WO2024085606A1 - Anticorps bispécifique comprenant un premier site de liaison à l'antigène qui se lie de maniere spécifique à l'angiopoïétine-2 humaine et son utilisation - Google Patents

Anticorps bispécifique comprenant un premier site de liaison à l'antigène qui se lie de maniere spécifique à l'angiopoïétine-2 humaine et son utilisation Download PDF

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WO2024085606A1
WO2024085606A1 PCT/KR2023/016063 KR2023016063W WO2024085606A1 WO 2024085606 A1 WO2024085606 A1 WO 2024085606A1 KR 2023016063 W KR2023016063 W KR 2023016063W WO 2024085606 A1 WO2024085606 A1 WO 2024085606A1
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seq
antibody
vegf
binding site
specifically binds
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Korean (ko)
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김용인
오인재
김희곤
김예진
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(주)니오테스바이오
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    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention relates to a bispecific antibody comprising a first antigen binding site that specifically binds to human angiopoietin-2 and its applications.
  • Angiopoietin-2 (hereinafter also referred to as 'ANG-2') binds to the receptor Tie2 present on vascular endothelial cells, but acts as an antagonistic ligand, producing angiophore, an agonist of Tie2. It acts to inhibit signal transduction by Tie2 by competing with Angiopoietin-1 (Ang1) for binding to Tie2. Due to this mechanism of action, when vascular endothelial growth factor (Vascular Epidermal Growth Factor, hereinafter referred to as 'VEGF') is overexpressed or in a state of inflammation, vascular endothelial cells are activated and vascular permeability increases.
  • vascular endothelial growth factor Vascular Epidermal Growth Factor
  • Ang1 While it induces stabilization of endothelial cells and reduces vascular permeability, increased Ang2 in activated vascular endothelial cells competes with Ang1, thereby inhibiting stabilization of vascular endothelial cells by Ang1. Therefore, Ang2 inhibits Ang1-Tie2 binding and signaling through Ang1-Tie2, which maintains the stability of vascular endothelial cells in the presence of VEGF, and ultimately promotes angiogenesis in blood vessels, resulting in increased angiogenesis, destabilization of blood vessels, and vascular permeability.
  • Ang2 has been reported to have agonist properties that induce the activity of the Tie2 receptor in some specific situations, including the formation and maintenance of lymphatic vessels. Therefore, it functions as an antagonist and a weak agonist. It is believed to have all of the following functions and perform various functions depending on the situation.
  • neovascularization Since the process of neovascularization is an essential factor in the growth of cancer, there have been attempts to prevent further growth of cancer by inhibiting neovascularization by inhibiting the Tie2-dependent function of Ang2 as described above. However, in most cases, these are It is known to have the function of inhibiting binding and preventing its role as an antagonist. In addition to antibodies that interfere with the binding of Ang2 to Tie2, recombinant proteins or antibodies that directly bind to the Tie2 receptor and induce phosphorylation and activation have been reported.
  • Ranibizumab brand name Lucentis®
  • bevacizumab Avastin
  • this antibody has been affinity matured to provide stronger binding to VEGF-A (International Patent Application Publication No. WO 98/45331).
  • VEGF-A blockade may be associated with some systemic toxicity, it is known that ranibizumab is losing its Fc portion to reduce serum half-life and consequently systemic toxicity.
  • the object of the present invention is a bispecific antibody comprising a site that specifically binds to human ANG-2 and a site that specifically binds to human VEGF, TGF-beta, TIM-3 ligand or PEDF-R (PNPLA2, ATGL). It provides antibodies.
  • Another object of the present invention is to provide a pharmaceutical composition containing the bispecific antibody as an active ingredient.
  • the present invention provides a first antigen binding site that specifically binds to human angiopoietin (ANG)-2, human vascular endothelial growth factor (VEGF), transforming growth factor, Hereinafter referred to as 'TGF')-beta, the ligand of T-cell immunoglobulin and mucin-domain containing protein (T-cell immunoglobulin and mucin-domain containing, hereinafter referred to as 'TIM')-3, and pigment epithelium-derived factor receptor.
  • a bispecific antibody comprising a second antigen binding site that specifically binds to one antigen selected from the group consisting of (pigment epithelium-derived factor receptor, hereinafter referred to as 'PEDF-R'),
  • the first antigen binding site that specifically binds to the ANG-2 is the CDRH1 region of SEQ ID NO: 1 or 7 or 13, the CDRH2 region of SEQ ID NO: 2 or 8 or 14, and the CDRH3 of SEQ ID NO: 3 or 9 or 15 comprising a region within a heavy chain variable domain, a CDRL1 region of SEQ ID NO: 4 or 10 or 16, a CDRL2 region of SEQ ID NO: 5 or 11 or 17 and a CDRL3 region of SEQ ID NO: 6 or 12 or 18 within a light chain variable domain;
  • the second antigen binding site specifically binding to VEGF comprises a VEGFR1 D2 domain
  • the second antigen binding site specifically binding to TGF-beta comprises TGF-beta RII ECD
  • the second antigen binding site that specifically binds to the ligand of TIM-3 includes TIM-3 ECD
  • the second antigen binding site that specifically binds to the PEDF-R includes a 44-mer.
  • the antibody provides a bispecific antibody that induces Tie2 activation.
  • the VEGFR1 D2 domain is the VEGFR1 D2 domain of SEQ ID NO: 21
  • the TGF-beta RII ECD is the TGF-beta RII ECD of SEQ ID NO: 24
  • the TIM-3 ECD is the TIM of SEQ ID NO: 27 -3 ECD and the 44-mer PEDF preferably include the amino acid sequence of SEQ ID NO: 30, but are not limited thereto.
  • the VEGFR1 D2 domain, the TGF-beta RII ECD, the TIM-3 ECD, and the 44-mer PEDF are preferably connected to an immunoglobulin by a linker, and the linker It is more preferable that the linker is SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, or SEQ ID NO: 29, respectively, but is not limited thereto.
  • the present invention provides a pharmaceutical composition for the treatment of cancer containing the antibody according to the present invention.
  • the present invention provides a pharmaceutical composition for the treatment of vascular diseases containing the antibody according to the present invention.
  • the present invention also provides a nucleic acid encoding the bispecific antibody according to the present invention.
  • the present invention also provides a method for producing a bispecific antibody according to the present invention, comprising expressing a nucleic acid encoding the bispecific antibody in a prokaryotic or eukaryotic host cell and recovering the bispecific antibody from the cell or cell culture supernatant.
  • a method for producing a bispecific antibody comprising expressing a nucleic acid encoding the bispecific antibody in a prokaryotic or eukaryotic host cell and recovering the bispecific antibody from the cell or cell culture supernatant.
  • a “bispecific antibody” according to the invention is an antibody with two different antigen-binding specificities. If an antibody has more than one specificity, the epitope recognized may be associated with a single antigen or more than one antigen.
  • the antibodies of the invention are specific for two different antigens, ANG-2 as the first antigen and VEGF, TGF-beta, the ligand of TIM-3 or PEDF-R as the second antigen.
  • Bispecific antibodies of the invention include, for example, multivalent single chain antibodies, diabodies and triabodies, as well as additional antigen binding sites (e.g., single chain Fv, It includes antibodies having the constant domain structure of a full-length antibody to which a VH domain and/or a VL domain, Fab, or (Fab)2) are linked.
  • Antibodies can be full length from a single species, chimeric or humanized. For antibodies with two or more antigen binding sites, some of the binding sites may be identical, as long as the protein has binding sites for two different antigens. That is, the first binding site is specific for ANG-2, while the second binding site is specific for VEGF, TGF-beta, TIM-3 ligand, and PEDF-R, and vice versa.
  • the antibodies of the invention further comprise immunoglobulin constant regions of one or more immunoglobulin classes.
  • Immunoglobulin classes include IgG, IgM, IgA, IgD and IgE isotypes and, in the case of IgG and IgA, their subtypes.
  • the antibodies of the invention have the constant domain structure of an IgG type antibody, but have four antigen binding sites.
  • chimeric antibody refers to an antibody comprising a variable region, i.e., a binding region, from one source or species and at least a portion of a constant region from a different source or species, typically produced by recombinant DNA technology. Chimeric antibodies comprising murine variable regions and human constant regions are preferred. Other preferred forms of “chimeric antibodies” encompassed by the present invention are those wherein the constant portions are altered or varied from the constant portions of the original antibody, thereby producing properties according to the present invention, particularly with regard to C1q binding and/or Fc receptor (FcR) binding. will be. Such chimeric antibodies are also referred to as “class-switched antibodies”.
  • Chimeric antibodies are the product of expressed immunoglobulin genes comprising a DNA segment encoding an immunoglobulin variable region and a DNA segment encoding an immunoglobulin constant region.
  • Methods for producing chimeric antibodies include conventional recombinant DNA and gene transfection techniques well known in the art. For example, Morrison, S.L., et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; See US 5,202,238 and US 5,204,244.
  • humanized antibody refers to an antibody whose framework or “complementarity determining regions” (CDRs) have been altered to include the CDRs of an immunoglobulin of different specificity compared to the parent immunoglobulin.
  • murine CDRs are grafted into the framework region of a human antibody to produce a “humanized antibody.”
  • CDRs complementarity determining regions
  • human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • Human antibodies are well known in the art (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001) 368-374).
  • Human antibodies can also be produced in transgenic animals (e.g., mice) that can produce the full repertoire or selection of human antibodies upon immunization in the absence of endogenous immunoglobulin production. Transfer of a human germline immunoglobulin gene sequence into such germline mutant mice will result in the production of human antibodies upon challenge (e.g. Jakobovits, A., et al., Proc. Natl. Acad.
  • Boerner, P., et al.'s techniques are also available (Cole, A., et al., Monoclonal Antibodies and Cancer Therapy, Liss, A.L., p. 77 (1985); and Boerner, P., et al. al., J. Immunol. 147 (1991) 86-95).
  • the term “recombinant human antibody” refers to any human antibody that is manufactured, expressed, produced or isolated by recombinant methods, such as from a host cell such as NS0 or CHO cells or from an animal transgenic for human immunoglobulin genes (e.g. It is intended to include antibodies isolated from (e.g., mouse), or antibodies expressed using recombinant expression vectors transfected into host cells.
  • the recombinant human antibody has variable and constant regions in rearranged form.
  • CDR complementarity determining region
  • variable domain refers to each of the pairs of light and heavy chains that are directly involved in antibody binding to antigen.
  • the domains of the variable human light and heavy chains have the same general structure, with each domain consisting of four framework (FR) regions of broadly conserved sequence, linked by three "hypervariable regions” (or complementarity determining regions, CDRs). Includes.
  • the framework region has a ⁇ -sheet conformation, and the CDRs can form loops connecting the ⁇ -sheet structures.
  • the CDRs within each chain maintain their three-dimensional structure by framework regions and, together with CDRs from other chains, form an antigen-binding site.
  • hypervariable region or “antigen-binding site of an antibody” refers to the amino acid residues of an antibody that are responsible for antigen-binding. Hypervariable regions include amino acid residues from the “complementarity determining region” or “CDR”.
  • the “framework” or “FR” region is the variable domain region excluding the hypervariable region residues as defined herein. Therefore, the light and heavy chains of the antibody comprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • the CDRs on each chain are separated by the framework amino acids.
  • CDR3 of the heavy chain is the region that most contributes to antigen binding.
  • CDR and FR regions are determined according to the standard definitions of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).
  • Bispecific antibodies according to the invention also include said antibodies with “conservative sequence alterations” (this means “variants” of bispecific antibodies).
  • amino acids with uncharged polar side chains e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • amino acids with nonpolar side chains e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • amino acids with beta-branched side chains e.g. threonine, valine, isoleucine
  • amino acids with aromatic side chains e.g. tyrosine, phenylalanine, tryptophan, histidine.
  • a predicted non-essential amino acid residue in a bispecific antibody may be replaced with another amino acid residue, preferably from the same side chain family.
  • a "variant" bispecific antibody is one whose amino acid sequence differs from the "parent" bispecific antibody amino acid sequence by at most 10, preferably about 2 to about 5, additions, deletions, and deletions within one or more variable or constant regions of the parent antibody. /or refer to a different molecule by substitution. Amino acid substitutions are as described in Riechmann, L., et al., Nature 332 (1988) 323-327 and Queen, C, et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033 may be performed by mutagenesis based on molecular modeling. “Variant” bispecific antibodies according to the invention also include bispecific antibody forms in which the linker (if present) therein is modified or replaced by another linker.
  • binding refers to the binding of an antibody to an epitope of an antigen (human VEGF, ANG-2, TGF-beta, TIM-3 ligand or PEDF-R) in an in vitro assay. Mention.
  • epitope includes any polypeptide determinant capable of specifically binding to an antibody.
  • epitope determinants include amino acids, sugar side chains, chemically active surface groups of molecules such as phosphoryl or sulfonyl, and, in certain embodiments, may have specific three-dimensional structural properties and/or specific charge properties. there is.
  • An epitope is a region of an antigen that is bound by an antibody.
  • linker refers to a peptide having an amino acid sequence, preferably of synthetic origin. Typically, the peptides link a) VH-CH1 to VL-CL, b) VL-CL to VH-CH1, c) VH-CL to VL-CH1, or d) VL-CH1 to VH-CL.
  • the linker according to the present invention is used to connect human immunoglobulin G4 and human VEGFR1 domain 2, TGF-beta RII ECD, TIM-3 ECD, and the PEDF sequence of the 44-mer.
  • pharmaceutical carrier includes any and all physiologically compatible solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion).
  • composition of the present invention can be administered by various methods known in the art. As will be appreciated by those skilled in the art, the route and/or mode of administration will vary depending on the desired results. To administer an antibody of the invention by a particular route of administration, it may be necessary to coat the antibody with or co-administer the antibody with a material that will prevent its inactivation.
  • the antibody can be administered to a subject in a suitable carrier, such as liposomes or a diluent.
  • suitable carrier such as liposomes or a diluent.
  • Pharmaceutically acceptable diluents include saline and aqueous buffered solutions.
  • Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media or agents for pharmaceutically active ingredients is known in the art.
  • parenteral administration and “administered parenterally” refer to any mode of administration other than enteral and topical administration, typically by injection, including intravenous, intramuscular, intraarterial, intrathecal, and intraarticular. Including, without limitation, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, intraepithelial, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injections and infusions.
  • cancer refers to proliferative diseases such as lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin and intraocular melanoma, and uterine cancer.
  • NSCL non-small cell lung
  • bronchioloalveolar cell lung cancer bone cancer
  • pancreatic cancer skin cancer, head and neck cancer
  • skin and intraocular melanoma and uterine cancer.
  • ovarian cancer rectal cancer, anal area cancer, stomach cancer, gastrointestinal cancer, colon cancer, breast cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, Thyroid cancer, parathyroid cancer, adrenal cancer, soft sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or urethral cancer, renal cell cancer, renal pelvis cancer, mesothelioma, hepatocellular cancer, biliary tract cancer, and tumors of the central nervous system (CNS) , spine tumor, brainstem tumor, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma and Ewing'
  • Another aspect of the invention is the bispecific antibody or said pharmaceutical composition according to the invention as an anti-angiogenic agent.
  • the anti-angiogenic substances can be used for the treatment of cancer, especially solid tumors, and other vascular diseases.
  • One embodiment of the invention is a bispecific antibody according to the invention for the treatment of vascular diseases.
  • Another aspect of the present invention is the pharmaceutical composition for treating vascular diseases.
  • Another aspect of the invention is the use of the antibody according to the invention for the manufacture of a medicament for the treatment of vascular diseases.
  • Another aspect of the invention is a method of treating a patient suffering from vascular disease by administering an antibody according to the invention to the patient in need of treatment.
  • vascular disease includes cancer, inflammatory disease, atherosclerosis, ischemia, trauma, sepsis, COPD, asthma, diabetes, AMD, retinopathy, stroke, adiposity, acute lung injury, hemorrhage, e.g. cytokine-induced Vascular effusion, allergy, Graves' disease, Hashimoto's autoimmune thyroiditis, idiopathic thrombocytopenic purpura, giant cell arteritis, rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, Crohn's disease, multiple sclerosis, ulcerative colitis, especially solid tumors.
  • cytokine-induced Vascular effusion allergy
  • Graves' disease Hashimoto's autoimmune thyroiditis, idiopathic thrombocytopenic purpura, giant cell arteritis, rheumatoid arthritis, systemic lupus erythematosus (S
  • intraocular neovascular syndromes such as proliferative retinopathy or age-related macular degeneration (AMD), rheumatoid arthritis and psoriasis
  • AMD age-related macular degeneration
  • rheumatoid arthritis rheumatoid arthritis
  • psoriasis psoriasis
  • the composition may also include adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured by the above sterilization procedures and by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic substances such as sugars, sodium chloride, etc. into the composition. Additionally, sustained absorption of injectable pharmaceutical forms can be obtained by the inclusion of substances that delay absorption, such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured by the above sterilization procedures and by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic substances such as sugars
  • the antibodies of the invention which can be used in suitable hydrated forms and/or pharmaceutical compositions of the invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.
  • the actual dosage levels of the active ingredient in the pharmaceutical compositions of the invention may vary to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, while not being toxic to the patient.
  • the dosage level selected will depend on the activity of the particular composition of the invention employed, the route of administration, the time of administration, the rate of excretion of the particular antibody to be employed, the duration of treatment, the other drugs, compounds and/or substances to be combined with the particular composition to be employed, the treatment. It will vary depending on various pharmacokinetic factors, including factors well known in the medical field, such as the patient's age, gender, weight, condition, general health, and past medical history.
  • compositions of the present invention must be fluid and sterile to the extent that the compositions can be delivered by syringe.
  • the carrier is preferably isotonic buffered saline. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintenance of the required particle size in case of dispersion, and by the use of surfactants.
  • isotonic substances such as sugars, polyhydric alcohols such as mannitol or sorbitol, and sodium chloride in the composition.
  • the present invention proposes an antibody that promotes downstream signaling by inhibiting Ang2, VEGF, TGF-beta, and TIM-3 ligands or activating PEDF-R while simultaneously activating the Tie2 receptor, thereby inhibiting neovascularization and improving vascular permeability. Suggests a new way to reduce it.
  • the antibody proposed in the present invention is expected to have applicability in the diagnosis and treatment of cancer, diseases related to abnormal angiogenesis, and/or diseases caused by increased vascular permeability.
  • Figure 1 is a schematic diagram of a bispecific antibody comprising a first antigen binding site that specifically binds to human angiopoietin-2 developed in the present invention.
  • Figure 2 shows the SDS-PAGE results of purified anti-Ang2/VEGF antibody.
  • Figures 3 and 4 show the results of SDS-PAGE purified anti-Ang2/TGF- ⁇ antibody ( Figure 3) and the results of Western blot confirmation of the TGF- ⁇ binding region, the second antigen binding site ( Figure 4), respectively.
  • Figures 5 and 6 show ELISA results analyzing the binding affinity of anti-Ang2/VEGF antibodies (Figure 5: 2C8-D2, Figure 6: 1D3-D2) to human Ang2, respectively.
  • Figures 7 and 8 are ELISA results showing the binding ability of anti-Ang2/VEGF antibody (Figure 7, 2C8-D2, Figure 8, 1D3-D2) to human VEGF or mouse VEGF, respectively.
  • Figure 9 shows ELISA results showing that anti-Ang2/VEGF antibody can bind to VEGF and Ang2 simultaneously.
  • Figure 10 is a Western blot result showing that anti-Ang2/VEGF antibody and anti-Ang2/TGF- ⁇ antibody phosphorylate AKT and ERK 42/44 in HUVEC along with Ang2.
  • Figure 11 is a result showing that anti-Ang2/VEGF antibody binds to VEGF competitively with soluble VEGFR2.
  • Figure 12 shows the results of anti-Ang2/VEGF antibody inhibiting HUVEC proliferation caused by VEGF.
  • Figures 13 to 15 show the inhibition of neovascularization by anti-Ang2/VEGF antibodies in the mouse laser-induced CNV (wet AMD) model (Figure 13. representative FFA images & CTF, Figure 14. representative OCT images & CNV lesion) and improvement in vision, respectively. This is a result showing the effect ( Figure 15. ERG).
  • Example 1 Construction of a bispecific antibody comprising a first antigen binding site that specifically binds to human angiopoietin-2
  • the heavy chain of the anti-Ang2/VEGF antibody is a heavy chain CDR sequence of 1A9 or 2C8 or 1D3 (SEQ ID NOs: 1 to 3 or 7 to 9 or 13 to 15) and C- The human immunoglobulin G4 heavy chain sequence and linker sequence (SEQ ID NO: 20) and VEGFR1 D2 sequence (SEQ ID NO: 21), in which terminal lysine was replaced with alanine, were composed into one and synthesized in GenScript.
  • the light chain of the anti-Ang2/VEGF antibody used was the light chain CDR sequence (SEQ ID NO: 4 to 6 or 10 to 12 or 16 to 18) of 1A9 or 2C8 or 1D3 described in Korean Patent Application No. 10-2022-0028751. .
  • the heavy chain of the anti-Ang2/TGF- ⁇ antibody is the heavy chain CDR sequence of 1A9 or 2C8 or 1D3 (SEQ ID NOs: 1 to 3 or 7 to 9 or 13 to 15) described in Korean Patent Application No. 10-2022-0028751.
  • the human immunoglobulin G4 heavy chain sequence and linker sequence (SEQ ID NO: 23), in which lysine at the C-terminal was replaced with alanine, and the TGF- ⁇ extracellular domain (SEQ ID NO: 24) were constructed as one, and then synthesized in GenScript.
  • the light chain of the anti-Ang2/TGF- ⁇ antibody has the light chain CDR sequence (SEQ ID NO: 4 to 6 or 10 to 12 or 16 to 18) of 1A9 or 2C8 or 1D3 described in Korean Patent Application No. 10-2022-0028751. used.
  • the heavy chain of the anti-Ang2/TIM-3 ligand antibody is the heavy chain CDR sequence of 1A9 or 2C8 or 1D3 (SEQ ID NOs: 1 to 3 or 7 to 9 or 13 to 15) described in Korean Patent Application No. 10-2022-0028751.
  • the human immunoglobulin G4 heavy chain sequence and linker sequence SEQ ID NO: 26
  • lysine at the C-terminal was replaced with alanine
  • the human TIM-3 extracellular domain SEQ ID NO: 27
  • the light chain of the anti-Ang2/TIM-3 ligand antibody is the light chain CDR sequence of 1A9 or 2C8 or 1D3 (SEQ ID NOs: 4 to 6 or 10 to 12 or 16 to 18) described in Korean Patent Application No. 10-2022-0028751. was used.
  • the heavy chain of the anti-Ang2/PEDF-R antibody is the heavy chain CDR sequence of 1A9 or 2C8 or 1D3 (SEQ ID NOs: 1 to 3 or 7 to 9 or 13 to 15) described in Korean Patent Application No. 10-2022-0028751, and The human immunoglobulin G4 heavy chain sequence and linker sequence (SEQ ID NO: 29), in which lysine at the C-terminal was replaced with alanine, and the human PEDF 44-mer peptide (SEQ ID NO: 30) were combined into one, and then synthesized in GenScript.
  • the light chain of the anti-Ang2/PEDF-R antibody has the light chain CDR sequence (SEQ ID NO: 4 to 6 or 10 to 12 or 16 to 18) of 1A9 or 2C8 or 1D3 described in Korean Patent Application No. 10-2022-0028751. used.
  • Table 1 is the CDR sequence of mouse anti-Ang2 antibody 1A9
  • Table 2 shows the CDR sequence of anti-Ang2 antibody 2C8
  • Table 3 shows the CDR sequence of anti-Ang2 antibody 1D3
  • Table 4 shows the linker connecting human immunoglobulin G4 and the (129-229) sequence containing human VEGFR1 domain 2, and the (129-229) sequence containing human VEGFR1 domain 2,
  • Table 5 shows the linker connecting human immunoglobulin G4 and human TGF- ⁇ RII extracellular domain sequence and human TGF- ⁇ RII extracellular domain sequence
  • Table 6 shows the linker connecting human immunoglobulin G4 and human TIM-3 extracellular domain sequence and human TIM-3 extracellular domain sequence
  • Table 7 shows the linker connecting human immunoglobulin G4 and human PEDF 44-mer peptide sequence and human PEDF 44-mer peptide sequence
  • the ExpiCHO expression system (Thermo Fisher) was used to produce a bispecific antibody containing a first antigen binding site that specifically binds to human angiopoietin-2.
  • ExpiCHO was cultured in a 125 mL flask (Corning) using 30 mL of ExpiCHO expression medium (Thermo Fisher) until the cell number reached 6
  • a total of 25 ⁇ g of heavy chain and light chain DNA prepared in Example 1 was prepared at a 1:1.5 ratio (w/w) using 1 mL of OptiPRO SFM (Thermo Fisher).
  • 80 ⁇ L of ExpiFectamine CHO reagent (Thermo Fisher) was prepared by adding it to 920 ⁇ L of OptiPRO SFM.
  • transfection was performed by adding it to 25 mL of the ExpiCHO culture medium prepared earlier within 5 minutes, and cultured at 37°C, 8% CO 2 , and 125 rpm.
  • 150 ⁇ L of ExpiFectamine CHO Enhancer (Thermo Fisher) and 4 mL of ExpiCHO Feed (Thermo Fisher) were added to the culture medium, and the conditions were changed to 32°C, 5% CO 2 , and 125 rpm.
  • an additional 4 mL of ExpiCHO Feed was added.
  • the culture was transferred to a conical tube (SPL) and centrifuged at 3,500 g for 30 minutes at 4°C. The supernatant was collected and filtered using a 0.22 ⁇ m CA syringe filter (Sartorius), and -70 Stored in a deep freezer at °C.
  • Amicon 30 kDa (Millipore) was used to change the buffer of the purified bispecific antibody to PBS. 5 mL of PBS was added to Amicon, centrifuged at 4°C, 5,000 rpm, and cleaned. After centrifugation, all of the solution was discarded, 10 mL of the sample eluted earlier was added, and then centrifuged at 4°C and 5,000 rpm to concentrate to about 3 mL. After adding 12 mL of PBS to the concentrated 3 mL, it was centrifuged again and concentrated to 3 mL. This process was repeated four additional times and the buffer was exchanged for PBS. Bispecific antibodies were prepared by performing sterile filtration on the sample after buffer exchange was completed using a 0.22 ⁇ m syringe filter (Millipore).
  • Example 3 SDS-PAGE analysis of purified anti-Ang2/VEGF antibody samples
  • Example 2 To confirm the purity and integrity of the anti-Ang2/VEGF antibody purified in Example 2, it was analyzed by SDS-PAGE. To 15 ⁇ L of a sample of appropriate protein concentration, add SDS-Sample Buffer 4X reducing (Gendepot) or Native SDS-Sample Buffer 4X non reducing (Gendepot) to 1X each, and heat the reducing sample at 98°C for 10 minutes using a heat block. Samples were prepared by heat treatment. NuPAGE 4-12%, Bis-Tris mini protein gel (Invitrogen) was installed in the XCell SureLock Mini-Cell (Invitrogen), and NuPage 1X MOPS SDS running buffer (Invitrogen) was added to prepare for electrophoresis.
  • SDS-Sample Buffer 4X reducing Gendepot
  • Native SDS-Sample Buffer 4X non reducing Gendepot
  • Example 4 SDS-PAGE and Western blot analysis of purified anti-Ang2/TGF- ⁇ antibody samples
  • Example 2 To confirm the purity and integrity of the anti-Ang2/TGF- ⁇ antibody purified in Example 2 in the same manner as in Example 3, it was analyzed by SDS-PAGE. As shown in Figure 3, a band of the desired size was confirmed under reducing (R) and non-reducing (NR) conditions, and it was confirmed that there was almost no impurity.
  • TGF- ⁇ binding region of the anti-Ang2/TGF- ⁇ antibody Western blot analysis was performed using anti-hTGF- ⁇ RII antibody. 60 ng and 600 ng of purified anti-Ang2/TGF- ⁇ antibody were subjected to SDS-PAGE electrophoresis in the same manner as in Example 3.
  • the gel was mounted on the After the transfer, the membrane was placed in 0.05% TBST (Tris Buffered Saline containing 0.05% (v/v) Tween-20) containing 5% (w/v) skim milk and blocked for 1 hour. After washing the blocked membrane three times with 0.1% TBST, it was added to primary antibody buffer (2 ⁇ g/mL Goat anti-hTGF- ⁇ RII antibody (R&D systems), 5% (w/v) skim milk, 0.05% TBST). A membrane was added and allowed to bind for 2 hours.
  • TBST Tris Buffered Saline containing 0.05% (v/v) Tween-20
  • primary antibody buffer 2 ⁇ g/mL Goat anti-hTGF- ⁇ RII antibody (R&D systems), 5% (w/v) skim milk, 0.05% TBST.
  • ELISA was performed to confirm the binding ability of the anti-Ang2/VEGF antibody with human Ang2, human VEGF, or mouse VEGF.
  • a 96-well MaxiSorpTM flat-bottom plate was coated with 1 ⁇ g/mL of human Ang2 (Sino Biological), human VEGF 165 (Sino Biological), or mouse VEGF 164 (Sino Biological). Then, the plate was washed five times with PBST (Phosphate Buffer Saline containing 0.05% (v/v) Tween-20) and then washed with PBST containing 1% (v/v) skim milk at room temperature for 2 hours. Blocked.
  • PBST Phosphate Buffer Saline containing 0.05% (v/v) Tween-20
  • ELISA was performed to confirm the ability of the anti-Ang2/VEGF antibody to simultaneously bind Ang2 and VEGF.
  • 1 ⁇ g/mL human VEGF 165 (Sino Biological) was coated on a 96-well MaxiSorpTM flat-bottom plate. The coated plate was washed five times with PBST and then blocked with PBST containing 1% (v/v) skim milk at room temperature for 2 hours. 60 nM of human Ang2 (Sino Biological) was mixed in advance with 11 different concentrations of anti-Ang2/VEGF antibody, serially diluted 3 times from 300 nM, and left at room temperature for 30 minutes.
  • the anti-Ang2/VEGF antibody developed in the present invention can bind to Ang2 and VEGF at the same time.
  • Bio-layer interferometry (BLI) analysis was performed to confirm the binding ability of the anti-Ang2/TGF- ⁇ antibody to Ang2 or TGF- ⁇ .
  • Octet Amine Reactive Second-Generation (AR2G) Biosensor (Sartorius) was placed in 1X kinetics buffer included in the AR2G kit and hydrated for 10 minutes. After completing hydration, the biosensor was placed in a 0.5 mL LightSafe micro centrifuge tube (Sigma) containing 1X kinetics buffer, and the initial baseline was measured for 60 seconds.
  • the biosensor was placed in a 0.5 mL LightSafe micro centrifuge tube containing 225 ⁇ L of distilled water and 12.5 ⁇ L each of 400 mM EDC and 200 mM S-NHS and activated for 300 seconds. After loading 4 ⁇ L of 10 mM acetate buffer (pH 5.0) containing 20 ⁇ g/mL TGF- ⁇ (Acrobiosystems) into the drop holder, the activated biosensor was added and immobilized for 300 seconds. The immobilized biosensor was placed in a 0.5ml LightSafe micro centrifuge tube containing 250 ⁇ L of 1 M ethanolamine (pH 8.5) and quenched for 300 seconds. After quenching was completed, the biosensor was placed back in 1 proceeded. After the association was completed, the biosensor was placed in 1X kinetics buffer and dissociation was measured for 600 seconds. After completing the analysis, ka, kd, and KD were calculated by global fitting.
  • Example 7 Western blot analysis to confirm activation of Tie2 signaling by anti-Ang2/VEGF antibody or anti-Ang2/TGF- ⁇ antibody
  • Ang2 binds to the Tie2 receptor expressed on vascular endothelial cells and acts as a weak agonist or antagonist.
  • the anti-Ang2/VEGF antibody or anti-Ang2/TGF- ⁇ antibody developed in the present invention can bind to Ang2 and form a complex with Ang2-Tie2, thereby activating the Tie2 receptor and promoting downstream signaling.
  • ERK and AKT phosphorylation experiments were performed in HUVEC. In order to compare the degree of activation of Tie2 downstream signaling, the same test was performed on the group treated only with Ang2 (Sino Biological).
  • HUVEC Longza cells
  • 60 nM of anti-Ang2/VEGF antibody or anti-Ang2/TGF- ⁇ antibody was mixed with 40 nM of Ang2 protein (Sino Biological) and left for 30 minutes. Then, the cultured cells were treated and incubated for another 10 minutes. For comparison, a group treated with only 40 nM Ang2 without antibody was also prepared. After washing the cells using PBS, lysis buffer (Ripa buffer (Biosesang), 0.15M Sodium chloride, 1% Triton , and 2 mM EDTA), centrifuged at 13,000 rpm for 15 minutes, and the supernatant was recovered to obtain a cell lysate.
  • anti-Ang2/VEGF antibody and anti-Ang2/TGF- ⁇ antibody showed AKT and ERK activation signals compared to the negative control group.
  • Example 8 Competitive VEGF binding of anti-Ang2/VEGF antibody to VEGFR2
  • VEGF is a representative pro-angiogenic cytokine that promotes blood vessel formation.
  • VEGF is known to regulate angiogenesis by binding to VEGFR2 and activating downstream signals.
  • the anti-Ang2/VEGF antibody designed in the present invention can bind to VEGF competitively with VEGFR2 and inhibit VEGF from activating the VEGFR2 downstream signal.
  • ELISA analysis was performed to confirm competitive VEGF binding of anti-Ang2/VEGF antibody and VEGFR2.
  • Greiner Bio-One 96-well Half Area ELISA Microplates were coated with 2 ⁇ g/mL human VEGFR2 ECD (Sino Biological). The plate was washed five times with PBST (Phosphate Buffer Saline containing 0.05% (v/v) Tween-20) and then blocked with PBST containing 1% (w/v) skim milk at room temperature for 2 hours. After washing the plate 5 times with PBST, pre-incubate 150 ⁇ g/mL of human VEGF (Sino Biological) with AVI tag and 11 different concentrations of anti-Ang2/VEGF antibody serially diluted 3 times from 1,500 nM for 10 minutes at room temperature.
  • PBST Phosphate Buffer Saline containing 0.05% (v/v) Tween-20
  • VEGF is a representative pro-angiogenic cytokine that promotes blood vessel formation, and VEGF is known to promote HUVEC proliferation.
  • Anti-Ang2/VEGF antibodies can inhibit HUVEC proliferation by binding to VEGF in the culture medium and inhibiting VEGF from binding to VEGFR2 of HUVEC. To confirm this, an experiment was performed to check the proliferation of HUVEC by adding VEGF or VEGF and anti-Ang2/VEGF antibody to HUVEC.
  • EBM-2 media (Lonza) containing 0.5% FBS
  • HUVEC HUVEC
  • SPL 96 well cell culture plate
  • EBM-2 media containing 0.5% FBS was prepared 30 minutes before treating the cells with VEGF 30 ng/mL, VEGF 30 ng/mL + anti-Ang2/VEGF antibody 2.5 nM.
  • 100 ⁇ L of the previously prepared sample was added to each well and cultured in an incubator (37°C, 5% CO 2 ) for 72 hours.
  • Example 10 Inhibition of neovascularization and improvement of visual acuity of anti-Ang2/VEGF antibody in Mouse Laser-induced CNV model
  • IVT intravitreal injection of CNV control group and positive control group, IgG and aflibercept
  • IgG and aflibercept positive control group
  • the test group, anti-Ang2/VEGF antibody was administered as a single IVT at a dose of 5.8 ⁇ g/ ⁇ L/eye (about 35 ⁇ M).
  • FFA fundus fluorescein angiography
  • OCT optical coherence tomography
  • Electroretinogram evaluation was performed on the scotopic ERG (electroretinogram) 12 days after CNV induction. Electroretinogram changes in response to mono-flash (0.9 log cds/m2) stimulation were evaluated using B-wave amplitude.
  • the fused cells were cultured in a medium (HAT mediaum) supplemented with hypoxantin, aminopterine, and thymidine, and cells (hybridoma) in which B lymphocytes and sp2/0 cells were fused were selectively selected.
  • the obtained hybridoma cells were separated into positive and negative cells using a serial dilution method, and the cloning process was repeated to prepare monoclonal cells that produce antibodies that react to the antigen.
  • the obtained antibody-producing hybridoma cells were cultured in DMEM (Dulbeco's Modified Eagle's Mediaum) containing 10% (v/v) FBS at 37°C and 5% CO 2 conditions, and then centrifuged to obtain antibody-producing cells.
  • the culture medium in which the antibodies were secreted was separated, and the antibodies were purified using an affinity column (Protein A/G agarose column, Protein A/G (GenDEPOT)).

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Abstract

La présente invention concerne un anticorps bispécifique comprenant un premier site de liaison à l'antigène qui se lie de manière spécifique à l'angiopoïétine-2 humaine (ANG-2), et un second site de liaison à l'antigène qui se lie de manière spécifique à au moins un antigène choisi dans le groupe constitué par le facteur de croissance endothéliale vasculaire (VEGF), le facteur de croissance transformant (TGF)-beta, l'immunoglobuline de cellule T et le ligand de protéine contenant le domaine mucine (TIM)-3, et le récepteur de facteur dérivé de l'épithélium pigmentaire (PEDF-r), le premier site de liaison à l'antigène qui se lie de manière spécifique à l'ANG-2 comprenant, dans le domaine variable de chaîne lourde, une région CDRH1 de SEQ ID NO : 1 ou 7 ou 13, une région CDRH2 de SEQ ID NO : 2 ou 8 ou 14, et une région CDRH3 de SEQ ID NO : 3 ou 9 ou 15, et comprend, dans le domaine variable de chaîne légère, une région CDRL1 de SEQ ID NO : 4 ou 10 ou 16, une région CDRL2 de SEQ ID NO : 5 ou 11 ou 17, et une région CDRL3 de SEQ ID NO : 6 ou 12 ou 18.
PCT/KR2023/016063 2022-10-20 2023-10-17 Anticorps bispécifique comprenant un premier site de liaison à l'antigène qui se lie de maniere spécifique à l'angiopoïétine-2 humaine et son utilisation WO2024085606A1 (fr)

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KR10-2022-0135696 2022-10-20
KR20220135696 2022-10-20
KR1020230136843A KR20240056422A (ko) 2022-10-20 2023-10-13 인간 안지오포이에틴-2에 특이적으로 결합하는 제1 항원 결합 부위를 포함하는 이중특이적 항체 및 그 응용
KR10-2023-0136843 2023-10-13

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150079112A1 (en) * 2013-09-17 2015-03-19 Samsung Electronics Co., Ltd. Use of an anti-ang2 antibody
KR20190039577A (ko) * 2016-08-23 2019-04-12 메디뮨 리미티드 항-ｖｅｇｆ-ａ 및 항-ａｎｇ2 항체 및 이의 용도
US20210324062A1 (en) * 2018-10-29 2021-10-21 Hoffmann-La Roche Inc. Antibody formulation
US20220073599A1 (en) * 2020-09-04 2022-03-10 Hoffmann-La Roche Inc. Antibody that binds to vegf-a and ang2 and methods of use
WO2022212360A1 (fr) * 2021-03-30 2022-10-06 Abpro Corporation Méthodes de traitement de la néovascularisation choroïdienne à l'aide d'anticorps multi-spécifiques anti-ang2 x vegf

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150079112A1 (en) * 2013-09-17 2015-03-19 Samsung Electronics Co., Ltd. Use of an anti-ang2 antibody
KR20190039577A (ko) * 2016-08-23 2019-04-12 메디뮨 리미티드 항-ｖｅｇｆ-ａ 및 항-ａｎｇ2 항체 및 이의 용도
US20210324062A1 (en) * 2018-10-29 2021-10-21 Hoffmann-La Roche Inc. Antibody formulation
US20220073599A1 (en) * 2020-09-04 2022-03-10 Hoffmann-La Roche Inc. Antibody that binds to vegf-a and ang2 and methods of use
WO2022212360A1 (fr) * 2021-03-30 2022-10-06 Abpro Corporation Méthodes de traitement de la néovascularisation choroïdienne à l'aide d'anticorps multi-spécifiques anti-ang2 x vegf

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