times with 0.1% TBST (5 mM CaCl 2 ) and then 2% BSA blocking buffer 200 After adding each, incubate for 2 hours at 25 O C. After blocking, wash 3 times with 0.1% TBST (5 mM CaCl 2 ). In each well, wash down with 4% BSA 50 and keep at 4 O C. After mixing 50, shake for 1 hour at room temperature.
  • TMB #BD TMB substrate reagent set 555214
  • Phage binding (phage binding) At the end, after washing 3 times with 0.05% PBST, react with anti-Ang2 mouse antibody (RND, MAB098) at room temperature with 0.5 gM for 1 hour. After antibody binding is complete, wash 3 times with 0.05% PBST and then HRP-conjugated mouse -Add 100 IgG antibody 1:2000 (HRP-conjugated Goat anti-mlgG Ab) (RND, HAF007) at a time and react for 1 hour at 25°C.
  • RBD anti-Ang2 mouse antibody
  • Phagemid was extracted from the selected E. Coli clone and the variable region was amplified using a pooling method.
  • the amplified heavy chain variable region was an expression vector containing a heavy chain constant region (Invivogen, pfusess-hchgl).
  • the light chain variable region which was inserted into and amplified, was inserted into an expression vector (Invivogen, pfuse2ss-hclk) containing the light chain constant region to complete the IgG form of DNA cloning.
  • the transient expression of IgG is the Expi293F expression system kit:
  • Thermo Fisher Scientific, US was used.
  • the Expi293 cells included in the kit were cultured on a 125 rpm orbital shaker in an environment at 37°C and 5% CO2 using a dedicated medium. Subcultured to 3 X 10 5 cells/ml every 3 days, expression vector
  • the number of cells was adjusted to be 3 X 10 6 cells/ml and used.
  • an exclusive reagent Expifectamine was used, and a Lipid-DNA complex containing the expression vector DNA 1 and Expifectamine 2.7 per ml of the cell suspension was used. It was prepared and added to the cell suspension, and expression was induced by adding Enhancer 1/2 16-18 hours after the introduction. After that, after incubation for 3-4 days under the same conditions, centrifugation was performed to take an IgG-containing supernatant.
  • each antibody was treated in a non-reducing and reducing LDS sample buffer (Non-reducing and Reducing LDS sample buffer: Thermo Fisher Scientific) and subjected to electrophoresis using a NuPAGE System (Thermo Fisher Scientific).
  • a non-reducing and reducing LDS sample buffer Non-reducing and Reducing LDS sample buffer: Thermo Fisher Scientific
  • 50 kDa IgG having a total molecular weight of about 150 nos including heavy chain and 25 no light chain was obtained.
  • Ang2 protein is human (R&D systems, 923-AN/CF), mouse (sino, 50300-M07H) Add 100 or 1 Angl solution to 4 O C It was allowed to stand overnight and adsorbed. The next day, it was washed three times with PBS containing 0.05% Tween-20 (hereinafter, PBST), and a 2% BSA/PBST solution was added to each well, and then allowed to stand at room temperature for 2 hours to block.
  • PBS containing 0.05% Tween-20 hereinafter, PBST
  • BSA/PBST solution was added to each well, and then allowed to stand at room temperature for 2 hours to block.
  • each test antibody solution was added by each concentration of WO pl and bound for 1 hour at room temperature, washed three times with PBST, diluted in a ratio of 1:2000, and HRP-conjugated goat anti-human IgG (kappa ) (bethyl lab #A80-115P) was added to WO ⁇ il and reacted for 1 hour at room temperature to induce binding, and after washing three times with PBST, color development was performed using 100 TMB substrate reagent. 2 Stopped by adding S0 4 , Sunrise
  • the measurement was performed at 2000 seconds, and the mouse Ang2 was analyzed with a binding interval of 300 seconds and a dissociation interval of 1000 seconds.
  • the analysis model was analyzed using a 1:1 binding model. 5].
  • the resulting streptavidin (R&D systems, DY998) was added to 100 [ xl and reacted at room temperature for 1 hour to induce binding, and after washing 4 times with PBST, 100 TMB substrate reagent was used to develop color.
  • the color reaction was 50 of 2N H 2
  • S0 4 was stopped, and specific absorbance OD 45 ° was measured using a Sunrise microplate reader (TECAN, CH) (FIGS. 4 and 5).
  • the selected antibodies were human and human.
  • Ang2/Tie2 binding was neutralized in all mice.
  • the neutralization ability was quantified by obtaining 1C value, and it is shown in [Table 6].
  • the binding of integrin/Ang2 was also neutralized.
  • the neutralization ability of integrin/Ang2 was digitized by obtaining an IC value of 5. [ It is indicated in the notation.
  • Phospho-Tie2 Duoset IC ELISA (R&D systems, DYC2720-5) was used. After adding 4 human Tie2 capture proteins to each well of a 96-well immunoplate (Nunc, US)
  • the lysis buffer of (PMSF) was resuspended using W ml buffer per gram of cell weight. Power: 20W, rest: 3Sec, Work: 3Sec, Time:
  • the cells were crushed under the condition of 5 Min.
  • the crushed cells were centrifuged for 1 hour in 1 WOOrpm to separate the supernatant and the precipitated material.
  • ScFv was expressed in an insoluble form, and pellet wash was performed to proceed with refolding. 50 mM Tris-Hcl pH 7.4, 150 mM NaCl buffer
  • the pellets were washed twice by centrifuging lOOOrpm and lh. E. coli-derived substances remaining in the pellet were removed using 50 mM Tris-Hcl pH 7.4, 150 mM NaCl, 2 M Urea, and 0.5% Triton X-100 buffer, and 50 mM Tris-Hcl pH 7.4, 150 mM NaCl buffer. Washing was repeated 3 times.
  • the inclusion body After resuspension of the inclusion body with 50 mM Tris-Hcl pH 7.4, 150 mM NaCl, 8 M Urea, and 10 mM DTT buffer, the inclusion body was reacted for about 30 min to make the ScFv unfolded and separated from the sediment by lOOOrpm and lh Centrifuge.
  • Dialysis buffer is based on 50 mM Tris-Hcl pH 7.4 and 150 mM NaCl.
  • the Urea concentration was decreased by 1/2.
  • 0.1 M L-Arginine was added to suppress the formation of aggregation.
  • the system was used for purification, and 5 ml HisTrap Packing Column was used.
  • the ScFv antibody spring water flowed through the Histrap column at a flow rate of 5 ml/min to bind to the resin in the column. .
  • the concentration gradient was made so that the ScFv antibody was elution.
  • the anti-Ang2 antibody was immobilized on a biosensor, and the binding kinetics of human Ang2 by concentration were measured to measure the binding rate constant (k a ), the dissociation rate constant (k dis ), and the binding constant ( K D ) was calculated (Table 12).
  • Example 12 In vivo efficacy analysis of selected anti- Ang2 ScFv antibodies (CNV mouse model)
  • the efficacy was tested with the commercial drug aflibercept as a control.
  • anesthetic eye drops are instilled into the eye for additional local anesthesia, and mydriatic is instilled.
  • the mouse is placed on the target and the CNV induction condition (wavelength 532 nm, using Micron-IV) is used.
  • the CNV induction condition wavelength 532 nm, using Micron-IV
  • laser burn is induced to destroy Bruch's membrane.
  • Lesions where bubbling was not observed during laser burn induction were classified as unsuccessful laser bums, and were excluded from the result analysis and statistical processing based on exclusion criteria that modified the criteria proposed by Gong Y. et al.
  • ERG retinal potential test was performed. Mice were induced to acclimate in the dark from 12 hours before ERG evaluation. On the day of evaluation (11 days after CNV induction), after systemic anesthesia with Erumpon® and Ketamine®, Alkine® was instilled into the eye to induce additional local anesthesia. The mice were placed on the ERG preparation and the ERG probes were in contact with the tail, head, and cornea, respectively. ERG was immediately determined as the change in retinal potential for a single flash stimulus (0.9 log cds/m2 (10 responses/intensity)). When the ERG evaluation was completed, a drop of Tobrex was instilled in the mouse eye. ERG analysis was conducted using the'LabScribeERG (iWorx DataAcquisition Software)' program, except that eyes that meet the exclusion criteria proposed by Gong Y. et al. were excluded from the final result and statistical analysis process.
  • Example 13 Analysis of antitumor efficacy of selected anti-Ang2 antibodies (TNBC model)
  • MDA-MB-231 was transplanted to the left flank of NSG mice, and the anticancer efficacy of isotype control, nesvacumab, and anti-Ang2 antibody treatments were evaluated by intravenous administration. The experiment was conducted by requesting the US graduates Oncology.
  • Tumor volume was measured twice a week for 20 days after drug administration. No clinical toxic reaction was observed in mice during the measurement process.
  • Anti-Tumor effect of anti-Ang2 antibody was 10 In the mg/kg treatment group, about 70% of the tumor growth inhibitory effect was shown, and the anti-tumor effect was approximately twice that of the control drug Nesvacumab 10 mg/ml treatment group.
  • the anti-show 13 ⁇ 42 antibody or antigen-binding fragment thereof according to the present invention exhibits a binding force targeted for Show 13 ⁇ 42, and is intended to inhibit cancer/tumor or angiogenesis and

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Abstract

본 발명은 항-Ang2 항체 또는 이의 항원 결합 단편, 이를 코딩하는 핵산, 상기 핵산을 포함하는 벡터, 상기 벡터로 형질전환된 세포, 상기 항체 또는 그의 항원 결합 단편의 제조방법, 이를 포함하는 혈관신생 저해제 및 엔지오포이에틴-2 활성화 및 또는 과생성과 관련된 질병의 치료용 조성물, 및 상기 항체를 포함하는 엔지오포이에틴-2 활성화 및 또는 과생성과 관련된 질병의 진단용 조성물, 상기 항체를 포함하는 안질환, 또는 암 예방 또는 치료용 약학적 조성물 및 Ang2에 결합하는 항체 이외의 조성물과의 병용투여용 조성물에 관한 것이다.

Description

2020/175886 1»（：1/10公020/002687 명세서
발명의명칭 :항- ANG2항체및이의용도 기술분야
[1] 본발명은엔지오포이에틴- 2(Angiopoietin-2; Ang-2)에특이적으로결합하여 기능을저해하는항체와관련된것으로,항- Ang2항체또는이의항원결합단편, 이를코딩하는핵산,상기핵산을포함하는벡터,상기벡터로형질전환된세포, 상기항체또는그의항원결합단편의제조방법 ,이를포함하는혈관신생 저해제및엔지오포이에틴- 2활성화및또는과생성과관련된질병의치료용 조성물,및상기항체를포함하는엔지오포이에틴- 2활성화및또는과생성과 관련된질병의진단용조성물,상기항체를포함하는안질환,또는암예방또는 치료용약학적조성물및 Ang2에결합하는항체이외의조성물과의병용투여용 조성물에관한것이다.
四
배경기술
[3] 혈관신생 (angiogenesis)은기존혈관으로부터새로운혈관이생성되는기작을 의미하며 ,기관의형성 ,정상적인생리학적성장,상처치유등에중요한역할을 하는것으로알려져 있다.또한,종양의성장과전이에중요한역할을한다고 알려져 있으며,비정상적인혈관신생은종양의성장과전이,연령관련 황반변성,당뇨병성망막병증,건선,류마티스성관절염,만성염증과같은 질병에결정적인역할을하는것으로알려져있다.
[4] 따라서혈관신생에관여하는인자들이암등의질병의새로운치료제의
개발을위한중요한타깃이되고있으며,고령화와서구화된식습관으로 당뇨질환환자수가급격히증가됨에따라신생혈관안질환환자가급격히증가 하고있다.주요안질환으로서는연령관련황반변성증 (AMD;Age-related Macular Degeneration,이하‘황반변성증’이라함),당뇨병성망막증 (DR; Diabetic
Retinopathy),당뇨병성황반부종 (DME; Diabetic Macular Edema)등이있다.특히 황반변성증과당뇨병성망막증은전세계적으로주요실명원인질환이다.
[5] 노인성황반변성의진행과관련한요인은여러가지가있겠으나산화적
스트레스,염증반응,그리고신생혈관형성과관련이있는것으로알려져 있다. 그러나가장주요한인자로는 Vascular Endothelial Growth Factor (VEGF)가가장 널리알려져있다.치료제로서단일클론항체,항체단편,혹은융합단백질을 이용한 VEGF억제제개발이시도돼왔고,현재황반변성치료제로널리쓰이고 있는대표적인약물은아일리아 (Aflibercept)루센티스 (Ranibizumab)가사용되고 있다.이들약물의작용기전은 VEGF신호억제에의한혈관형성억제를 유도하는것으로알려져 있다.그러나,이들약물의투여환자가운데 10-15%는 기존치료법에반응하지않는것으로알려져있다.그이유로는,기존의항 VEGF 치료는병리적인혈관형성만을억제한반면,또다른경로의혈관형성요인이 질병의진행에영향을주기때문인것으로알려져 있다.엔지오포이에틴 2 (ANG2)는혈관벽의내피세포에있는 Tie2수용체에결합하여신생혈관형성을 촉진하는사이토카인으로알려져 있다.앞서동물실험과임상시험등을통하여 ANG2신호억제를억제함으로써종양내혈관형성을억제하여항암효과를 발휘하는것으로알려져 있다.더욱이 ANG의발현은노인성황반변성 환자안구의방수액내에서높게존재하는것으로알려져있다.따라서항 VEGF 치료제와함께항- ANG2치료제의개발과병행요법이황반변성치료에도움을 줄수있을것으로기대된다.이에당사는노인성황반변성및당뇨병성 망막병증의치료제를개발하기위하여 Ang-2에주목하였다.
[6] 안지오포이에틴- 2 (Angiopoietin-2; Ang2)는혈관내피세포에존재하는수용체 Tie2의길항적인리간드 (antagonistic ligand)로서 , Tie2의작용물질 (agonist)인 안지오포이에틴- l(Angiopoietin-l; Angl)과 Tie2결합에대해경쟁함으로써 Tie2에의한신호전달을억제하는작용을하며 , Tie2수용체를활성화시키는 리간드인 Angl은혈관내피세포의장벽기능 (barrier function)을유지시킴으로써 혈관의안정화 (stabilization)를유지하는주요조절자 (key regulator)로작용한다. VEGF의과발현또는염증 (inflammation)상태에서는혈관내피세포가
활성화되며,혈관투과성 (vascular permeability)이증가한다.
[7] 이때 , Angl은혈관내피세포의접합부통합 (junctional integrity)을족진함으로써 혈관내피세포의안정화를유도하고혈관투과성을감소시키는반면,활성화된 혈관내피세포에서증가된 Ang2는 Tie-2에결합함으로써혈관내피세포의이동과 팁형성 (tip formation)에관여한다.결과적으로신생혈관의형성을촉진하게 된다.
[8] 당뇨병성망막증의경우, PDGF시그널링 (signaling)이혈관주변세포를
조절함으로써 blood-retinal barrier의형성과성숙에필수적임이규명되었고,성체 망막혈관에서혈관주변세포의소실이 VEGF-A에대한혈관내피세포의 반응성을증가시켜 F0X01-Ang2 loop를활성화시킴으로써당뇨망막병증을 악화시킴이증명되었다.즉 Ang2차단및 Tie2활성화를유도하면당뇨병성 망막증의새로운치료법개발이가능할것으로판단된다.
[9] 또한, Ang-2는암조직에서의신생혈관형성에도기여한다.암조직에서신생 혈관형성을위해암세포가기존의혈관을선택하는혈관공용 (cooption)이 발생한다.그후, Ang-2경로에의해기존의혈관의기능을파괴시키는혈관 퇴화가일어난다.기존혈관의퇴화로인하여암조직내의환경은
저산소 (hypoxia)환경이되어신생혈관이형성될수있는조건을제공한다.상기 조건하에서혈관내피세포성장인자 (VEGF)의과발현이유도되어,앞서 언급한바와같이신생혈관이유도된다.이러한이유로 Ang-2는혈관신생 억제제를통한함암제개발의주요타깃이되어왔다.
[1이 이러한기술적배경하에서 ,본출원의발명자들은항- Ang2항체를개발하기 2020/175886 1»（：1^1{2020/002687
3 위하여노력하였다.그결과,본발명자들은쇼1¾2에목적하는결합력을 나타내는항-쇼1¾2항체를개발하고,이러한항-쇼1¾2항체가목적하는
면역항암제또는안과질환치료제의역할을할수있음을확인하고,본발명을 완성하였다.
[11]
[12] 발명의요약
[13] 본발명의목적은쇼1¾2에대한신규항체또는이의항원결합단편을제공하는 데있다.
[14] 본발명의다른목적은상기항체또는이의항원결합단편을코딩하는핵산을 제공하는데있다.
[15] 본발명의다른목적은상기핵산을포함하는벡터,상기벡터로형질전환된 세포및이의제조방법을제공하는데있다.
[16] 본발명의또다른목적은상기항체또는그의항원결합단편을포함하는 혈관신생저해제및엔지오포이에틴- 2활성화및또는과생성과관련된질병의 치료용조성물을제공하는데있다.
[17] 본발명의또다른목적은상기항체또는그의항원결합단편을포함하는 혈관신생저해제및엔지오포이에틴- 2활성화및또는과생성과관련된질병의 진단용조성물을제공하는데있다.
[18] 본발명의또다른목적은상기항체또는그의항원결합단편을포함하는 안질환의 예방또는치료용조성물을제공하는데있다.
[19] 본발명의또다른목적은상기항체또는그의항원결합단편을포함하는종양 또는암의예방또는치료용조성물을제공하는데있다.
[2이 본발명의또다른목적은상기항체또는그의항원결합단편을포함하는 쇼1¾2항체또는쇼1¾2항체와병용투여하기위한조성물을제공하는데있다.
[21] 상기목적을달성하기위하여,본발명은서열번호 1, 7, 13, 19및 25로구성된 군에서선택되는중쇄
Figure imgf000005_0001
[22] 서열번호 2, 8, 14, 20및 26으로구성된군에서선택되는중쇄 0이12,및
[23] 서열번호 3, 9, 15, 21, 27, 51, 52및 53로구성된군에서선택되는중쇄 00113를 포함하는중쇄가변영역 ,및
[24] 서열번호 4, 10, 16, 22,및 28로구성된군에서선택되는경쇄 0이11,
[25] 서열번호 5, 11, 17, 23및 29으로구성된군에서선택되는경쇄 00112,및
[26] 서열번호 6, 12, 18, 24및 30로구성된군에서선택되는경쇄 00113을포함하는 경쇄가변영역을포함하는,쇼1¾2（쇼^^01）0 1111-2）에결합하는항체또는이의 항원결합단편을제공한다.
[27] 본발명은또한,상기항체또는이의항원결합단편을코딩하는핵산을
제공한다.
[28] 본발명은또한,상기핵산을포함하는벡터를제공한다.
[29] 본발명은또한,상기벡터로형질전환된세포를제공한다. [3이 본발명은또한,다음단계를포함하는상기항체또는이의항원결합단편의 제조방법을제공한다: (a)상기세포를배양하는단계 ;및 (b)상기배양된 세포에서항체또는그의항원결합단편을회수하는단계.
[31] 본발명은또한,상기항체또는이의항원결합단편을포함하는혈관신생 저해제및엔지오포이에틴- 2활성화및또는과생성과관련된질병의치료용 조성물을제공한다.본발명은또한,상기항체또는그의항원결합단편을 포함하는혈관신생저해제및엔지오포이에틴- 2활성화및또는과생성과 관련된질병의진단용조성물을제공한다.본발명은또한,상기항체또는그의 항원결합단편을포함하는종양또는암의 예방또는치료용조성물을 제공한다.본발명은또한,상기항체또는그의항원결합단편을포함하는 안질환의 예방또는치료용조성물을제공한다.본발명은또한,상기항체또는 그의항원결합단편을포함하는 Ang2항체또는 Ang2항체와병용투여하기 위한조성물을제공한다.
[32]
도면의간단한설명
[33] 도 1은선별된단클론 scFv파지가 Ang2/Tie2결합을저해하는능력이 있음을 확인한결과이다.
[34] 도 2는선별한항- Ang2항체를일시발현및정제후산물에대한환원조건및 비환원조건에서의 SDS-PAGE결과이다.
[35] 도 3은일시발현및정제한항- Ang2항체의인간및마우스 Ang2와 Angl에 대한결합을평가한 ELISA결과이다.
[36] 도 4는선별된항- Ang2항체의인간및마우스 Ang2ATie2결합을중화할수있는 능력을보여주는결과이다.
[37] 도 5는선별된항- Ang2항체의 Ang2/integrin의결합을중화할수있는능력을 보여주는결과이다.
[38] 도 6은선별된항- Ang2항체가 Ang2/Tie2신호전달을저해할수있음을
보여주는결과이다.
[39] 도 7은선별된항- Ang2 scFv항체를대장균에서발현하여정제단계별순도를 확인한결과이다.
[4이 도 8은대장균에서발현한항- Ang2 scFv항체의인간 Ang2단백질에대한
결합을평가한 ELISA결과이다.
[41] 도 9내지도 11은선별된항- Ang2 ScFv항체의 in vivo효능을 CNV mouse 모델에서확인한결과이다.
[42] 도 12은인간유래삼중음성유방암모델을이용한항- Ang2항체의항종양 효과에대한결과이다.
[43]
[44] 발명의상세한설명및바람직한구현예 2020/175886 1»（：1^1{2020/002687
5
[45] 다른식으로정의되지않는한,본명세서에서사용된모든기술적및과학적 용어들은본발명이속하는기술분야에서숙련된전문가에의해서통상적으로 이해되는것과동일한의미를갖는다.일반적으로,본명세서에서사용된 명명법은본기술분야에서잘알려져있고통상적으로사용되는것이다.
[46] 본발명은일관점에서,서열번호 1, 7, 13, 19및 25로구성된군에서선택되는 중쇄 00111,
[47] 서열번호 2, 8, 14, 20및 26으로구성된군에서선택되는중쇄 0이12,및
[48] 서열번호 3, 9, 15, 21, 27, 51, 52및 53로구성된군에서선택되는중쇄 00113를 포함하는중쇄가변영역 ,및
[49] 서열번호 4, 10, 16, 22,및 28로구성된군에서선택되는경쇄 0이11,
[5이 서열번호 5, 11, 17, 23및 29으로구성된군에서선택되는경쇄 00112,및
[51] 서열번호 6, 12, 18, 24및 30로구성된군에서선택되는경쇄 00113을포함하는 경쇄가변영역을포함하는,쇼1¾2（쇼^^01）0 1111-2）에결합하는항체또는이의 항원결합단편에관한것이다.
[52] 본명세서에서사용된용어, "항체 "는쇼1¾2에특이적으로결합하는 항-쇼1¾2항체를의미한다.본발명의범위에는쇼1¾2에특이적으로결합하는 완전한항체형태뿐아니라,상기항체분자의항원결합단편도포함된다.
[53] 완전한항체는 2개의전체길이의경쇄및 2개의전체길이의중쇄를가지는 구조이며각각의경쇄는중쇄와다이설파이드결합으로연결되어있다.중쇄 불변영역은감마外）,뮤（나）,알파（«）,델타（6）및엠실론四타입을가지고 서브클래스로감마 1外1）,감마 2外2）,감마 3（作）,감마 4外4）,알파 1（（x1）및 알파 2（（x2）를가진다.경쇄의불변영역은카파（幻및람다（X）타입을가진다.
[54] 항체의항원결합단편또는항체단편이란항원결합기능을보유하고있는 단편을의미하며 ,므此,므（此’）,므（此’）2및 등을포함한다.항체단편중므此는 경쇄및중쇄의가변영역과경쇄의불변영역및중쇄의첫번째
불변영역（0¾1）을가지는구조로 1개의항원결합부위를가진다.므此’는중쇄 011도메인의（:-말단에하나이상의시스테인잔기를포함하는힌지영역（ 1¾
[55] 011）을가진다는점에서므 와차이가있다. 此’ 항체는므此’의힌지
영역의시스테인잔기가디설파이드결합을이루면서생성된다. 는중쇄 가변영역및경쇄가변영역만을가지고있는최소의항체조각이다.이중쇄 1 0；\¥0-（;1 111 1 ）는비공유결합으로중쇄가변영역과경쇄가변영역이연결되어 있고단쇄 1 （ 1¾16- 11 1 , 1今）는일반적으로펩타이드링커를통하여 중쇄의가변영역과경쇄의가변영역이공유결합으로연결되거나또는 （:-말단에서바로연결되어있어서이중쇄 와같이다이머와같은구조를이룰 수있다.이러한항체단편은단백질가수분해효소를이용해서얻을수 있고（예를들어,전체항체를파파인으로제한절단하면므 를얻을수있고 펩신으로절단하면므（ ’）2단편을얻을수있다）,유전자재조합기술을통하여 제작할수도있다. [56] 하나의실시예에서,본발명에따른항체는 Fv형태 (예컨대, scFv)이거나, 완전한항체형태이다.또한,중쇄불변영역은감마 (Y),뮤 (나),알파 (a),델타 (6) 또는엠실론四중의어느한이소타입으로부터선택될수있다.예를들어, 불변영역은감마 l(IgGl),감마 3(IgG3)또는감마 4(IgG4)이다.경쇄불변영역은 카파또는람다형일수있다.
[57] 본명세서에서사용되는용어,”중쇄”는항원에특이성을부여하기위한
충분한가변영역서열을갖는아미노산서열을포함하는가변영역도메인 VH및 3개의불변영역도메인 CH1, CH2및 CH3을포함하는전체길이중쇄및이의 단편을모두의미한다.또한,본명세서에서사용되는용어 ,’’경쇄’’는항원에 특이성을부여하기위한충분한가변영역서열을갖는아미노산서열을 포함하는가변영역도메인 VL및불변영역도메인 CL을포함하는전체길이 경쇄및이의단편을모두의미한다.
[58] 본발명의항체는단일클론항체,다특이적항체,인간항체,인간화항체, 키메라항체,단쇄 Fvs(scFV),단쇄항체, Fab단편, F(ab')단편,
다이설파이드-결합 Fvs(sdFV)및항-이디오타입 (항- Id)항체,또는상기항체들의 에피토프-결합단편등을포함하나,이에한정되는것은아니다.
[59] 상기단일클론항체는실질적으로동질적항체집단으로부터수득한항체,즉 집단을차지하고있는개개의항체가미량으로존재할수있는가능한천연 발생적돌연변이를제외하고는동일한것을지칭한다.단일클론항체는고도로 특이적이어서,단일항원부위에대항하여유도된다.전형적으로상이한 결정인자 (에피토프)에대해지시된상이한항체를포함하는통상의
(폴리클로날)항체제제와는대조적으로,각각의모노클로날항체는항원상의 단일결정인자에대해지시된다.
[6이 ”에피토프”은항체가특이적으로결합할수있는단백질결정부위
(determinant)를의미한다.에피토프는통상화학적으로활성인표면분자군,예를 들어아미노산또는당측쇄로구성되며,일반적으로특정한 3차원의구조적 특징뿐만아니라특정한전하특성을갖는다.입체적에피토프및비입체적 에피토프는변성용매의존재하에서전자에대한결합은소실되지만후자에 대해서는소실되지않는다는점에서구별된다.
[61] 상기 "인간화”형태의비-인간 (예:뮤린)항체는비-인간
면역글로불린으로부터유래된최소서열을함유하는키메라항체이다.
대부분의경우,인간화항체는,수용자의초가변영역으로부터의잔기를 목적하는특이성,친화성및능력을보유하고있는비-인간종 (공여자항체), 예를들어마우스,랫트，토끼또는비-인간영장류의초가변영역로부터의 잔기로대체시킨인간면역글로불린 (수용자항체)이다.
[62] 상기“인간항체”는인간면역글로불린으로부터유래하는분자로서상보성 결정영역 ,구조영역을포함한항체를구성하는모든아미노산서열전체가 인간의면역글로불린으로구성되어 있는것을의미한다. 2020/175886 1»（：1^1{2020/002687
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[63] 중쇄및/또는경쇄일부가특별한종으로부터유래되거나특별한항체부류 또는아부류에속하는항체내의상응하는서열과동일하거나이와상동성인 반면,나머지쇄（들）는또다른종으로부터유래되거나또다른항체부류또는 아부류에속하는항체내의상응하는서열과동일하거나이와상동성인
"키메라”항체（면역글로불린）뿐아니라목적하는생물학적활성을나타내는 상기항체의단편이포함된다.
[64] 본원에사용된바와같은’’항체가변도메인’’은상보성결정영역（0011;즉, 00111, 00112,및 00113）,및골격영역 의아미노산서열을포함하는항체 분자의경쇄및중쇄부분을지칭한다. \¾는중쇄의가변도메인을지칭한다. 은경쇄의가변도메인을지칭한다.
[65] "상보성결정영역”片011;즉, 00111, 00112,및 00113）은항원결합을위해 필요한존재인,항체가변도메인의아미노산잔기를지칭한다.각가변 도메인은전형적으로,
Figure imgf000009_0001
00112및 00113으로서확인된 3개의 영역을 갖는다.본발명은서열번호 1의중쇄 00113를포함하는중쇄가변영역및 서열번호 2의경쇄 00요3를포함하는경쇄가변영역을포함한다.
[66] 본발명에 있어,상기쇼1¾2에결합하는항체또는이의항원결합단편은
서열번호 1의중쇄 0이11,서열번호 2의중쇄 0이12및서열번호 3의중쇄 00113을포함하는중쇄가변영역,서열번호 4의
쇄 00113를포함하는
Figure imgf000009_0002
[67]
Figure imgf000009_0003
,서열번호 8의중쇄 0이12및서열번호 9의중쇄
00113을포함하는중쇄가변영역,서열번호 의경쇄 00111,서열번호 11의
12의경쇄 00113를포함하는경쇄가변영역 ,
[68]
Figure imgf000009_0004
서열번호 14의중쇄 00112및서열번호 15의중쇄
00113을포함하는중쇄가변영역,서열번호 16의경쇄 00111,서열번호 17의
18의경쇄 00113를포함하는경쇄가변영역 ,
[69]
Figure imgf000009_0005
서열번호 20의중쇄 00112및서열번호 21의중쇄
00113을포함하는
Figure imgf000009_0006
경쇄 00112,및서열번호 24의경쇄 00113를포함하는경쇄가변영역 , 이 서열번호 25의중쇄 서열번호 26의중쇄 00112및서열번호 27의중쇄
00113을포함하는중쇄가변영역,서열번호 28의경쇄 서열번호 29의
Figure imgf000009_0007
포함하는경쇄가변영역 , 1] 서열번호 13의중쇄 00111,서열번호 14의중쇄 00112및서열번호 51의중쇄
Figure imgf000009_0008
2020/175886 1»（：1^1{2020/002687
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00113을포함하는중쇄가변영역,서열번호 16의경쇄 00111,서열번호 17의
Figure imgf000010_0001
5] "IV’단편은완전한항체인식및결합부위를함유하는항체단편이다.이러한
Figure imgf000010_0002
단단하게사실상공유적으로연합된이량체로이루어진다.
6] ”므此”단편은경쇄의가변및불변도메인과,중쇄의가변및제 1불변도메인
(0¾1)을함유한다.므( ’)2항체단편은일반적으로그들사이에힌지시스테인에 의해그들의카복시말단근처에공유적으로연결되는한쌍의므 단편을 포함한다.
7]
Figure imgf000010_0003
단편은
Figure imgf000010_0004
포함하는데, 이들도메인은단일폴리펩티드쇄내에존재한다. 폴리펩티드는 가항원 결합을위해목적하는구조를형성할수있도록
Figure imgf000010_0005
사이에폴리펩티드링커를추가로포함할수있다.
8] 쇼1¾2항체는단쇄또는이중쇄를포함할수있다.기능적으로,쇼1¾2항체의 결합친화성은 10 ^내지 W-12 M범위내에있다.예를들어,쇼1¾2항체의결합 친화성은 106 M내지 10 12 107 M내지 10 12 108 M내지 10 12 109 M내지
Figure imgf000010_0006
10 10 107 M내지 10 10 10 8 M내지 10 10 109 M내지 1010 105 M내지 109 106 M내지 109 107 M내지 109 10 8 M내지 109 105 M내지 108 106 M내지 10 8 107 M내지 10 8 105 M내지 107 106 M내지 107 M 또는 105 M내지 106 M이다.
9] 상기쇼1¾2에결합하는항체또는이의항원결합단편은서열번호 32, 36, 40, 44, 48, 55, 57, 59및 61로구성된군에서선택되는중쇄가변영역을포함할수있다. 또한,상기쇼1¾2에결합하는항체또는이의항원결합단편은서열번호 34, 38,
42, 46및 50으로구성된군에서선택되는경쇄가변영역을포함할수있다.
[8이 특히 ,항-쇼1¾2항체의생산성및고농축제형개발을위하여생산성및용해도 개선을목표로중쇄가변영역의프레임워크부분에돌연변이를유도하였다. 돌연변이는중쇄가변영역 12번아미노산
Figure imgf000010_0007
변환하여 제작하였다.이에따라,서열번호 61의중쇄가변영역을포함하는항체를 실험하였다.그결과,향상된생산성및용해도를나타냄을확인하였다.
[81] 본발명에따른구체적실시예에서,서열번호 32의중쇄가변영역및서열번호 34의경쇄가변영역 ;
[82] 서열번호 36의중쇄가변영역및서열번호 38의경쇄가변영역 ;
[83] 서열번호 40의중쇄가변영역및서열번호 42의경쇄가변영역 ; 2020/175886 1»（：1^1{2020/002687
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[84] 서열번호 44의중쇄가변영역 및서열번호 46의경쇄가변영역 ;
[85] 서열번호 48의중쇄가변영역 및서열번호 50의경쇄가변영역 ;
[86] 서열번호 55의중쇄가변영역 및서열번호 42의경쇄가변영역 ;
[87] 서열번호 57의중쇄가변영역 및서열번호 42의경쇄가변영역 ;
[88] 서열번호 59의중쇄가변영역 및서열번호 42의경쇄가변영역 ;또는서열번호
61의중쇄가변영역 및서열번호 42의경쇄가변영역을포함할수있다.
[89] "파지 디스플레이 "는변이체폴리펩티드를파지，예를들어섬유상파지
입자의표면상에 외피단백질의 적어도일부와의융합단백질로서
디스플레이하는기술이다.파지디스플레이의유용성은무작위화단백질 변이체의큰라이브러리를대상으로하여,표적 항원과고친화도로결합하는 서열을신속하고도효율적으로분류할수있다는사실에 있다.펩티드및단백질 라이브러리를파지상에 디스플레이하는것은특이적결합특성을지닌 폴리펩티드를알아보기위해수백만개의폴리펩티드를스크리닝하는데 사용되어 왔다.
[9이 파지디스플레이 기술은특정 리간드（예 :항원）와결합하는신규단백질을 생성 및선별하기 위한강력한도구를제공하였다.파지 디스플레이기술을 사용하여，단백질변이체의큰라이브러리를생성시키고,표적 항원과고 친화성으로결합하는서열을신속하게분류할수있다.변이체폴리펩티드를 암호화하는핵산을바이러스성 외피단백질,예를들어유전자 III단백질또는 유전자 VIII단백질을암호화하는핵산서열과융합시킨다.단백질또는 폴리펩티드를암호화하는핵산서열을유전자 III단백질의 일부를암호화하는 핵산서열과융합시킨 1가파지 디스플레이시스템이 개발되었다. 1가파지 디스플레이시스템에서는,유전자융합물이 저수준으로발현되고야생형 유전자 III단백질이또한발현되어 입자감염성이유지된다.
[91] 섬유상파지표면상에서의 펩티드의발현과 E. 0센의주변세포질에서의
기능성 항체단편의 발현을입증하는것이항체파지디스플레이 라이브러리를 개발하는데에 있어중요하다.항체또는항원결합성폴리펩티드의
라이브러리는수많은방식 ,예를들어무작위 DNA서열을삽입함으로써단일 유전자를변경시키는방법또는관련유전자계열을클로닝하는방법으로 제조하였다.라이브러리를대상으로하여,목적하는특징을수반한항체또는 항원결합성단백질의 발현에관하여스크리닝할수있다.
[92] 파지디스플레이 기술은목적하는특징을지닌항체를제조하기 위한통상적인 하이브리도마및재조합방법에비해몇가지 이점을지니고있다.이러한 기술은동물을사용하지 않고서도짧은시간에다양한서열을지닌큰항체 라이브러리를생성시킬수있도록한다.하이브리도마의 제조나인간화항체의 제조는수개월의 제조기간을필요로할수있다.또한,면역이 전혀요구되지 않기 때문에,파지항체 라이브러리는독성이거나항원성이낮은항원에 대해서도항체를생성시킬수있다.파지 항체라이브러리를또한사용하여 2020/175886 1»（：1^1{2020/002687
10 신규한치료적항체를생성 및확인할수있다.
[93] 파지디스플레이 라이브러리를사용하여 면역시킨,비-면역시킨인간,
생식세포계서열,또는미감작 6세포 ¾레퍼토리 (repertory)로부터 인간항체를 생성시키는기술을사용할수있다.각종림프계조직을사용하여 ,미감작또는 비면역 항원결합성 라이브러리를제조할수있다.
[94] 파지디스플레이 라이브러리로부터고친화성 항체를확인및분리할수있는 기술은치료용신규항체분리에중요하다.라이브러리로부터고친화성 항체를 분리하는것은라이브러리의크기,세균성세포중에서의 생산효율및
라이브러리의다양성에좌우될수있다.라이브러리의크기는항체또는항원 결합성 단백질의부적절한폴딩과정지코돈의존재로인한비효율적 생산에 의해감소된다.세균성세포에서의 발현은항체또는항원결합성도메인이 적절하게폴딩되지 않는경우에는억제될수있다.발현은가변/불변계면의 표면이나선별된 잔기에서의잔기를교대로돌연변이시킴으로써 개선시킬 수있다.골격 영역의서열은세균성 세포에서항체파지 라이브러리를
생성시키는경우에 적절한폴딩을제공하기 위한하나의요소이다.
[95] 고친화성 항체분리에서 항체또는항원결합성 단백질의다양한
라이브러리를생성시키는것이중요하다. 00113영역은이들이종종항원 결합에 참여하는것으로밝혀졌다.중쇄상의 0이13영역은크기,서열및구조적 입체 형태면에서상당히다양하므로,이를이용하여다양한라이브러리를 제조할수있다.
[96] 또한,각위치에서 20개아미노산모두를사용하여
Figure imgf000012_0001
영역을무작위화함으로써다양성을발생시킬수있다. 20개의모든아미노산을 사용하면다양성이큰변이체항체서열이 생성되고신규한항체를확인할 기회가증가할수있다.
[97] 본발명의 항체또는항체단편은쇼1¾2를특이적으로인식할수있는범위
내에서 ,본명세서에기재된본발명의 항-쇼1¾2항체의서열뿐만아니라,이의 생물학적균등물도포함할수있다.예를들면,항체의결합친화도및/또는기타 생물학적특성을보다더 개선시키기 위하여항체의 아미노산서열에추가적인 변화를줄수있다.이러한변형은예를들어,항체의 아미노산서열잔기의결실, 삽입 및/또는치환을포함한다.이러한아미노산변이는아미노산곁사슬 치환체의상대적유사성,예컨대,소수성,친수성,전하,크기등에기초하여 이루어진다.아미노산곁사슬치환체의크기,모양및종류에 대한분석에 의하여 ,아르기닌,라이신과히스티딘은모두양전하를띤잔기이고;알라닌, 글라이신과세린은유사한크기를가지며 ;페닐알라닌,트림토판과타이로신은 유사한모양을갖는다는것을알수있다.따라서,이러한고려사항에 기초하여, 아르기닌,라이신과히스티딘;알라닌,글라이신과세린;그리고페닐알라닌, 트림토판과타이로신은생물학적으로기능균등물이라할수있다.
[98] 상술한생물학적균등활성을갖는변이를고려한다면,본발명의 항체또는 이를코딩하는핵산분자는서열번호에기재된서열과실질적인
동일성 (substantial identity)을나타내는서열도포함하는것으로해석된다.
상기의실질적인동일성은,상기한본발명의서열과임의의다른서열을최대한 대응되도록얼라인하고,당업계에서통상적으로이용되는알고리즘을이용하여 얼라인된서열을분석한경우에 ,최소 90%의상동성 ,가장바람직하게는최소 95%의상동성, 96%이상, 97%이상, 98%이상, 99%이상의상동성을나타내는 서열을의미한다.서열비교를위한얼라인먼트방법은당업계에공지되어 있다. NCBI Basic Local Alignment Search Tool(BLAST)은 NCBI등에서접근가능하며 , 인터넷상에서 blastp, blasm, blastx, tblastn및 tblastx와같은서열분석
프로그램과연동되어이용할수있다. BLSAT는
www.ncbi.nlm.nih.gov/BLAST/에서접속가능하다.이프로그램을이용한서열 상동성비교방법은 www.ncbi.nlm.nih.gov/BLAST/blast_ help.html에서확인할수 있다.
[99] 이에기초하여，본발명의항체또는그의항원결합단편은명세서에기재된 명시된서열또는전체와비교하여 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,또는그이상의상동성을가질수있다.이러한상동성은당업계에 공지된방법에의한서열비교및/또는정렬에의해결정될수있다.예를들어, 서열비교알고리즘 (즉, BLAST또는 BLAST 2.0),수동정렬,육안검사를 이용하여본발명의핵산또는단백질의퍼센트서열상동성을결정할수있다.
[100] 본발명은다른관점에서 ,상기항체또는그의항원결합단편을코딩하는
핵산에관한것이다.
[101] 본발명의항체또는그의항원결합단편을코딩하는핵산을분리하여항체 또는그의항원결합단편을재조합적으로생산할수있다.핵산을분리하고, 이를복제가능한벡터내로삽입하여추가로클로닝하거나 (DNA의증폭)또는 추가로발현시킨다.이를바탕으로,본발명은또다른관점에서상기핵산을 포함하는벡터에관한것이다.
[102] ’’핵산’’는 DNA(gDNA및 cDNA)및 RNA분자를포괄적으로포함하는의미를 가지며,핵산에서기본구성단위인뉴클레오타이드는자연의뉴클레오타이드 뿐만아니라,당또는염기부위가변형된유사체 (analogue)도포함한다.본 발명의중쇄및경쇄가변영역을코딩하는핵산의서열은변형될수있다.상기 변형은뉴클레오타이드의추가,결실,또는비보존적치환또는보존적치환을 포함한다.
[103] 본발명에따른구체적실시예에서,상기핵산은중쇄가변영역을코딩하는 서열번호 31, 35, 39, 43, 47, 54, 56, 58, 60로구성된군에서선택되는중쇄 가변영역을포함할수있다.또한,상기핵산은경쇄가변영역을코딩하는 서열번호 33, 37, 41, 45및 49로구성된군에서선택되는경쇄가변영역을포함할 수있다.
[104] 구체적으로,중쇄가변영역을코딩하는서열번호 31의핵산및경쇄 2020/175886 1»（：1^1{2020/002687
12 가변영역을코딩하는서열번호 33의핵산;
[105] 중쇄가변영역을코딩하는서열번호 35의핵산및경쇄가변영역을코딩하는 서열번호 37의핵산;
[106] 중쇄가변영역을코딩하는서열번호 39의핵산및경쇄가변영역을코딩하는 서열번호 41의핵산;
[107] 중쇄가변영역을코딩하는서열번호 43의핵산및경쇄가변영역을코딩하는 서열번호 45의핵산;
[108] 중쇄가변영역을코딩하는서열번호 47의핵산및경쇄가변영역을코딩하는 서열번호 49의핵산;
[109] 중쇄가변영역을코딩하는서열번호 54의핵산및경쇄가변영역을코딩하는 서열번호 41의핵산;
[110] 중쇄가변영역을코딩하는서열번호 56의핵산및경쇄가변영역을코딩하는 서열번호 41의핵산;
[111] 중쇄가변영역을코딩하는서열번호 58의핵산및경쇄가변영역을코딩하는 서열번호 41의핵산;또는
[112] 중쇄가변영역을코딩하는서열번호 60의핵산및경쇄가변영역을코딩하는 서열번호 41의핵산을포함할수있다.
[113] 상기항체를암호화하는 DNA는통상적인과정을사용하여（예를들어 ,항체의 중쇄와경쇄를암호화하는 DNA와특이적으로결합할수있는
올리고뉴클레오티드프로브를사용함으로써）용이하게분리또는합성한다. 많은벡터가입수가능하다.벡터성분에는일반적으로,다음중의하나이상이 포함되지만,그에제한되지않는다:신호서열,복제기점,하나이상의마커유 전자,증강인자요소,프로모터 ,및전사종결서열.
[114] 본명세서에서사용되는용어,”벡터”는숙주세포에서목적유전자를
발현시키기위한수단으로플라스미드벡터;코즈미드벡터;박테리오파지벡터, 아데노바이러스벡터,레트로바이러스벡터및아데노-연관바이러스벡터같은 바이러스벡터등을포함한다.상기벡터에서항체를코딩하는핵산은
프로모터와작동적으로연결되어 있다.
[115] “작동적으로연결”은핵산발현조절서열（예:프로모터,시그널서열,또는
전사조절인자결합위치의어레이）과다른핵산서열사이의기능적인결합을 의미하며，이에의해상기조절서열은상기다른핵산서열의전사및/또는 해독을조절하게된다.
[116] 원핵세포를숙주로하는경우에는,전사를진행시킬수있는강력한
프로모터（예컨대,
Figure imgf000014_0001
프로모터 , 1 프로모터 , 1 11¥5프로모터 , lpp프로모터,
Figure imgf000014_0002
프로모터 , 8?6 프로모터, 프로모터및 17프로모터등）,해독의개시를위한라이보좀결합 자리및전사/해독종결서열을포함하는것이일반적이다.또한,예를들어,진핵 세포를숙주로하는경우에는,포유동물세포의지놈으로부터유래된 2020/175886 1»（：1^1{2020/002687
13 프로모터（예:메탈로티오닌프로모터 ,（3 -액틴프로모터,사람헤로글로빈 프로모터 및사람근육크레아틴프로모터）또는포유동물바이러스로부터 유래된프로모터（예:아데노바이러스후기프로모터,백시니아바이러스 7.5 프로모터 , 프로모터 ,사이토메갈로바이러스（CMV）프로모터 , 의
Figure imgf000015_0001
프로모터 ,마우스유방종양바이러스（MMTV）프로모터 , 의 1그¾프로모터 , 몰로니 바이러스의프로모터엠스타인바바이러스田6\0의프로모터 및로우스 사코 바이러스（1«\0의프로모터）가이용될수있으며,전사종결서열로서 폴리아데닐화서열을일반적으로갖는다.
[117] 경우에따라서，벡터는그로부터발현되는항체의정제를용이하게하기
위하여다른서열과융합될수도있다.융합되는서열은,예컨대글루타티온 결합단백질
Figure imgf000015_0002
시등이 있다.
[118]
Figure imgf000015_0003
통상적으로이용되는항생제내성 유전자를포함하며 ,예를들어 암피실린,겐타마이신,카베니실린,
클로람페니콜,스트렙토마이신,카나마이신,게네티신,네오마이신및 테트라사이클린에 대한내성유전자가있다.
[119] 본발명은또다른관점에서,상기 언급된벡터로형질전환된세포에관한 것이다.본발명의항체를생성시키기위해사용된세포는원핵생물,효모또는 고등진핵생물세포일수있으며,이에 제한되는것은아니다.
[120] 에스케리치아콜라이 0011）,바실러스서브틸리스및바실러스
쥬린겐시스와같은바실러스속균주,스트렙토마이세스（와 마이고 , 슈도모나스（ 8 산011101 8）（예를들면,슈도모나스푸티다（ 8 산011101 8 ]31번（뇨））, 프로테우스미라빌리스 11山¾1 8）및
스타필로코쿠스（ 81）11）400^018）（예를들면,스타필로코쿠스
카르노수스（ 81）11）40대8。표미예애））와같은원핵숙주세포를이용할수있다.
[121] 다만,동물세포에 대한관심이 가장크며,유용한숙주세포주의 예는（：0 7,
Figure imgf000015_0004
있으나,이에 제한되는것은아니다.
[122] 본발명은또다른관점에서 ,如상기세포를배양하는단계 ;및 ）상기 배양된 세포에서 항체또는그의항원결합단편을회수하는단계를포함하는상기 항체 또는그의항원결합단편의 제조방법에관한것이다.
[123] 상기세포는각종배지에서 배양할수있다.시판용배지중제한없이 배양 배지로서사용할수있다.당업자에게공지되어 있는기타모든필수보충물이 적당한농도로포함될수도있다.배양조건,예를들어온도,
Figure imgf000015_0005
등이 발현을 위해선별된숙주세포와함께 이미사용되고있고,이는당업자에게 명백할 것이다. [124] 상기항체또는그의항원결합단편의회수는예를들어원심분리또는 한외여과에의해불순물을제거하고,그결과물을예를들어친화
크로마토그래피등을이용하여정제할수있다.추가의기타정제기술예를 들어음이온또는양이온교환크로마토그래피,소수성상호작용
크로마토그래피,히드록실아파타이트크로마토그래피등이사용될수있다.
[125] 본발명은또다른관점에서 ,상기항체를유효성분으로포함하는종양의예방 또는치료용조성물에관한것이다.상기항체는 IgG또는가변영역을포함한 단편즉 ScFv, Fab일수있다.또한중쇄의가변영역은 IgGl, IgG2, IgG3, IgG4일 수있다.
[126] 본발명은예를들어 ,（a）본발명에따른 Ang2에대한항체또는그의항원결합 단편의약제학적유효량;및（b）약제학적으로허용되는담체를포함하는 안질환의 예방또는치료용약제학적조성물일수있다.본발명은또한,상기 항체또는이의항원결합단편을종양환자에투여하는단계를포함하는 안질환의 예방또는치료방법일수있다.본발명은더욱이,상기항체또는이의 항원결합단편의 Ang2의기작방해용도및이를통한안질환의 예방또는치료 용도일수있다.
[127] 안질환과관련하여,각막은무혈관조직으로시력보존을위해항상투명성을 유지해야한다.그러나,신생혈관생성은눈에서도나타나안구의신생혈관관련 질환을유발하는것으로알려져있다.즉,각막에서의신생혈관생성은안구의 투명성을저해하여시력의손실을가져오게하며,망막에서의신생혈관생성은 비정상적인혈관이생성됨으로써혈액의삼출현상이일어나망막세포의변성을 통한실명을유도한다.
[128] 이를바탕으로,본발명은안질환예를들어,미숙아망막병증,
각막신생혈관생성증,당뇨병성망막증,맥락막신생혈관질환,황반변성 （예컨대,연령관련황반변성）등의예방또는치료에사용될수있다.
[129] 본발명은예를들어 ,（a）본발명에따른 Ang2에대한항체또는그의항원결합 단편의약제학적유효량;및（b）약제학적으로허용되는담체를포함하는종양 또는암의예방또는치료용약제학적조성물일수있다.본발명은또한,상기 항체또는이의항원결합단편을종양또는암환자에투여하는단계를 포함하는종양또는암의예방또는치료방법일수있다.본발명은더욱이 ,상기 항체또는이의항원결합단편의 Ang2의기작방해용도및이를통한종양또는 암의 예방또는치료용도일수있다.
[130] 상기조성물에적용되는질환인종양또는암은전형적으로 Ang2를
과발현하는종양또는암,및 Ang2를과발현하지않은종양또는암을포함한다. 치료용으로바람직한종양또는암의비-제한적인예는흑색종（예를들면, 전이성악성흑색종）,신장암（예를들면,투명세포암종）,전립선암（예를들면, 호르몬불응전립선샘암종）,췌장샘암종,유방암（경우에따라서삼중음성 유방암（Triple Negative Breast Cancer）,결장암,폐암（예를들면,비 -소세포폐암）, 2020/175886 1»（：1^1{2020/002687
15 식도암,두경부편평세포암종,간암,난소암,자궁경부암,갑상샘암,
아교모세포종,신경아교종,백혈병,림프종,및기타신생물암종을포함한다. 추가로,본발명은본발명의항체를사용하여치료할수있는불응또는 재발암을포함한다.
[131] 본발명은또다른관점에서 ,상기항-쇼1¾2항체또는이의항원결합단편을 유효성분으로포함하는혈관신생저해용약학조성물에관한것이다.또다른 예는상기항-쇼1¾2항체또는이의항원결합단편을유효성분으로포함하는 앤지오포이에틴- 2활성화및/또는과생성과관련된질병의 예방및/또는치료용 약학조성물을제공한다.
[132] 본발명은예를들어,상기항-쇼1¾2항체또는이의항원결합단편의치료적 유효량을혈관신생저해를필요로하는환자에게투여하는단계를포함하는, 혈관신생저해방법이제공된다.상기혈관신생저해방법은상기투여단계 이전에혈관신생저해를필요로하는환자를확인하는단계를추가로포함할수 있다.또다른예에서,상기항-쇼1¾2항체또는이의항원결합단편의치료적 유효량을앤지오포이에틴- 2활성화및/또는과생성과관련된질병의예방 및/또는치료를필요로하는환자에게투여하는단계를포함하는,
앤지오포이에틴- 2활성화및/또는과생성과관련된질병의 예방및/또는치료 방법이제공된다.상기예방및/또는치료방법은상기투여단계이전에 앤지오포이에틴- 2활성화및/또는과생성과관련된질병의 예방및/또는치료를 필요로하는환자를확인하는단계를추가로포함할수있다.
[133] 상기약학조성물은약학적으로허용가능한담체를추가로포함할수있으며 , 상기담체는약물의제제화에통상적으로이용되는것으로서 ,락토스, 덱스트로스,수크로스,솔비톨,만니톨,전분,아카시아고무,인산칼슘, 알기네이트,젤라틴,규산칼슘,미세결정성셀룰로스,폴리비닐피롤리돈, 셀룰로스,물,시럽,메틸셀룰로스,메틸히드록시벤조에이트,
프로필히드록시벤조에이트,활석，스테아르산마그네슘,미네랄오일등으로 루어진군에서선택된 1종이상일수있으나,이에한정되는것은아니다.상기 약학조성물은또한약학조성물제조에통상적으로사용되는희석제,부형제, 윤활제,습윤제,감미제,향미제,유화제,현탁제,보존제으로이루어진군에서 선택된 1종이상을추가로포함할수있다.
[134] 상기약학조성물,또는상기항체또는이의항원결합단편의유효량은경구 또는비경구로투여할수있다.비경구투여인경우에는정맥내주입,피하주입, 근육주입,복강주입,내피투여,국소투여,비내투여,폐내투여및직장내투여 등으로투여할수있다.경구투여시 ,단백질또는펩타이드는소화가되기 때문에경구용조성물은활성약제를코팅하거나위에서의분해로부터 보호되도록제형화될수있다.또한,상기조성물은활성물질이표적세포로 이동할수있는임의의장치에의해투여될수있다.
[135] 상기약학조성물내의항-쇼1¾2항체또는이의항원결합단편의함유량은 2020/175886 1»（：1^1{2020/002687
16 제제화방법，투여방식，환자의 연령 ,체중,성 ,병적상태，음식 ,투여시간,투여 간격,투여 경로,배설속도및반응감응성과같은요인들에의해다양하게 처방될수있다.예컨대,상기 항-쇼1¾2항체또는이의 항원결합단편의 1일 투여량은 0.001내지 10001¾/1¾,구체적으로 0.01내지 1001¾/1¾,보다구체적으로 0.1
Figure imgf000018_0001
적으로 0.1내지 20 !¾/!¾범위일수있으나이에 제한되는것은아니다.상기 1일투여량은단위용량형태로하나의 제제로 제제화되거나,적절하게분량하여제제화되거나,다용량용기내에 내입시켜 제조될수있다.
[136] 상기 약학조성물은다른혈관신생저해제또는앤지오포이에틴- 2활성화 및/또는과생성과관련된질병의치료제와같은다른약물과병용투여 가능하며,그투여량,투여방법 및다른약물의종류는환자의상태에 따라서 적절하게처방될수있다.
[137] 상기 약학적조성물은오일또는수성 매질중의용액,현탁액,시럽제또는 유화액 형태이거나엑스제,산제,분말제,과립제,정제또는캅셀제등의 형태로 제형화될수있으며,제형화를위하여분산제또는안정화제를추가적으로 포함할수있다.
[138] 특히 ,상기항-쇼1¾2항체또는그항원결합단편을포함하는약학조성물은 항체또는항원결합단편을포함하므로,면역 리포좀으로제형화될수있다. 항체를포함하는리포좀은당업계에 널리 알려진방법에 따라제조될수있다. 상기 면역 리포좀은포스파티딜콜린,콜레스테롤및
폴리에틸렌글리콜-유도체화된포스파티딜에탄올아민을포함하는지질 조성물로서 역상증발법에 의해제조될수있다.（공개특허 10-2015-0089329） 예를들어,항체의므此’단편은디설파이드-교체반응을통해리포좀에 접합될수 있다.
[139] 한편,상기항-쇼1¾2항체또는이의항원결합단편은앤지오포이에틴- 2에 특이적으로결합하므로,이를이용하여 앤지오포이에틴- 2의활성화및/또는 과생성 여부를확인할수있다.따라서,본발명의또다른예는상기항-쇼1¾2 항체또는이의 항원결합단편을포함하는앤지오포이에틴- 2활성화및/또는 과생성 및/또는앤지오포이에틴- 2활성화및/또는과생성과관련된질병의 진단용약학조성물을제공한다.또다른예에서,환자로부터 얻어진생물 시료에상기항-쇼1¾2항체또는이의항원결합단편을처리하는단계;항원-항체 반응여부를확인하는단계;및항원-항체반응이탐지되는경우상기환자를 앤지오포이에틴- 2활성화및/또는과생성증상이존재하거나,
앤지오포이에틴- 2의 활성화및/또는과생성 관련질병을갖는것으로판단하는 단계를포함하는,진단방법또는진단에 정보를제공하는방법을제공한다. 상기 생물시료는환자로부터 얻어진세포,조직,체액등으로이루어진군에서 선택된것일수있다.
[140] 상기항원-항체반응여부를확인하는단계는당업계에 공지된다양한방법을 통하여수행할수있다.예컨대,통상적인효소반응,형광,발광및/또는방사선 검출을통하여하여측정될수있으며,구체적으로,
면역크로마토그래피 (Immunochromatography),
역조직화학염색 (Immunohistochemistry),효소결합면역톱착분석 (enzymeliked immunosorbent assay: ELISA),방사선면역즉정법 (radioimmunoassay: RIA),효소 면역분석 (enzy meimmunoas s ay : EIA),형광면역분석 (Floresence immunoassay:
FI A),발광면역분석 (luminescence immunoassay: LIA),웨스턴블라팅 (Western bloting)등으로이루어진군으로부터선택된방법에의하여측정될수있으나, 이에제한되는것은아니다.
[141] 상기약학조성물의투여또는진단대상환자는인간,원숭이등을포함하는 영장류,마우스,래트등을포함하는설치류등을포함하는포유류일수있다.
[142] 상기앤지오포이에틴- 2활성화및/또는과생성과관련된질병은암;암전이; 미숙아망막병증,각막신생혈관생성증,당뇨병성망막증,맥락막신생혈관질환, 황반변성 (예컨대,연령관련황반변성)등의안질환;천식;류마티스성관절염; 건선;폐렴,만성염증등의염증성질환;고혈압,동맥경화등의심혈관질환또는 패혈증등일수있다.상기암은앤지오포이에틴- 2를과발현하는것일수있고, 고형암또는혈액암일수있으며 ,이에제한되지않지만,편평상피세포암, 소세포폐암,비소세포폐암,폐의선암,폐의편평상피암,복막암,피부암,피부 또는안구내흑색종,직장암,항문부근암,식도암,소장암,내분비선암, 부갑상선암,부신암,연조직육종,요도암,만성또는급성백혈병,림프구 림프종,간세포암,위장암,췌장암,교아종,경부암,난소암,간암,방광암,간종양, 유방암 (경우에따라서삼중음성유방암 (Triple Negative Breast Cancer),결장암, 대장암,자궁내막또는자궁암,침샘암,신장암,간암,전립선암,음문암, 갑상선암,간암,두경부암,뇌암,골육종등으로이루어진군에서선택된 1종 이상일수있다.상기암은원발성암또는전이성암일수있다.
[143]
[144] 실시예
[145] 이하,실시예를통하여본발명을더욱상세히설명하고자한다.이들실시예는 오로지본발명을예시하기위한것으로서,본발명의범위가이들실시예에 의해제한되는것으로해석되지는않는것은당업계에서통상의지식을가진 자에게있어서자명할것이다.
[146]
[147] 실시예 1. Ang2에결합하는항체의선별
[148] Ang2에결합하는항체를선별하기위한항체라이브러리및라이브러리의
준비는한국특허출원 (공개특허 10-2008-0109417호)와같은자체의인간 미감작 scFv (human naive ScFv)라이브러리를이용하였다. 96 -웰면역
늘레。1트에항원 (hAng2-his : RND systems. Cat. No 623-AN/CF, hAng2-Fc :
phamiabcine)을 2 g/m모로 100 [xl씩웰에넣고 4°C오버나이트 (overnight)시킨다. 다음날항원코팅플레이트 (coating plate)는 5mM CaCl2 TBS로 3번워싱한후 2% BSA차단버퍼 (blocking buffer) 200 를넣고실온에서 2시간반응시킨다. 2x YT-TET(tetracycline 10 g/m公)성장배지 (growth medium) 2ml에 XL 1 -Blue스톡 (stock) 50 를넣고 37 °C, 200 rpm에서 2시간정도키운후 13 ml를더첨가하여 OD600이 0.5가될때까지키워준다.차단 (blocking) 2시간이지나면 IX 5mM CaCl2 TBS로 3번워싱한다.워싱한각웰에파지라이브러리그룹 (phage library group)을합쳐파지라이브러리양과 4% BSA양을동일하게섞은후 200 씩 첨가하여실온에서 30분간로킹 (rocking)한후 2시간을반응시킨다.파지 라이브러리반응이끝나면상층액은버리고 0.1% TBST(5mM CaCl2)로 5번 워싱하고 TBS(5mM CaCl2)로 5번워싱해각웰에 100 mM TEA(trimethylamine) 100 를넣은후실온에서 10분간흔들어준다. 10분지나면각웰에 1M Tris(pH 7.5) 50 를넣고섞어준다.상등액 (supernatant)은 OD_ 0.5가된 XL 1 -blue 10ml에넣어 37OC에서 30분간감염 (infection)시킨다.감염이끝나면 100나1는 아웃풋타이터 (output titer)로사용하고나머지는 6,000 rpm, W분간원심분리 한다.상증액은버리고침전물은라지스퀘어늘레이트 (large square plate: CM 34 g/m公 + 1% Glucose)에스프레딩 (spreading)하여 30°C에서오버나이트 인큐베이션 (ovemingt incubation)한다.아웃풋타이터로남겨둔 100(x1는 1/10, 1/100, 1A000으로희석 (dilution)하여 CM플레이트에스프레딩하여 37OC에서 오버나이트한다.다음날스퀘어플레이트에자란콜로니 (colony)는 2x YT배지 50ml을넣은후룹 (loop)을사용하여긁어모은후 6000 rpm, 10분원심분리하여 상증액은버리고침전액 (precipitate)에대하여 1차패닝스톡 (panning stock)을 만들고 2x YT배양배지 (growth media: CM 34 g/m公 + 1% Glucose) 100 ml을 500 ml삼각플라스크에넣은후 0D6QQ 0.2되게세포를넣고 200 rpm, 37OC에서 0D 0.5가될때까지키워준다. OD_값이 0.5가될때까지세포를배양한후에헬퍼 파지 (helper phage: M13K07 mutant)을세포의 20배가되게넣어준다.헬퍼 파지를넣고 37°C, 30분감염 (infection)시킨후 6000 rpm, 10 min원심분리한다. 상층액을버리고세포는 2xYT배지 (CM 3A/ngM + Kan. 70/zg/m^ + ImM IPTG + 5mM MgCl2) 100ml로교체하여넣어준후 200 rpm, 30OC,오버나이트한다.
다음날자란세포는 7000 rpm, 10분,원심분리하고같은방법으로한번더 원심분리한다.모은상층액은상층액의 1/5 (v/v) 20% PEG/2.5m NaCl를넣고 아이스 (ice)에서 1시간침전시킨다.침전시킨후 9000 rpm, 1시간원심분리한다. 상증액은버리고 TBS 3ml로침전액 (precipitate)을풀어준후 0.45 [xm필터 (filter)에여과한후 4 에보관하여이를다음번패닝 (panning)과정에서 사용한다.이과정을 3~4번반복하여항원에결합하는항체를 ELISA를수행하여 확인하였다.
[149]
[150] 실시예 2. Ang2에특이적으로결합하며 Tie2와의결합을중화하는 Monoclonal ScFv phage선별 (binding ELIS A/competitive ELISA) [151] 패닝 (panning)과정이끝나면마지막라운드세포스톡 (round cell stock)을 CM 아가플레이트 (agar plate)에 200 500개의콜로니가형성될수있도록희석하여 깔아준후 37OC에서오버나이트한다.다음날콜로니 (colony)가자라면 96웰딥 늘레이트 (96 well deep plate)에 2xYT배지 (CM 34 g/m公 + 1% glucose) 200 를 넣고각웰에콜로니를하나씩넣은후 37OC, 3000rpm에서오버나이트한다. 다음날새로운 96웰딥플레이트에 2xYT배지 (CM 34/保/ M + 1% glucose)
200 를넣고각웰에전날키운세포를각웰에마다 20M씩넣은후 37OC, 3000rpm, 1시간 10분키운다.나머지세포는 50%글리세롤 100 씩첨가하여 -70OC에보관한다.세포가자라면헬퍼파지 1 와 2xYT배지 19 를섞은후각 웰에 20 씩첨가후 37OC에서 30분인큐베이션한다.인큐베이션이끝나면 3000rpm, 10분원심분리한다.상층액을버리고 2xYT배지 (CM 34/保/ M + Kan. lOim/mi + ImM IPTG + 5mM MgCl2) 200 를넣고메가그로우 (megagrow)에서 30°C, 3000rpm에서오버나이트한다.
[152] Ang2에특이적으로결합하는파지를선별하기위하여먼저, 96웰면역
늘레이트에 Ag (hAng2-Fc, hAngl-his: RND systems. Cat. No 923-AN/CF또는 mAngl-Fc, phmiabcine) 1 g/m모로만들어 100 Avell씩넣고 4°C에서
오버나이트한다.다음날전날키운세포는 3000rpm, 10분원심분리하여 4OC에 보관한다.깔아놓은 Ag은 0.1% TBST(5mM CaCl2)로 3번워싱한후에 2% BSA 차단버퍼 (blocking buffer) 200 씩넣은후 25OC 2시간인큐베이션한다.차단 (blocking)이끝나면 0.1% TBST(5mM CaCl2)로 3번워싱한다.각웰에 4% BSA 50 와다운 (down)하여 4OC에서보관했던파지 50 을섞은후실온에서 1시간 흔들어반응시킨다.파지결합 (phage binding)이끝나면 0.1% TBST(5mM CaCl2 )로 3번워싱한후에 HRP-접합마우스항- M13항체 1:3000 (HRP-conjugated mouse anti-M13 Ab) (Sino, 11973-MM05)을 100 씩넣은후 25°C 1시간 반응시킨다.반응이끝나면 0.1% TBST(5mM CaCl2)로 3번워싱한후에
TMB(#BD TMB substrate reagent set 555214) 100 씩넣은후 3~5분발색시킨후 정지용액 (stop solution)을 50 씩넣은후에 ELISA리더 (ELISA reader)로 분석한다.
[153]
2020/175886 1»（：1/10公020/002687
20
[154] [표 1]
義¾2 항원에 특이적으로결합하는
Figure imgf000022_0001
축정한 결과
Figure imgf000022_0002
[155]
[156] 선별된항체의염기서열은다음표 2,표 3과같다
[157]
2020/175886 1»(：1/10公020/002687
21
[158] [표 2]
^2항원에 특이적으로 결합하는항체의 051서열
Figure imgf000023_0001
[159]
Figure imgf000024_0001
[160]
Figure imgf000025_0001
[161]
[162] [S3]
Ang2 ¾-¾ofl fo]¾I 3«-S r 1-31] £1 7¾¾¾ A]¾
Figure imgf000026_0001
[163]
Figure imgf000027_0001
tt [164]
Figure imgf000028_0001
[165]
Figure imgf000029_0001
[166]
[167] Competitive ELISA에서는 Ang2/Tie2의결합을중화하는파지를선별하기
위하여, 96웰면역늘레이트에 Ag (hTie2-Fc : phrmabcine) 1 g/m모로만들어 lOO^/well씩넣고 4OC에서오버나이트한다.깔아놓은 Ag은 IX PBS로 2번 워싱한후에 3% BSA차단버퍼 (blocking buffer) 200 씩넣은후 25OC 2시간 인큐베이션한다.차단 (blocking)이끝나면 0.1% PBST로 2번워싱한다.각웰에 hAng2-his(RND, 623-AN/CF) 5/zg/M 20 와다운 (down)하여 4OC에서보관했던 파지를부피별로 (80 , 40M+1X PBS 40¹, 20¹+lX PBS 60¹)섞은후 실온에서 1시간흔들어반응시킨다.파지결합 (phage binding)이끝나면 0.05% PBST로 3번워싱한후에항- Ang2마우스항체 (RND, MAB098)를 0.5 gM로 실온에서 1시간반응시킨다.항체결합이끝나면, 0.05% PBST로 3번워싱한 후에 HRP-접합마우스항- IgG항체 1:2000 (HRP-conjugated Goat anti-mlgG Ab) (RND, HAF007)을 100 씩넣은후 25°C 1시간반응시킨다.반응이끝나면 0.05% PBST로 3번워싱한후에 TMB(#BD TMB substrate reagent set 555214) 100 씩 넣은후 3~5분발색시킨후정지용액 (stop solution)을 50 씩넣은후에 ELISA 리더 (ELISA reader)로분석한다.그결과를 [도 1]에나타내었다.도 1도면에서 보여지듯이선별된파지들은 Tie2/Ang2의결합을중화시키는능력이있음을 확인하였다.
[168]
[169] 실시예 3. Ang2에대한고친화력을가진항체선별 (Off-rate screening)
[17이 선별된항체의항원에대한결합력을 Octet (Fortebio)을이용하여측정하였다. 이를위하여 Ang2를바이오센서 (biosensor)에고정 (immobilize)한후 scFv 형태로발현된후보항체를넣고결합시킨후,해리속도상수를측정하였다.그 결과를 [표 4]에나타내었다.
[171]
[172] [표 4]
Ang2항원에 특이적으로결합하는항체의 해리속도상수
Figure imgf000030_0001
[173]
[174] 실시예 4.항- Ang2항체발현
[175] 선별한 scFv파지의 IgG형태로의전환은분자생물학적기법을사용하여
수행하였다.선별한 E. Coli클론에서파지미드 (Phagemid)를추출,모이기법을 사용하여가변영역을증폭하였다.증폭한중쇄가변영역을중쇄불변영역을 포함하는발현벡터 (Invivogen, pfusess-hchgl)에삽입하고,증폭한경쇄 가변영역은경쇄불변영역을포함하는발현벡터 (Invivogen, pfuse2ss-hclk)에 삽입하여, IgG형태의 DNA클로닝을완료하였다.
[176] IgG의일시발현은 Expi293F발현시스템키트 (Expi293F expression system kit:
Thermo Fisher Scientific, US)을사용하였다. Kit에포함된 Expi293세포를전용 배지를사용하여 37°C, 5% C02환경하에서 125 rpm오비탈쉐이커상부유배양 하였다. 3일마다 3 X 105 cells/ml되도록계대배양하였으며 ,발현벡터
도입시에는 3 X 106 cells/ml되도록세포수를조정한후사용하였다.유전자 도입은전용시약인 Expifectamine을사용하였으며 ,세포현탁액 1 ml당발현 벡터 DNA 1 과 Expifectamine 2.7 을함유하는 Lipid-DNA복합체를제작, 세포현탁액에첨가하였으며,도입 16-18시간후인헨서 (Enhancer) 1/2를 첨가하여발현을유도하였다.이후동일조건에서 3-4일간배양후원심분리하여 IgG함유상등액을취하였다.
[177]
[178] 실시예 5.항- Ang2항체의정제
[179] 취득한상등액을 Protein A컬럼 (GE Healthcare)에주입하여친화력 크로마토그래피를통해 IgG를정제하였다.컬럼을 20 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA (pH 7.0)으로평형화시킨후상등액을주입 , 50 mM Tris-HCl, 500 mM NaCl, 5 mM EDTA, 0.2% polysorbate 20 (pH 7.0)용액으로세정한후, 50 mM NaCl, 0.1 M glycine-HCl (pH 3.5)로용출후 1 M Tris로중화하였다.용출된 단백질은 MWCO 10,000 spectra/por dialysis membrane (Spectrum Labs, US)를 사용한투석과정을통해 PBS로용매를교체하였다.이후 Vivaspin (Satorius,
DE)을사용하여필요농도로농축하여분주후 -80OC에서보관하였다.
[180] 정제후각항체는비환원및환원 LDS샘플버퍼 (Non-reducing및 Reducing LDS sample buffer: Thermo Fisher Scientific)에처리하여 NuPAGE System (Thermo Fisher Scientific)를사용하여전기영동하였다.그결과 50 kDa의중쇄및 25 노 의경쇄사슬을포함하는총분자량약 150노 의 IgG를획득하였다. (도 2)
[181]
[182] 실시예 6.항- Ang2항체의결합특이성분석
[183] 결합특이성분석에는 ELISA법과 Biacore T200 system (GE Healthcare Life
Sciences)을사용하여결합상수를측정하였다.
[184] 96웰이뮤노플레이트 (Nunc, US)의각웰에 1收/ 의 His-tag된인간 (R&D
systems, 623-AN/CF),마우스 (sino, 50298-M07H) Ang2단백질는인간 (R&D systems, 923-AN/CF),마우스 (sino, 50300-M07H) Angl용액을 100나 1첨가후 4OC에서하룻밤정치하여흡착시켰다.다음날 0.05% Tween-20함유 PBS (이하 PBST)로 3회세정하고, 2% BSA/PBST용액을각웰당 200 첨가후상온에서 2시간정치하여블로킹하였다. PBST로 3회세정후각시험항체용액을 농도별로 WO pl씩첨가하여상온에서 1시간결합시킨후, PBST로 3회세정, 1:2000의비율로희석한 HRP-접합된 goat anti-human IgG(kappa) (bethyl lab #A80-115P)를 WO ^il첨가하여상온에서 1시간반응시켜결합을유도하였으며, PBST로 3회세정후 100 의 TMB기질시약을사용하여발색시켰다.발색 반응은 50 의 2N H2S04를첨가하는것으로정지시켰으며 , Sunrise
마이크로플레이트리더 (TECAN, CH)를사용하여특이적흡광도 OD45()-630을 측정하였다 (도 3).도 3에서보여지듯이선별된항체는인간과마우스 Ang2에 특이적으로결합하며,인간과마우스 Angl에는결합하지않는다.
[185] 선별된항 Ang2항체의결합력을분석하기위해인간 Ang2와마우스 Ang2에 대한친화도분석을 BIACORE® T200 (GE Healthcare)을사용하여분석하였다. protein A센서칩을사용하였으며,제조자의매뉴얼에따라실험하였다.분석의 세부적인조건은다음과같다. Porotein A고정은 2000 RU(Response Unit)이고, 항 Ang2항체후보는 25 RU로결합하였으며,인간 Ang2와마우스 Ang2를 다양한농도로결합하였다.분석시작농도는각각 100nM과 150nM이다.분석은 유속 30 /min으로진행하며인간 Ang2의결합구간은 300초,해리구간은
2000초로측정하였으며,마우스 Ang2는결합구간 300초,해리구간 1000초로 분석하였다.분석모델은 1:1결합모델을사용하여분석하였다.분석결과는 [표 5]에나타내었다.
[186]
[187] [표 5]
Figure imgf000032_0001
[188]
[189] 실시예 7.항- Ang2항체의중화능력확인 (Ang2/Tie2, Ang2/integrin)
[190] 96웰이뮤노플레이트 (Nunc, US)의각웰에 1收/ 의인간 Tie2-Fc (R&D
systems, 313-TI),마우스 Tie2-Fc(R&D systems, 762-T2-100),인테그린 a3/(31(R&D systems, 2840-A3-050)그리고인테그린 a5/(31(R&D systems, 3230-A5-050)용액을 100 ^il첨가후 4OC에서하룻밤정치하여흡착시켰다.다음날 0.05% Tween-20 함유 PBS (이하 PBST)로 4회세정하고, 2% BSA/PBST용액을각웰당 200나1 첨가후상온에서 1시간정치하여블로킹하였다.항 Ang2후보항체는
최고 1000nM에서 4배로단계희석하여일부는 biotin-결합된인간 Ang2단백질이 최종 100ng/mL이되도록만들고,일부는 biotin-결합된인간 Ang2단백질이최종 625ng/mL이되도록만들어서미리상온에서 1시간결합하였다.블로킹이끝난 플레이트는 PBST로 4회세정후미리항원항체반응을유도한시료를넣어 , 상온에서 1시간결합시킨다.위의플레이트를 PBST로 3회세정, 1:200의비율로 희석한 HRP-접합된 streptavidin (R&D systems, DY998)를 100 [xl첨가하여 상온에서 1시간반응시켜결합을유도하였으며, PBST로 4회세정후 100 의 TMB기질시약을사용하여발색시켰다.발색반응은 50 의 2N H2S04를첨가 하는것으로정지시켰으며, Sunrise마이크로플레이트리더 (TECAN, CH)를 사용하여특이적흡광도 OD45。을측정하였다 (도 4,도 5).도 4에서보여지듯이 선별된항체는인간과마우스모두에서 Ang2/Tie2결합을중화하였다.
중화능력은 1C 값을구하여수치화하였으며, [표 6]에나타내었다.또한 도 5에서보여지듯이인테그린/ Ang2의결합도중화하였다.인테그린/ Ang2의 중화능력은 IC5。값을구하여수치화하였으며，[표기에나타내었다.
[191] [192] [표 6]
Figure imgf000033_0001
[193]
[194] [표 7]
Figure imgf000033_0002
[195]
[196] 실시예 8.항- Ang2항체의 Ang2ATie2신호억제효과분석 (p-Tie2 assay)
[197] 인간 Tie2과발현세포 (lxlO5)를 96웰플레이트에넣고, 37OC이산화탄소가
공급되는인큐베이터에서하룻밤키운다.위의세포를혈청없는배지에서 하룻밤키워혈청기아상태를만든다.인간 Ang2 (5 g/M)와다양한농도의 항- Ang2를상온에서 1시간미리반응시킨후,세포가들어있는플레이트에넣고 20분간반응시킨다.이때항체가없고, Ang2단백질만들어있는웰을포함시켜 신호억제효과분석의기준값으로삼는다.세포는용해완충액을넣어용해시킨 후정량한다.인산화반응을즉정하기위하여 R&D systems사의 Human
Phospho-Tie2 Duoset IC ELISA(R&D systems, DYC2720-5)를사용하였다. 96웰 이뮤노플레이트 (Nunc, US)의각웰에 4 인간 Tie2캡쳐단백질을첨가후
4OC에서하룻밤정치하여흡착시켰다.다음날희석제를웰당 200 첨가후 상온에서 2시간정치하여블로킹하였다.세포용해물을 50 g씩넣고상온에서 2시간결합하였다.반응이끝난후,항포스포티로신항체를 2700:1로희석하여, 상온에서 2시간결합시킨다.반응이끝난플레이트는 TMB기질시약을 사용하여발색시켰다.발색반응은 50 의 2N H2S04를첨가하는것으로 정지시켰으며 , Sunrise마이크로플레이트리더 (TECAN, CH)를사용하여특이적 흡광도 OD45。을측정하였다 (도 6).도 6에서보여지듯이항체의농도가
높아질수록인산화가줄어드는것을확인할수있었다.인산화정도는 IC5Q값을 구하여수치화하였으며，[표 8]에나타내었다.
[198] [199] [표 8]
Figure imgf000034_0004
[200]
[201] 실시예 9:친화도증진을위한변이체제작및선별
[202] 항-쇼1¾2항체클론 No.8의친화도증진을위해항체최적화를수행하였다.
.8의원래 DNA서열을 70%보존하며 랜덤화（ 11 10111뇨句시키는
소프트-랜덤화（80 11（10111뇨 011）법을활용하여 , ^.8의경쇄 00113와중쇄 00113에무작위변이를도입한프라이머를제작하였다.이를사용한모01을통해 변이가도입된경쇄가변영역,중쇄가변영역코딩 DNA단편을확보하였다.이 DNA단편을각각 ^.8 파지파지
파지미드의중쇄가변영역과치환하여
Figure imgf000034_0001
파지 DNA라이브러리를제작하였다.
[203] 변이체 파지 DNA라이브러리를페놀-클로로포름정제후전기천공법을 사용하여대장균
Figure imgf000034_0002
질전환하였다.형질전환효율분석및, DNA 서열분석을통해다양성이확보된것을확인한후, 500 1111규모로배양하여파지 발현을유도하고,모묘（3 -침전법을이용하여경쇄와중쇄의 00113변이체 파지라이브러리를제작하였다.
[204] 각변이체 파지라이브러리를사용하여실시예 1에서제시한방법으로 바이오패닝을실시하였다.그후선별과정에서는결합을유지하는능력의 정량적평가지표로서
Figure imgf000034_0003
해리속도상수 1«1 를측정하였다.선별된최적화 클론 3종에대한아미노산서열（표 9, 10）과해리속도상수측정결과（표 11）를 도시하였다.
[205]
[206] [표 9]
Figure imgf000034_0005
[207]
[208] [3L10]
Figure imgf000035_0001
[209]
Figure imgf000036_0001
2020/175886 1»(：1^1{2020/002687
35
[21이
Figure imgf000037_0002
[211]
[212] [표 11]
Figure imgf000037_0003
[213]
[214] 실시예 10:
Figure imgf000037_0001
[215] 최적화된항 Ang2항체 (No.04)를대장균에서발현시키기위하여 pET-22b 벡터 (Novagen)에클로닝하였다. BL21(De3)에형질전환하여생성된콜로니를 선별하여 LB (Lysogeny broth)배지에 100；Mg/m公 Ampicillin을첨가하여 37°C, 200rpm조건에서전배양한대장균을 100 g/M의 Ampicillin항생제를포함한 LB배지에 1%접종하였다 . 37°C, 200rpm조건에서배양하여 OD600=0.6-0.8에 이르면배양기의온도를 20OC으로내리고 0.5 mM IPTG을첨가하여 16시간 배양하였다.
[216] 배양한대장균을 8000rpm, lOmin원심분리하여집균하였다.배지를제거한후 50 mM Tris-Hcl pH 7.4, 150 mM NaCl, 1 mM Phenylmethylsulfonyl fluoride
(PMSF)의 lysis buffer을 cell무게 g당 W ml buffer를이용하여 resuspension 하였다.초음파파쇄기를사용하여 Power:20W, rest: 3Sec, Work: 3Sec, Time:
5Min조건에서 Cell을파쇄하였다.파쇄된 cell을 l WOOrpm에서 1시간원심분리 하여상증액과침전된물질을분리하였다.
[217] ScFv는 insoluble한형태로발현되어 refolding을진행하기위해 Pellet wash를 진행하였다. 50 mM Tris-Hcl pH 7.4, 150 mM NaCl buffer를이용하여
Homogenizer으로균질화시킨후 l lOOOrpm, lh원심분리하는방법으로 pellet를 2번 wash하였다. 50 mM Tris-Hcl pH 7.4, 150 mM NaCl, 2 M Urea, 0.5% Triton X-100 buffer를이용하여 pellet내에남아있는대장균유래의물질들을제거 하였고 50 mM Tris-Hcl pH 7.4, 150 mM NaCl buffer으로 3번워싱을반복하였다. 50 mM Tris-Hcl pH 7.4, 150 mM NaCl, 8 M Urea, 10 mM DTT buffer으로 inclusion body를 resuspension시킨후 30 min정도반응시켜 ScFv를 unfolded한형태로 만들고 l lOOOrpm, lh Centrifuge하여침전물질과분리하였다.
[218] ScFv항체를 Step Dialysis를통해 Urea를제거하여 Refolding을유도하였다.
Dialysis buffer는 50 mM Tris-Hcl pH 7.4, 150 mM NaCl를기본으로하여
Urea농도를 1/2씩낮춰가면서진행하였다.구조가대부분형성되는 4-2-1 M Urea 농도구간에서 0.1 M L- Arginine를첨가하여 Aggregation형성을억제하여
Refolding을진행하였다. Refolding이완료된 ScFv항체는 HisTrap과 Capo L column을이용하여분리정제진행하였다.
[219] 분리정제는다음과같은순서로진행하였다. AKTA Prime(GE healthcare)
System을이용하여정제하였으며 , 5 ml HisTrap Packing Column을사용하였다.
50 mM Sodium Phosphate, 400 mM NaCl, 10 mM Imidazole pH 7.4 buffer으로 Histrap column Volume의 10 Column Volume를려평형화시킨후, ScFv항체 샘물을 5 ml/min의유속으로 Histrap Column에흐르게하여 Column내 Resin과 결합시켰다. 50 mM sodium Phosphate, 400 mM NaCl, 10 mM Imidazole pH 7.4 buffer으로 Resin에존제하는비특이적인결합물질을제거해주기위해약 10 Column Volume를려준후, 50 mM Sodium Phosphate, 400 mM NaCl, 300 mM Imidazole pH 7.4 buffer를농도 Gradient하게를려 ScFv항체가 Elution되어나오게 하였다. Elution된항체시료는 5 ml Capto L column을이용하여다음정제를 진행하였다· PB S (Pho sphate-buffered saline) pH 7.4 buffer으로 Capto L Column을 평형화시킨후,샘늘을 5 ml/min의유속으로 Capto L column에흐르게하여 Column내 Resin과결합시킨다. PBS Buffer를약 10 Column Volume를려 비특이적결합물질을제거하였다. 0.1 M Citric Acid, 0.2 M Na2HP04 pH 2.6 Buffer을 10 Column Volume를려 ScFv항체가 Elution되어나오게하였다. (도 7)은 최종적으로정제후얻은단백질을 SDS-PAGE를수행한결과이다.그결과 순도가높은 ScFv항체를얻을수있었다.
[22이
[221] 실시예 11 :최적화된항 Ang2 ScFv항체의결합특이성분석
[222] 결합특이성분석에는 ELISA법과 Octet system (Pall Fortebio LLC. US)을
사용하여결합상수를측정하였다.
[223] 96웰이뮤노플레이트 (Nunc, US)의각웰에 1收/ 의 His-tag된인간 Ang2 단백질 (R&D systems, 623-AN/CF)용액을 100 첨가후 4OC에서하룻밤 정치하여흡착시켰다.다음날 0.05% Tween-20함유 PBS (이하 PBST)로 3회 세정하고, 2% BSA/PBST용액을각웰당 200 첨가후상온에서 2시간 정치하여블로킹하였다. PBST로 3회세정후각시험항체용액을농도별로 100 씩첨가하여상온에서 1시간결합시킨후, PBST로 3회세정, 1:1000의비율로 희석한 HRP-접합된항- His태그모노클로날항체 (HRP-conjugated anti-His tag monoclonal antibody: MAB050H, R&D systems, US)를 100 [xl첨가하여상온에서 1시간반응시켜결합을유도하였으며, PBST로 3회세정후 100 의 TMB기질 시약을사용하여발색시켰다.발색반응은 50 의 2N H2S04를첨가하는것으로 정지시켰으며 , Sunrise마이크로플레이트리더 (TECAN, CH)를사용하여특이적 흡광도 OD45。을측정하였다 (도 8).도 8에서보여지듯이정제된항체는인간 Ang2에결합한다.
[224] 정제된 ScFv항체의인간 Ang2에대한결합력을 Octet system (Fortebio Inc.
US)를사용하여측정하였다.이를위하여항- Ang2항체를바이오센서에고정화 하고,인간 Ang2의농도별결합동역학을측정하여결합속도상수 (ka),해리 속도상수 (kdis)및결합상수 (KD)를산출하였다 (표 12).
[225]
[226] [표 12]
Figure imgf000039_0001
[227]
[228] 실시예 12:선별된항- Ang2 ScFv항체의 in vivo효능분석 (CNV mouse model)
[229] 항- Ang2 scFv항체의신생혈관형성의억제효능이있는지를확인하기위하여 , laser-induced choroidal neovascularization mouse모델에서약효시험을
실시하였다.그효력은상용약물인 aflibercept을대조군으로시험하였다. 마우스를케타민으로전신마취시킨후,마취점안제를안구에점안하여추가 국소마취시키고,산동제를점안하여산동유도한다.마우스를제물대위에 올려놓고 Micron-IV를이용하여 CNV유도조건 (wavelength 532 nm, diameter 50 [xm, duration 80 mS, power level 200 mW)에따라 laser burn을유도하여 Bruch’s membrane을파괴시킨다. Laser burn유도과정에서 bubbling이관찰되지않은 병변은 unsuccessful laser bum으로분류하고 Gong Y.등이제안한기준을 modification한 exclusion criteria에근거하여결과분석및통계처리과정에서 제외하였다.
[23이 CNV유도및약물에의한 angiogenesis억제효과를확인하기위해 CNV유도 후 W일에마우스를케타민으로전신마취시킨후,형광조영제를복강 주사하였다.마취점안제를안구에점안하여추가국소마취를시킨후, 산동제를점안하여산동을유도하였다.마우스를제물대위에올려놓고
Micron-IV의 imaging camera로 fundus에영상을맞준후,해당안구에윤활젤을 점안한뒤 OCT의렌즈를마우스의각막과접촉시켰다. FFA/OCT촬영을실시한 후마우스안구에항생제점안액을한방울점안하였다. FFA및 OCT이미지에 대한분석은 ¾nage-J，프로그램을이용하여실시하였다.단, Gong Y.등이 제안한 exclusion criteria에해당하는 CNV병변은최종결과및통계분석 과정에서제외되었다.
[231] 시신경회복효과를확인하기위해망막전위도 (ERG)검사를진행하였다. 마우스는 ERG평가 12시간전부터암실에서암순응을유도하였다.평가 당일 (CNV유도후 11일)에럼푼®과케타민®으로전신마취시킨후,알카인®을 안구에점안하여추가국소마취를유도하고산동제를점안하여산동을 유도하였다.마우스를 ERG제물대위에올려놓고 ERG의 probe를각각꼬리 , 머리및각막에접촉시켰다. ERG는단일 flash자극 (0.9 log cds/m2 (10 responses/intensity))에대한망막전위도변화로즉정하였다. ERG평가가 종료되면마우스안구에토브렉스를한방울점안하였다. ERG분석은 ‘LabScribeERG (iWorx DataAcquisition Software)’프로그램을이용하여 실시하였다.단, Gong Y.등이제안한 exclusion criteria에해당하는안구는최종 결과및통계분석과정에서제외되었다.
[232] CNV mouse model에대한항- Ang2 ScFv항체의신생혈관감소효능결과는 [도 9]에나타내었다 . FFA상에서관찰된 CNV병변의크기에있어서, ScFv를 10 / 투여한시험군에서 CNV대조군대비통계적으로유의한감소가 관찰되었으며 (P<0.01), aflibercept을투여한시험군과비교할때에도보다뛰어난 효과를나타내는것으로확인되었다. [도 1이에서 OCT측정으로병변의부피를 CNV대조군과비교할때에도, ScFv를투여한모든시험군에서통계적으로 유의하게병변의부피를농도의존적으로감소시키는것을확인하였다 (각각 P<0.01, P<0.0001, P<0.05).또한, [도 11]에서 scotopic-ERG로망막전위도를 CNV 대조군과비교하였을때,통계적으로유의한 B-wave amplitude의증가가 관찰되었다 (각각 P<0.0001).
[233]
[234] 실시예 13:선별된항- Ang2항체의항종양효능분석 (TNBC model)
[235] 항- Ang2항체의신생혈관형성의억제효능이 있는지를확인하기위하여 , human triple negative breast cancer (TNBC)모델 (MDA-MB-231)에서항종양 효력시험을실시하였다.항- Ang2항체의항종양활성을평가하기위해인체 유래유방암세포주인 MDA-MB-231을왼쪽옆구리에이식한 NSG마우스에서 isotype control, nesvacumab,항- Ang2항체치료제들의미정맥투여에의한항암 약효를평가하고자하였다.실험은미국 Champions Oncology에의뢰하여 진행하였다.
[236] 인체유래유방암세포주 MDA-MB-23 l(breast cancer cell line)를해동
(thawing)한후 C02인큐베이터 (Forma, USA)내에서온도 37X：와 C02농도 5%로 맞춰서배양하였다.배양최종일에모든암세포를수거하여계수하고무혈청 배지 (serum-free media)를이용하여세포농도를 lxlO8 cells/ml로조절하였다. 이렇게조절된세포배양액을마우스왼쪽옆구리에 0.1 ml(lxl07 cells/mouse)씩 주입하였다.마우스의군분리는종양의부피가 W0-150 _3가될때각 처리군당 8마리씩지그재그방식으로군분리를실시했다.군분리직후약물의 투여를시작했다.음성대조군에는 isotype control을 10 mg/ml,항- Ang2항체 처리군에는 3, 10 mg/ml, Nesvacumab처리군에는 3, 10 mg/ml을각각주 2회 빈도로 3주간정맥주사방식으로투여하였다.약물투여후주 2회씩 20일동안 종양부피를측정하였다.측정과정중 mouse의임상적독성반응은보이지 않았다.항- Ang2항체의항종양효과는 10 mg/kg처리군에서약 70%의종양성장 억제효과를보였고,대조약물인 Nesvacumab 10 mg/ml처리군대비약 2배의 항종양효과를보였다.종양부피의계산식은다음과같다. Tumor Volume (TV) = width2 x length x 0.52.각개체별무게는평균 5%정도의고른증가주세를 보임으로써특별히독성반응은없는것으로확인되었다 (도 12).
[237]
[238] [표 13]
항-쇼|¾2항체 투여에 의한 #¾=성장억제 효과
Tumor Volume - Mean
Figure imgf000042_0001
세
Figure imgf000042_0003
Figure imgf000042_0004
Figure imgf000042_0005
[239]
[24이 [표 14]
날짜별 마우스의 체중 변화 (8)
Body Weight - Mean
Figure imgf000042_0002
Figure imgf000042_0006
Figure imgf000042_0007
[241]
산업상이용가능성
[242] 본발명에따른항-쇼1¾2항체또는이의항원결합단편은쇼1¾2에목적하는 결합력을나타내며,목적하는암/종양또는혈관신생저해제및
엔지오포이에틴- 2활성화및또는과생성과관련된질병의 예방또는치료에 유용하게사용될수있다.본발명을통해기존의쇼1¾2를표적으로하는 치료제와다른표적포인트를가진치료제를개발함으로써,종양을치료하는데 있어서기존치료제와의병용치료및단독치료법을제공할수있다.
[243] [244] 이상으로본발명의내용의특정한부분을상세히기술하였는바,당업계의 통상의지식을가진자에게있어서,이러한구체적기술은단지바람직한 실시양태일뿐이며,이에의해본발명의범위가제한되는것이아닌점은 명백할것이다.따라서,본발명의실질적인범위는첨부된청구항들과그것들의 등가물에의하여정의된다고할것이다.
[245]
서열목록 Free Text
[246] 전자파일첨부하였음.

Claims

2020/175886 1»（：1/10公020/002687 42 청구범위
[청구항 1] 서열번호 1, 7, 13, 19및 25로구성된군에서선택되는중쇄 00111,
서열번호 2, 8, 14, 20및 26으로구성된군에서선택되는중쇄 0이12,및 서열번호 3, 9, 15, 21, 27, 51, 52및 53로구성된군에서선택되는중쇄 00113를포함하는중쇄가변영역 ,및
서열번호 4, 10, 16, 22,및 28로구성된군에서선택되는경쇄 0이11, 서열번호 5, 11, 17, 23및 29으로구성된군에서선택되는경쇄 00112,및 서열번호 6, 12, 18, 24및 30로구성된군에서선택되는경쇄 00113을 포함하는경쇄가변영역을포함하는,쇼1¾2（쇼1 0]30 1111-2）에결합하는 항체또는이의항원결합단편.
[청구항 2] 제 1항에 있어서,
서열번호 1의중쇄 00111,서열번호 2의중쇄 00112및서열번호 3의중쇄 0^13을포함하는중쇄가변영역,서열번호 4의경쇄 0^11,서열번호 5의 경쇄 00112,및서열번호 6의경쇄 00113를포함하는경쇄가변영역 , 서열번호 7의중쇄 00111,서열번호 8의중쇄 00112및서열번호 9의중쇄 0^13을포함하는중쇄가변영역,서열번호 10의경쇄 0^11,서열번호 11의경쇄 0이12,및서열번호 12의경쇄 0이13를포함하는경쇄 가변영역,
Figure imgf000044_0001
열번호 14의중쇄 00요2및서열번호 15의 중쇄 00113을포함하는중쇄가변영역,서열번호 16의경쇄 00111, 서열번호 17의경쇄 00112,및서열번호 18의경쇄 00113를포함하는경쇄 가변영역,
Figure imgf000044_0002
열번호 20의중쇄 00요2및서열번호 21의 중쇄 0이13을포함하는중쇄가변영역,서열번호 22의경쇄 0이11, 서열번호 23의경쇄 00112,및서열번호 24의경쇄 00113를포함하는경쇄 가변영역,
Figure imgf000044_0003
열번호 26의중쇄 00요2및서열번호 27의 중쇄 00113을포함하는중쇄가변영역,서열번호 28의경쇄 00111, 서열번호 29의경쇄 00112,및서열번호 30의경쇄 00113를포함하는경쇄 가변영역,
Figure imgf000044_0004
열번호 14의중쇄 00요2및서열번호 51의 중쇄 00113을포함하는중쇄가변영역,서열번호 16의경쇄 00111, 서열번호 17의경쇄 00112,및서열번호 18의경쇄 00113를포함하는경쇄 가변영역,
Figure imgf000044_0005
열번호 14의중쇄 00요2및서열번호 52의 중쇄 00113을포함하는중쇄가변영역,서열번호 16의경쇄 00111, 서열번호 17의경쇄 00112,및서열번호 18의경쇄 00113를포함하는경쇄 2020/175886 1»（：1^1{2020/002687
43 가변영역,
서열번호
Figure imgf000045_0001
열번호 14의중쇄 00요2및서열번호 53의 중쇄 00113을포함하는중쇄가변영역,서열번호 16의 경쇄 00111, 서열번호 17의경쇄 00112,및서열번호 18의 경쇄 00113를포함하는경쇄 가변영역을포함하는항체또는이의 항원결합단편.
[청구항 3] 제 1항에 있어서,서열번호 32, 36, 40, 44, 48, 55, 57, 59및 61로구성된 군에서선택되는중쇄가변영역을포함하는항체또는이의항원 결합단편.
[청구항 4] 제 1항에 있어서,서열번호 34, 38, 42, 46및 50으로구성된군에서
선택되는경쇄가변영역을포함하는항체또는이의 항원결합단편.
[청구항 5] 제 1항내지제 4항중어느한항의 항체또는이의항원결합단편을
코딩하는핵산.
[청구항 6] 제 5항에 있어서,중쇄가변영역을코딩하는서열번호 31, 35, 39, 43, 47,
54, 56, 58및 60로구성된군에서선택되는핵산.
[청구항 7] 제 5항에 있어서 ,경쇄가변영역을코딩하는서열번호 33, 37, 41, 45및
49로구성된군에서선택되는핵산.
[청구항 8] 제 5항의 핵산을포함하는발현벡터 .
[청구항 9] 제 8항의 발현벡터로형질전환된세포.
[청구항 10] 다음단계를포함하는쇼1¾2에결합하는항체또는이의항원결합단편의 제조방법:
知）제 9항의 세포를배양하는단계 ;및
여）상기 배양된세포에서 항체또는이의항원결합단편을회수하는 단계.
[청구항 11] 제 1항내지제 4항중어느한항의 항체또는이의항원결합단편을
유효성분으로포함하는엔지오포이에틴- 2활성화및/또는과생성과 관련된질병의 예방또는치료용조성물.
[청구항 12] 제 1항내지제 4항중어느한항의 항체또는이의항원결합단편을
유효성분으로포함하는혈관신생 저해용조성물.
[청구항 13] 제 1항내지제 4항중어느한항의 항체또는이의항원결합단편을
유효성분으로포함하는엔지오포이에틴- 2활성화및/또는과생성과 관련된질병의진단용조성물.
[청구항 14] 제 1항내지제 4항중어느한항의 항체또는이의항원결합단편을
유효성분으로포함하는안질환예방또는치료용조성물.
[청구항 15] 제 1항내지제 4항중어느한항의 항체또는이의항원결합단편을
유효성분으로포함하는종양또는암의 예방또는치료용조성물.
[청구항 16] 제 1항내지제 4항중어느한항의 항체또는이의항원결합단편을
포함하는병용투여용조성물.
PCT/KR2020/002687 2019-02-25 2020-02-25 항-ang2 항체 및 이의 용도 WO2020175886A1 (ko)

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CA3131250A CA3131250A1 (en) 2019-02-25 2020-02-25 Anti-ang2 antibody and use thereof
SG11202108832XA SG11202108832XA (en) 2019-02-25 2020-02-25 Anti-ang2 antibody and use thereof
US17/429,208 US20220204603A1 (en) 2019-02-25 2020-02-25 Anti-ang2 antibody and use thereof
JP2021550123A JP7177284B2 (ja) 2019-02-25 2020-02-25 抗ａｎｇ２抗体及びその用途
EP20762642.5A EP3932946A4 (en) 2019-02-25 2020-02-25 ANTI-ANG2 ANTIBODIES AND ITS USE
CN202080028107.0A CN113728004A (zh) 2019-02-25 2020-02-25 抗Ang2抗体及其用途
AU2020227580A AU2020227580A1 (en) 2019-02-25 2020-02-25 Anti-Ang2 antibody and use thereof

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EP3932946A1 (en) 2022-01-05
AU2020227580A1 (en) 2021-09-30
EP3932946A4 (en) 2023-03-22
JP7177284B2 (ja) 2022-11-22
JP2022522195A (ja) 2022-04-14
SG11202108832XA (en) 2021-09-29
KR102527315B1 (ko) 2023-05-03

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