WO2019221574A1 - Use for preventing and treating myeloid-derived suppressor cell-related diseases - Google Patents

Use for preventing and treating myeloid-derived suppressor cell-related diseases Download PDF

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Publication number
WO2019221574A1
WO2019221574A1 PCT/KR2019/006007 KR2019006007W WO2019221574A1 WO 2019221574 A1 WO2019221574 A1 WO 2019221574A1 KR 2019006007 W KR2019006007 W KR 2019006007W WO 2019221574 A1 WO2019221574 A1 WO 2019221574A1
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Prior art keywords
antibody
mdsc
cancer
seq
amino acid
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Application number
PCT/KR2019/006007
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French (fr)
Korean (ko)
Inventor
김솟을
홍정원
지길용
윤상순
송형근
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다이노나(주)
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Priority claimed from KR1020190055950A external-priority patent/KR102373502B1/en
Priority to AU2019268959A priority Critical patent/AU2019268959B2/en
Priority to MX2020012247A priority patent/MX2020012247A/en
Priority to IL278581A priority patent/IL278581B1/en
Priority to SG11202010948XA priority patent/SG11202010948XA/en
Priority to CN201980031966.2A priority patent/CN112118868A/en
Application filed by 다이노나(주) filed Critical 다이노나(주)
Priority to US17/052,591 priority patent/US20220119518A1/en
Priority to JP2020564084A priority patent/JP7165855B2/en
Priority to BR112020023265-3A priority patent/BR112020023265A2/en
Priority to CA3099968A priority patent/CA3099968A1/en
Priority to EP19802548.8A priority patent/EP3795175A4/en
Publication of WO2019221574A1 publication Critical patent/WO2019221574A1/en
Priority to PH12020551912A priority patent/PH12020551912A1/en

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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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • 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
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention provides an immune activity enhancer comprising an antibody against CD66c or an antigen-binding fragment thereof expressed in myeloi d-derived suppressor cells (MDSC), and prevention of MDSC-related diseases using the immune activity enhancer. Or to a use relating to treatment or amelioration.
  • the present invention uses a monoclonal antibody that specifically binds CD66c to induce the effect of reducing the immunosuppressive ability of MDSC by controlling the production, death or activity of MDSC, thereby preventing, treating or ameliorating MDSC-related diseases.
  • MDSC myeloi d-derived suppressor cells
  • Cancer cells reduce the activity of various immune cells to prevent their immune response and induce immune-suppressing cells such as inactive dendritic cells, regulatory T cells (Treg), and Tumor-associated macrophage (TAM).
  • immune-suppressing cells such as inactive dendritic cells, regulatory T cells (Treg), and Tumor-associated macrophage (TAM).
  • Treg regulatory T cells
  • TAM Tumor-associated macrophage
  • MDSC is defined as a collection of bone marrow-derived immature bone marrow cells with immunosuppressive functions. In healthy individuals, MDSC is limited, but accumulates in peripheral blood, lymphoid organs, spleen, and cancer tissues in pathological conditions such as chronic / acute infections and cancer. It is reported.
  • MDSCs can inhibit the immune response of T cells and cells and induce the production of Treg cells, which are immunosuppressive cells, thereby promoting cancer cell growth and also inducing distant metastasis of cancer cells. was also confirmed.
  • the immunosuppressive mechanisms of MDSC known to date are largely divided into four types. The first is how to deplete the nutrients lymphocytes need. The second form forms oxidative stress, which produces free radicals or nitrogen, which inhibits a variety of processes from the proliferation of T cells to function. The third is how it affects traf f icking and survival. Mechanisms such as inhibiting the reciprocal ion of T cells into lymph nodes, preventing T cells from moving to the center of the tumor, and inducing T cell death are known. Fourth, it is known to proliferate antigen-specific natural Treg cells and to accelerate the process of converting na ⁇ ve CD4 + T cells to Tregs.
  • MDSC One of the greatest features of MDSC is its diversity in form, phenotype, and function.
  • Lineage (-), HLA-DRL0W / (-), CDllb (+), and CD33C + are known as marker markers of MDSC.
  • marker markers are commonly expressed in several different types of myeloid cells, such as dendritic cells, macrophages, granulocytes and precursor cells, so that MDSC has been defined as a group of myeloid-derived cells with immunosuppressive functions.
  • MDSC multinucleated
  • IC multinucleated
  • IC mononuclear
  • Multinucleated MDSCs induce antigen-specific immunosuppression through contact with T cells via free radicals.
  • Mononuclear MDSCs exhibit immunosuppressive functions mainly through high expression of arginase and the mediation of various immunosuppressive cytokines.
  • One embodiment of the present invention is an immune enhancer, an immune activator, or a composition for reducing or eliminating immune suppression by 3 ⁇ 410 ⁇ comprising an antibody binding to 0 Example 6 (:) and antigen-binding fragment thereof expressed in 3 ⁇ 4 ⁇ ⁇ It is about.
  • One embodiment of the present invention relates to a pharmaceutical composition or the use thereof for the prevention, treatment or amelioration of 3 ⁇ 4 ⁇ ⁇ related diseases, including an antibody binding to 0066 (:) and an antigen-binding fragment thereof expressed in ⁇ ) ⁇ . .
  • One embodiment of the present invention is to increase or activate an immune response of a subject comprising administering to a subject in need thereof an antibody and antigen-binding fragment thereof that binds to Example 6 (; Provide a method.
  • An additional embodiment of the present invention is an antibody that binds to 0066 (:) expressed in i)% and an antigen-binding group thereof Contacting, It relates to a method of inhibiting activity.
  • one embodiment of the present invention relates to a method for preventing, treating, or ameliorating a related disease, comprising administering the immune enhancing agent or immune activator to a subject having a 3 ⁇ 4 ⁇ related disease.
  • One embodiment of the present invention comprising administering an immune enhancer or immune activator comprising an antibody that binds to 0066 (:) and an antigen-binding fragment thereof expressed in VII to a subject having a disease related to 3 ⁇ 40 ⁇ , A method for preventing, treating, or ameliorating a disease.
  • Antibodies and antigen-binding fragments thereof which bind to 0 ⁇ 6 (:) expressed in 3 ⁇ 40 ⁇ ( ⁇ ) in accordance with the present invention, eliminate or reduce the immunosuppressive ability of ,, or reduce the number of cells of 1 « ⁇ (: Specifically, it can be achieved by controlling the activity, production or killing of 3 ⁇ 4 ⁇ , or by inducing killing.
  • the present invention relates to an immune enhancing agent, an immune activating agent, or an agent for reducing or eliminating the immune suppression ability of the antibody, including an antibody that binds to 0 ⁇ 6 (expressed) and an antigen-binding fragment thereof.
  • a further embodiment of the present invention relates to the prevention, treatment or alleviation of ⁇ -associated diseases, including cancer, infectious diseases, including antibodies that bind 0066 (:) and antigen-binding fragments thereof expressed at 3 ⁇ 40 ⁇ . will be.
  • antibodies that bind specifically to 0 ⁇ 6 (: 2019/221574 1 »(: 1 ⁇ 1 ⁇ 2019/006007
  • the present invention relates to a disease prevention or treatment and diagnostic use by inducing an effect of reducing an immunosuppressive reaction by phototherapy.
  • the antibody may be a polyclonal antibody or a monoclonal antibody, and may be a mouse antibody, a chimeric antibody, or a humanized antibody. It may be an antibody.
  • nucleic acid molecules encoding antigen binding fragments.
  • Another example provides a recombinant vector comprising the nucleic acid molecule.
  • the recombinant vector may be used as an expression vector for expressing the nucleic acid molecule in a host cell.
  • Another example provides a recombinant cell comprising said nucleic acid molecule or said recombinant vector.
  • the recombinant cell may be obtained by transforming the nucleic acid molecule or the recombinant vector into a host cell.
  • the preparation method may include expressing the nucleic acid molecule in a host cell.
  • the expressing step may include culturing the recombinant cell, and optionally, may further include separating and / or purifying the antibody from the obtained cell culture.
  • the production method is a composition for reducing or eliminating immune suppression by an immune enhancer, immune activator, or ⁇ ⁇ comprising an antibody binding to 0066 (:) and antigen-binding fragment thereof expressed at 3 ⁇ 40. It is about.
  • MDSCs have been shown to promote cancer cell growth by inhibiting T-cell and cell immune responses and inducing the production of Treg cells, which are immunosuppressive cells, and may also induce distant metastasis of cancer cells. It became.
  • the immunosuppressive mechanisms of MDSCs known to date range from how the lymphocytes deplete the nutrients they need, how they affect the trafficking and survival of lymphocytes, and which produce free radicals or free radicals, from the proliferation of T cells to their functions.
  • Methods of forming oxidative stress that inhibits the process such as inhibiting the process of recirculation of T cells into lymph nodes, or preventing T cells from moving to the center of the tumor, inducing T cell death are known. It is also known to promote antigen-specific natural Treg cells and to promote the process of converting na_ve ⁇ 4+ T cells into Tregs.
  • MDSC is defined as a set of bone marrow-derived immature bone marrow cells that have immunosuppressive functions. In healthy individuals, MDSC is limited, but accumulates in peripheral blood, lymphoid organs, spleen, and cancer tissues in pathological conditions such as chronic / acute infection and cancer. It is reported. MDSC accumulation and immunosuppressive functions in carcinoma have been reported in colorectal cancer, fibrosarcoma, thymoma, lung cancer, mesothelioma, lymphoma, prostate cancer, head and neck cancer, and melanoma (Gabri lovich DI, et al., Coordinated regulation of myeloid cells by tumors, Nat Rev Immunol. 12 (4): 253-68 (2012)). In addition to cancers, MDSCs can be found in Trypanosoma cruzi, Listeria monocytogenes, Leishmania major, helminths, Candida albicans,
  • MDSC shows a nonlymphogenic HLA-DRLow / (-), CDllb +, and CD33 + phenotype
  • the MDSC expressing CD66c expressing the phenotype may be a target of an anti-CD66c antibody or antigen-binding fragment thereof according to the present invention.
  • the composition according to the present invention targets the disease in which CD66c-positive MDSCs accumulate among MDSCs exhibiting nonlymphogenic HLA-DRLow / (-), CDllb +, CD33 + phenotypes. Suggest ways to improve or treat the problem.
  • the present invention relates to monocytes, except lymphocytes, according to the size of cells in a dot plot. 2019/221574 1 »(: 1 ⁇ 1 ⁇ 2019/006007
  • a group with no or low expression of-can be selected, and the group positive in 0) 1113 and 33 can be designated as ⁇ ⁇ .
  • the effect of lysing on the whole blood ( ⁇ 13100 (1) and Sah) can be reduced or reduced the number of cells in cells which are positive for both seedlings and positive cells, preferably in a new way. Reduction or cell death.
  • Whole blood contains a mixture of neutrophils positive for ⁇ ⁇ 6 target antigen and ⁇ ) ⁇ , although it is difficult to selectively dissolve the bay, the peripheral blood mononuclear cells produced by removing neutrophils are produced. Selective dissolution of 3 ⁇ 40 ⁇ by the antibody is possible.
  • ⁇ per unit volume of a sample may be the case where the number of cells has increased compared to a normal person,
  • the disease may be an increase in cell number compared to normal cell number.
  • the MDSC-related diseases include chronic / acute infections and cancers, and specifically, may be chronic / acute infections and cancers that exhibit immunosuppressive effects by MDSC, for example, non-lymphogenic HLA-DRLow / (-), CDllb +, CD33 + phenotype, MD66 accumulation of CD66c-positive MDSC in the disease, or MD66 CD66c-positive MDSC accumulation in the disease and cancer and the like.
  • the MDSC-related infectious diseases may be infections of Trypanosoma cruzi, Lister ia monocytogenes, Lei shmania major, helminths, Candida albicans, Porphyromonas gingival i s, or toxoplasmosis, multi-septic sepsis.
  • the MDSC-related cancer may be a CD66c positive MDSC-increased cancer, including solid cancer and hematologic cancer, and examples of the solid cancer include colorectal cancer, fibrosarcoma, thymoma, lung cancer, mesothelioma, lymphoma, prostate cancer, head Cervical cancer, melanoma, gastric cancer, liver cancer, or breast cancer, preferably colon cancer, stomach cancer or liver cancer.
  • the use of the prophylaxis, inhibition, or treatment of cancer and cancer metastasis can inhibit cancer cell growth, for example.
  • hematopoiet ic mal ignancy examples include acute myeloid leukemi a, acute lymphoblast ic leukemia, acute monocyt ic leukemi a, or Hodgkin's lymphoma. Hodgkin 1 s lymphoma and non-Hodgkin 1 s lymphoma.
  • the present invention relates to an antibody that binds to CD66c expressed in MDSC and antigen-binding fragment thereof, wherein CD66c (Cluster of Diferent i at ion 66c) is CEACAM 6 (carcinoembryonic ant igen-related cel l adhesion molecule 6) or NCA. (non-speci f ic cross-reacting glycoprotein ant i gen) -90, also known as an important protein associated with cell adhesion (cel l adhesion), but is not limited to this preferably the amino acid sequence of SEQ ID NO: 1 (Genbank Protein No. AAH05008).
  • CD66c Cluster of Diferent i at ion 66c
  • CEACAM 6 carcinoembryonic ant igen-related cel l adhesion molecule 6
  • NCA non-speci f ic cross-reacting glycoprotein ant i gen
  • the term "antibody” refers to a substance produced by stimulation of an antigen in the immune system, and the kind thereof is not particularly limited.
  • the antibody may be an animal antibody (eg, a mouse antibody, etc.), a chimeric antibody, a humanized antibody, or a human antibody, and the antibody may be a monoclonal antibody or a polyclonal antibody.
  • the anti-CD66c antibody or antigen-binding fragment specifically binds to a specific epitope of CD66c as described above, and is composed of animal antibodies (eg, mouse antibodies), chimeric antibodies, humanized antibodies, and antigen-binding fragments thereof. It may be selected from the group consisting of.
  • the animal antibody may be derived from an animal species other than human.
  • the animal antibody may be derived from a rat, a mouse, a goat, a guinea pig, a donkey, a rabbit, a horse, a llama, a camel, a bird (eg, a chicken, a duck, etc.). But it is not limited thereto. Techniques for making chimeric and / or humanized antibodies from such animal antibodies are well known in the art.
  • the humanized antibody may be any suitable isotype, such as IgG (IgGl, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE or any subc ass, and may preferably be an IgGl or IgG2 isotype. , More preferably afucoseized IgGl or IgG2 isotype.
  • the antibody herein may be understood to include antigen-binding fragments of an antibody having antigen-binding ability, unless otherwise specified.
  • 11 complementarity determining regions (Complementar i ty-determining regions (CDRs)) 1 'means a site that provides binding specificity with the antigen among the variable sites of the antibody.
  • the antigen-binding fragment of the antibody described above may be an antibody fragment comprising one or more complementarity determining regions.
  • complement i ty determining region (CDR) refers to the amino acid sequence of the hypervar i able region of the heavy and light chains of an immunoglobulin.
  • the heavy and light chains may each comprise three CDRs ( CDRH1, ⁇ RH2, CDRH3, and 1 RL1, CDRL2, CDRL3)
  • the CDRs may provide major contact residues for the antibody to bind antigen or epitope.
  • the term “specifically Binding 1 'or "specifically recognized” is the same as commonly known to those skilled in the art and means that the antigen and the antibody specifically interact to produce an immunological response.
  • antigen binding fragment refers to a portion of a polypeptide that includes a portion to which an antigen can bind, as a fragment thereof for the entire structure of an immunoglobulin.
  • it may be, but is not limited to, scFv, (scFv) 2, scFv-Fc, Fab, Fab ', or F (ab') 2.
  • Anti-CD66c specifically recognizes and / or binds to CD66c and the antibody comprises a mouse antibody, chimeric antibody or humanized antibody.
  • chimeric antibodies are antibodies in which variable region sequences are derived from one species and constant region sequences are derived from another species, eg, variable region sequences are derived from mouse antibodies and constant region sequences are derived from human antibodies.
  • the humanized antibody refers to an antibody that retains the activity of a non-human antibody while having low immunity in humans. It can be prepared, for example, by maintaining non-human CDR regions and replacing the rest of the antibody with human counter parts. For example, reference is made to Morr i son et al, Proc. Nat l.
  • the antibody fragment may include, but is not limited to, an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), as long as it can selectively recognize a CD66c epitope, Fab, Fab ', F (ab') 2.
  • scFv, dsFv and CDR may be selected from the group consisting of.
  • the scFv is an antibody fragment made of a single chain by connecting the heavy chain variable region (VH) and light chain variable region (VL) with a linker polypeptide.
  • hinge region refers to a region that is included in the heavy chain of an antibody, which exists between CH1 and CH2 regions, and which functions to provide f lexibi li ty of antigen binding sites in the antibody.
  • the hinge may be derived from a human antibody, specifically, may be derived from IgA, IgE, or IgG such as IgGl, IgG2, IgG3, or IgG4.
  • the anti-VIII 66c antibody may be a polyclonal antibody or a monoclonal antibody, for example a monoclonal antibody.
  • Monoclonal antibodies can be prepared according to methods well known in the art. For example, it may be prepared using a phage di splay technique.
  • the humanized antibody showed more than 10 times higher stability than chimeric 8F5 antibody in terms of stability in addition to the difference in greatly reducing the cause of immunogenicity in human administration.
  • the fluorescence value variation rate due to ANS binding at high temperature, for example, 62 ° C. is less than 200% stability is high.
  • chimeric 8F5 antibody showed 1,406% ANS reactivity change due to variation of antibody properties, while humanized antibody 114%, 133% Relatively minor changes in degree suggest that they have been significantly stabilized in terms of protein.
  • the chimeric 8F5 and humanized antibodies increase the activation of T cells, which indicates that activation is increased by T cell activators and even when T cell activity conditions are due to the mixing of T cells and homologous dendritic cells of different humans. Can be mentioned.
  • T cell activation induced death of cancer cells in co-culture with cancer cells, and may induce T cell activation in various cancer cells and co-culture conditions.
  • Antibodies or fragments thereof according to the invention have a tumor regression activity and a direct inhibitory effect on tumor cell lines.
  • Degeneration of a tumor herein includes inducing or facilitating a decrease in tumor size and / or inhibition, interruption or reduction of growth of tumor cells.
  • Tumor size reduction is, for example, based on 100% prior to treatment of a composition comprising an antibody or fragment thereof, the size of the tumor obtained when the composition comprising the antibody or fragment thereof is administered. Or less than 97%, 95% or less, 90% or less, 85% or less, 80% or less, or 75% or less.
  • Antibodies according to the present invention have antibody-dependent cell-mediated cytotoxicity (ADCC, Ant i body-dependent cel 1 -mediated cytotoxici ty) and complement-dependent cytotoxi ci ty (CDC), preferably It has ADCC characteristics.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxi ci ty
  • the antibody or antigen-binding fragment thereof according to the present invention can ameliorate or treat MDSC-related diseases by using a natural cell or cell-derived cell therapeutic agent.
  • the anti-CD66c antibody according to the present invention increases the cancer cell killing ability by the use with natural killer cells, and through this, for the effective removal of not only CEACAM6-positive cancer cells, but also CEACAM6-positive MDSC cells ⁇ or cell therapy The effect of combined treatment with is excellent.
  • Anti-CD66 C antibody according to the present invention respectively, MDSC and cancer cells show ADCC against other target cells, but in the case of cancer patients in which the two types of cells are actually increased together two kinds of anti-CD66c antibody according to the present invention Targets can be removed at the same time, and ⁇ the combination with cell therapy improves the efficacy of simultaneously removing cancer cells and MDSC targets.
  • the antibody according to the present invention may be a part or all of the FUC0 ′ which is a sugar residue bound to the antibody.
  • the fucose-removing antibody according to the present invention has a cell killing effect of MDSC, and in one embodiment, the antibody according to the present invention is characterized by low fucose or defucose type antibodies against MDSC killing compared to fucose type antibodies. It is excellent in efficacy and has a great effect on enhancing immune activity.
  • "normal fucose" or ⁇ normal fucose content '' refers to an antibody that typically has a fucose content of at least 90%, low fucose or tal fucose form according to the present invention.
  • the antibody of may be an antibody having a fucose content of about 10% or less, about 7% or less, or about 5% or less, for example, in the range of 0 to about 10%, 0 to about 7%, or 0 to about 5%. Can be.
  • the antibody applied to the present invention may be an anti-CD66c antibody or antigen binding fragment thereof, comprising the following complementarity determining regions (CDRs):
  • CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9
  • CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10
  • CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3
  • CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 11 or SEQ ID NO: 12,
  • CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5
  • ⁇ R-L3 comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 13.
  • the heavy chain variable region of the antibody comprises a framework comprising the amino acid sequence of SEQ ID NO: 22, 23, 24, 25, 26 or 27 (V-FR1), amino acid sequence of SEQ ID NO: 32, 33, 34, 35, 36 or 37 Framework comprising the amino acid sequence of SEQ ID NO: 42, 43, 44, 45, 46 or 47 (V-FR3), and SEQ ID NO: 52, 53, 54, 55, 56 Or an anti-CD66c antibody or antigen binding fragment thereof comprising at least one framework selected from the group consisting of a framework comprising the amino acid sequence of 57 (V-FR4). 2019/221574 1 »(: 1 ⁇ 1 ⁇ 2019/006007
  • the light chain variable region of the antibody comprises a framework comprising the amino acid sequence of SEQ ID NO: 28, 29, 30 or 31, a framework comprising the amino acid sequence of SEQ ID NO: 38, 39, 40 or 41
  • the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, 14, 15, 16, 17 or 18; And a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8, 19, 20, or 21 Or an antigen binding fragment thereof.
  • mouse antibody or chimeric antibody is an amino acid sequence that determines 001? Of the region comprising the amino acid sequence of SEQ ID NOs: 1 to 3 and an amino acid sequence of the amino acid sequence of SEQ ID NOs: 4 to 6 It may be an antibody or antigen-binding fragment thereof comprising at least one amino acid sequence selected from the group consisting of amino acid sequences to be determined.
  • sequences and variable region sequences are summarized in Table 1 below.
  • one example of the antibody of the present invention is the The amino acid sequence to be determined is SEQ ID NO: 1 (00 ⁇ ), SEQ ID NO: 2 ⁇ 2) and SEQ ID NO: 301 «3) and / or the region of I Amino acid sequences of SEQ ID NO: 4 ′ 1), SEQ ID NO: 501 «2) and SEQ ID NO: 6 ′ 3), which are amino acid sequences to be determined.
  • the mouse antibody or chimeric antibody may include a region including the amino acid sequence of SEQ ID NO: 7 and one region including the amino acid sequence of SEQ ID NO: 8.
  • Mouse antibody or chimeric antibody according to the invention as an active ingredient
  • the present invention uses the mouse antibody or chimeric antibody to prepare the amino acid sequence and the human antibody sequence of anti-CD66c antibody 8F5 based on the framework sequence.
  • the expression of humanized candidate antibodies is normally expressed on the basis of the expression level, the presence or absence of aggregat ion, and the degree of cell binding among the candidate humanized recombinant antibodies, the aggregat ion formed by the instability of the protein itself is small, and the ability to bind to the target antigen-positive cells.
  • Humanized antibody candidates similar to this chimeric antibody are selected.
  • the cell binding pattern is similar to that of the chimeric antibody, multiplying the antibody positive rate (% gated) and the mean fluorescence (mean), and comparing it with the chimeric antibody to select candidate antibodies included in the range of ⁇ 20%. Therefore, the antibody group selected in the present invention is very considerably in consideration of the result that the antibody binding ability is drastically reduced due to the change in the original protein structure when the mouse antibody CDR region sequence is inserted into the framework region of the humanized antibody. Good humanized antibodies can be selected.
  • ком ⁇ онент ⁇ exhibiting high avidity based on cell binding capacity as compared to chimeric antibodies are selected, and these are subjected to avidity analysis for CD66c antigen and similar CD66 antigens by ELISA method.
  • the humanized antibody according to the present invention exhibits excellent stability compared to chimeric antibodies, for example, an antibody having a stable ANS reactivity variation of less than 200%, and less than 200% is considered to be very insignificant. Beyond that yirwojyeo the protein structure, which change can be interpreted as meaning the ANS reactivity observed. Therefore, the humanized antibody according to the present invention has similar antigen binding properties and cell binding capacity as compared to chimeric antibodies, and the physical stability of the antibody protein itself is increased, which is very excellent in terms of druggabi li ty of therapeutic antibodies. have.
  • the fluorescence value variation rate of the antibody against the ANS reagent is determined by the difference between the fluorescence value measured at low temperature conditions (eg, 4 ° C) and the fluorescence value measured at high temperature conditions (eg, 62 ° C), Means the value divided by the fluorescence value measured at low temperature conditions. 2019/221574 1 »(: 1 ⁇ 1 ⁇ 2019/006007
  • Fluorescence value variation rate (fluorescence value measured at high temperature condition-fluorescence value measured at low temperature condition) / (fluorescence value measured at low temperature condition)
  • a humanized antibody according to the present invention comprises a heavy or light chain variable region comprising the amino acid sequence of SEQ ID NOs.
  • It may include one or more amino acid sequence selected from the group consisting of the amino acid sequence to be determined, and further one example of a mouse antibody or chimeric antibody of the region comprising the amino acid sequence of SEQ ID NO: 1 to 3 It may be an antibody comprising at least one amino acid sequence selected from the group consisting of the amino acid sequence to be determined and the amino acid sequence to determine the region comprising the amino acid sequence of SEQ ID NO: 4 to 6.
  • a humanized antibody is an amino acid sequence that determines 0 1 of an ass region comprising an amino acid sequence of SEQ ID NO: 1 or 9, and an amino acid sequence that determines 0 2 of a region comprising an amino acid sequence of SEQ ID NO: 2 or 10 It may be a heavy chain variable region comprising an amino acid sequence that determines 0 3 of the region comprising the amino acid sequence of SEQ ID NO: 3.
  • one example of a humanized antibody is an amino acid sequence determining 0 example 1 of a region comprising the amino acid sequence of SEQ ID NO: 4, 11 or 12, a region of I region comprising the amino acid sequence of SEQ ID NO: 5 It may be a heavy chain variable region comprising an amino acid sequence that determines 0 3 of the VI region including the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 13 to determine.
  • a humanized antibody is a heavy chain variable region selected from the group consisting of a heavy chain variable region comprising SEQ ID NO: 7 and amino acid sequences of SEQ ID NOs: 14-18, and a light chain variable comprising the amino acid sequence of SEQ ID NO: 8 and SEQ ID NOs: 19-21 It may include a light chain variable region selected from the group consisting of regions, except that the antibody comprising SEQ ID NO: 7 and SEQ ID NO: 8.
  • An example framework sequence of the humanized antibody according to the present invention is shown in Tables 2 and 3, wherein the antibody is selected from the group consisting of frameworks 1 to 4 of the heavy chain variable region, and frameworks 1 to 4 of the light chain variable region. It may be an antibody comprising at least a framework.
  • the amino acid sequence of the framework 1 in the heavy chain variable region may include SEQ ID NO: 23 to 27, the amino acid sequence of the framework 2 may include SEQ ID NO: 32 to 37, the amino acid sequence of the framework 3 SEQ ID NOs: 43-47, and the amino acid sequence of Framework 4 may include SEQ ID NOs: 53-57.
  • the amino acid sequence of Framework 1 may include SEQ ID NOs: 29 to 31
  • the amino acid sequence of Framework 2 may include SEQ ID NOs: 39 to 41
  • the amino acid sequence of framework 3 may be SEQ ID NO: 49 To 51
  • the amino acid sequence of Framework 4 may include SEQ ID NOs: 59-61. Framework sequences according to one example of such humanized antibodies are shown in the table below.
  • the humanized antibody may include a region selected from the group consisting of amino acid sequences of SEQ ID NOs: 14 to 18 and one region selected from the group consisting of amino acid sequences of SEQ ID NOs: 19 to 21.
  • examples of the humanized antibody include antibody 03 ⁇ 48 + ⁇ 3 ⁇ 46) comprising an amino acid sequence of SEQ ID NO: 15 and a region selected from the group consisting of the amino acid sequence of SEQ ID NO: 21, and an amino acid sequence of SEQ ID NO: 18.
  • the antibody include an antibody comprising a VH region comprising an amino acid sequence of SEQ ID NO: 15 and a VL region comprising an amino acid sequence of SEQ ID NO: 21 (Vk8 + VH6), a open region comprising an amino acid sequence of SEQ ID NO: 18 And an antibody (Vk8 + VH11) comprising a VL region selected from the group consisting of the amino acid sequence of SEQ ID NO: 21, a VH region comprising the amino acid sequence of SEQ ID NO: 16, and a VL region selected from the group consisting of the amino acid sequence of SEQ ID NO: 19;
  • An antibody (Vk7 + VH6) comprising an antibody (Vk5 + VH7), a region comprising the amino acid sequence of SEQ ID NO: 17, and a VL region selected from the group consisting of the amino acid sequence of SEQ ID NO: 20, an amino acid sequence of SEQ ID NO: 15; It may be an antibody (Vk7
  • CD66C antibody or antibody fragments can be combined with various labeling agents, toxic substances or antitumor agents. It is apparent to those skilled in the art that antibodies of the present invention can be combined with the labeling group, toxin, or antitumor agent by methods well known in the art. Such binding may be performed chemically at the attachment site after the expression of the antibody or antigen, or the binding product may be manipulated into the antibody or antigen of the present invention at the DNA level. Next, in the appropriate host system described below in the present specification, DNA is expressed and the expressed protein is recovered and regenerated as necessary. The linkage may be accomplished through a linker known in the art.
  • linkers which release toxins or anti-tumor agents under acidic or reducing conditions or upon exposure to certain proteases.
  • Antibodies or antibody fragments against the antigenic region of CD66c are conventionally used as antigens using cells expressing the CD66c protein, the antigenic region of CD66c, a portion of the CD66c protein including the antigenic region of CD66c, or the antigenic region of CD66c. It can be manufactured by the method.
  • the method for producing a CD66c antibody includes (a) a CD66c protein, an antigenic determining site of CD66c, and an antigenic determining site of CD66c.
  • the producer may isolate the antibody by culturing in vitro or in vivo. For example, the mouse is inserted into the abdominal cavity and separated from the ascites and purified.
  • Isolation and purification of monoclonal antibodies can be carried out using affinity chromatography, such as ion exchange chromatography (DEAE or DE52), anti-immunoglobulin column or protein A column, in culture supernatant or ascites.
  • affinity chromatography such as ion exchange chromatography (DEAE or DE52), anti-immunoglobulin column or protein A column, in culture supernatant or ascites.
  • the epitope to which the antibody according to the present invention binds exhibits MDSC-specific expression. Therefore, anti-CD66c antibodies can be usefully used for detection of MDSCs, and can also specifically poison MDSCs by including toxic substances.
  • Another example provides the use of an antibody according to the invention as a marker for the detection of MDSCs comprising CD66c, wherein the antibody or antigen-binding fragment thereof for CD66c is used to detect MDSC and to diagnose or diagnose MDSC-related diseases. It can also be used for informational purposes.
  • the present invention provides a composition for detecting MDSC, including a substance interacting with an antigen-determining site of the antibody by using an antibody against CD66c or an antigen-binding fragment thereof.
  • the interacting substance is composed of all substances capable of interacting with the epitope located at CD66c, such as a chemical molecular molecule, an antibody, an antigen-binding fragment of the antibody, and an aptamer. It may be one or more selected from the group.
  • the diagnostic composition of the present invention detects undesirable or overexpression of CD66c in various cells, tissues or other suitable samples, comprising contacting the sample with the antibody of the invention and detecting the presence of CD66c in the sample. It is useful for detection including. Therefore, the diagnostic composition of the present invention may be used for evaluating the onset or disease state defined below.
  • MDSC expressing CD66c may be targeted to the antibody, antibody fragment or derivative of the present invention.
  • the cells bound by the antibodies of the present invention are therefore attacked by immune system functions such as the complement system or by cell mediated cytotoxicity, thus reducing the number of cells exhibiting undesirable or overexpression of CD66C or killing such cells. .
  • the present invention provides a method or diagnostic composition for diagnosing MDSC-related diseases using an antibody against CD66c or an antigen-binding fragment thereof.
  • infiltrated MDSCs around cancer tissues can be used for diagnosis and treatment as targets, regardless of the expression of CEACAM6 antigen in cancer tissues or cancer cells themselves.
  • the antibody against CD66c according to the present invention not only binds to CD66c expressed in solid cancer cells, but also in cancers that do not express CD66c in solid cancer, and detects the increased state of MDSC caused by cancer by targeting CD66c expressed in MDSC to cancer. Can be detected.
  • MDSC tends to increase in cancer patients, which can detect and confirm the MDSC infiltrating into the microtumor environment as shown in Example 8.
  • This in combination with the results of Example 5.2 showing that MDSCs can be selectively dissolved, indicates that MDSCs can be used for diagnostic and therapeutic purposes, regardless of the degree of CEACAM6 positivity on cancer cells.
  • the presence or absence of CEACAM6 expression on the surface of cancer cells may vary. Since MDSC is increased in carcinomas, MDSC can be used for general purpose diagnostic and therapeutic purposes for most carcinomas through targeted therapy using the CD66c antibody according to the present invention.
  • the antibody, antibody fragment or derivative of the present invention is bound to a labeling group.
  • Such antibodies are particularly suitable for diagnostic applications.
  • composition according to the invention can be administered as the sole active agent or in combination with other agents.
  • a CD66c antibody is reacted with a sample containing MDSC, and (b) a sample having a positive reaction with the antibody is determined as MDSC.
  • the sample may be, but is not limited to, lymph, bone marrow, blood or blood cells.
  • the CD66c antibody may be labeled with a substance capable of confirming antigen-antibody reactivity.
  • the materials that can be used include radioisotopes, fluorescent materials, luminescent materials, chromogens, or other dyeing materials.
  • the CD66c antibody of the present invention can be provided as a diagnostic kit for diagnosing MDSC disease.
  • the diagnostic kit may include antigen-antibody reaction detection means in addition to the CD66c antibody.
  • the detection means may be flow cytometry, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay ( Floresion immunoassay (FIA) and luminescence immunoassay (LIA) may be a conventional material for carrying out the method selected from the group consisting of.
  • the therapeutic effect of the solid cancer is not only growth inhibition (quantitative reduction), apoptosi s effect of cancer cells (especially cancer stem cells) or cancer tissues including the same, but also migration (migrat ion), invasion, metastasis (metastasi). s) and the like to inhibit the exacerbation of cancer thereby.
  • the antibody according to the present invention may be expected to obtain a higher effect when combined with the antibody of the present invention in order to maximize the effect by co-treatment with STING agoni st or 5_Fu.
  • subject or ⁇ patient '' refers to a patient in need of alleviation, prevention and / or treatment of MDSC-related diseases and includes all mammals such as primates such as humans, monkeys, mice, rats, and the like. May be a rodent of MDSC The patient may be suffering from, having symptoms of, or at risk of developing a MDSC-related disease.
  • a therapeutic agent containing a CD66c antibody as an active ingredient is administered orally or parenterally, preferably parenterally, to a subject having an MDSC-related disease, that is, a human or an animal.
  • the therapeutic agent may include a pharmacologically acceptable excipient, and the dosage thereof may be appropriately adjusted according to the condition of the patient, but may be, for example, 3 mg to 6, 000 mg per day.
  • the formulation of the therapeutic agent may be, but is not limited to, a liquid, powder, emulsion, suspension or injection.
  • the onset provides a method for treating MDSC related diseases using antibodies selected from the group consisting of antibodies against CD66c epitopes, antibody fragments (F (ab ′) 2, Fab and Fv, etc.), and ligands.
  • the antibody or antibody fragment is preferably selected from the group consisting of monoclonal or polyclonal antibodies, preferably from humans and animals.
  • the CD66c antibody or antibody fragment may further contain the above-mentioned toxin. Toxins can be fused, conjugated, bound, or linked to an antibody, which can be implemented by known techniques.
  • the pharmaceutical composition of the present invention may be administered as the sole active agent or in combination with other agents, preferably in combination with those known to be suitable for the treatment of the disease in question.
  • the method of administering the antibody of the present invention can be carried out in parallel with other anticancer therapies, such as chemotherapy, radiation therapy, and cell therapy.
  • therapeutic agents known to treat various MDSC related diseases used in the chemotherapy or cell therapy may be used.
  • the present invention provides an immune activity enhancer comprising an antibody against CD66c or an antigen-binding fragment thereof expressed in myeloid-derived suppressor cells (MDSC), and MDSC-related diseases using the immune activity enhancer.
  • an immune activity enhancer comprising an antibody against CD66c or an antigen-binding fragment thereof expressed in myeloid-derived suppressor cells (MDSC), and MDSC-related diseases using the immune activity enhancer.
  • [Brief Description of Drawings] 1 shows the result of cloning the antibody gene from mouse 8F5 antibody and expressing it with chimeric recombinant antibody, binding to A549 cell surface that is CD66c antigen positive.
  • 2A to 2C show HPLC analysis results of eight humanized recombinant antibodies selected first from 96 humanized recombinant antibodies.
  • 3A to 3E show CD66c antigen positive cell surface binding to eight humanized recombinant antibodies selected first from 96 humanized recombinant antibodies, and show similar levels of cell surface binding compared to chimeric antibodies. .
  • Figures 4a and 4b is a result confirmed by ELISA binding to CD66c antigen to five humanized recombinant antibodies selected from 96 humanized recombinant antibodies
  • Figure 4a is a result for the CECACAM6 (66c) antigen
  • 4b is the result for CEACAM1 (66a) antigen.
  • 5A and 5B show the results of antibody stability under harsh temperature conditions for five humanized recombinant antibodies selected from 96 humanized recombinant antibodies.
  • 6A to 6D show the cell surface binding of CD66c antigen positive cells A549 of the humanized recombinant antibody expressed at CH0.
  • the viscosity table is a schematic diagram of MDSC selected from the group that is positive for CDllb and CD33 in the group, and the bottom is the result of confirming the positive rate of DNP002 in the designated MDSC group.
  • CD66b is expressed in granulocyt i c MDSC but not in monocyt i c MDSC.
  • FIG. 8 since most of the specific MDSCs are CD66b-positive, they can be classified as granulocyt i c MDSCs, and granulocytic MDSCs are significantly reduced by DNP002 treatment.
  • Figure 9 shows the effect of MDSC killing after treatment with fucose DNP002 in 5 patients
  • the percentage of the MDSC group was decreased by DNP002 compared to the control group in all 5 patients.
  • P # 1 on the horizontal axis in the graph means pat ient 's whole blood # 1. Represents the relative MDSC viabi li ty% change.
  • Figure 10 shows the results of analyzing the effect of MDSC killing by flow cytometry after treatment with DNP002 antibody to PBMC isolated from the blood of gastric cancer patients.
  • Figure 11a is a result of comparing the MDSC killing effect according to the I sotype of DNP002, a fucosylated IgGl type of DNP002 is the result confirming the phenomenon that most effectively induces MDSC killing in the blood.
  • Lib compares MDSC killing effect according to i sotype of DNP002 in 5 patients with gastric cancer. In all patients, afucosyl ated IgGl type DNP002 showed the highest MDSC killing in blood.
  • P # 1 in the horizontal axis means patient 's whole blood # 1 and the vertical axis indicates relative MDSC vi a bi li ty% change.
  • 12A and 12B show the results of apoptosis effects analyzed in combination with DNP002 antibody alone, NK cells alone, and DNP002 antibody in gastric cancer cell line A549 and pancreatic cancer cell line AsPC-1 positive for DNP002, which is CEACAM6 positive. to be.
  • FIG. 13 shows lung adenocarcinoma positive for CEACAM6 in cancer cells and lung squamous cancer (Ur inary bladder cancer), urinary tract (Melanoma mal ignancy) tissue for CEACAM6 negative in cancer cells themselves.
  • the result of CEACAM6 immunostaining showed that there was a CEACAM6 positive MDSC at the non-tumor site of cancer tissue.
  • the 8F5 antibody gene was cloned using Mouse Ig-Pr imer Set (Mill ipore, Cat. #: 69831). Isolated from 8F5 hybridomas PCR was performed using a Mouse Ig-Primer Set from RNA, inserted into a pGem-T vector (Promega, Cat. #: A3600), and DNA sequencing was confirmed by sequencing.
  • the IMGT site www.imgt) .org
  • the heavy and light chain variable region sequences of the analyzed 8F5 antibody are as follows.
  • heavy chain expression plasmids and Light chain expression plasmids were prepared, respectively.
  • the light chain expression plasmid was used as a pOptiVEC (Invitrogen) vector
  • the heavy chain expression plasmid was used as a pcDNA3.3 (Invitrogen) vector.
  • variable region coding cDNA and the constant region coding cDNA of each antibody were synthesized (Bioneer).
  • the heavy and light chain expression genes thus synthesized were cut with restriction enzymes Xho I and Sal I, and the light chain fragments were ligation to the pOptiVec vector and the heavy chain fragments to the pcDNA3.3 vector to prepare a complete antibody expression plasmid ( pcDNA3.3-ant i-CD66c heavy chain expression plasmid and p0ptiVEC-anti-CD66c light chain expression plasmid).
  • the transformation process was performed by transfecting DG44 cells (Invitrogen).
  • DG44 cells were transformed into adherent cells by adapting to MEMa medium containing 5% FBS to increase transformation efficiency. Transformation was performed on 6wel 1 plate using ViaFect transfection regent (Pr omega, Cat. #: E4981).
  • DG44 cells were prepared by attachment at a concentration of 1 X 105 cells / well, and the amount of DNA used for transformation was pcDNA3.3-ant i-CD66c heavy chain expression plasmid and p0ptiVEC-anti_CD66c.
  • Light chain expression plasmids were used in a combination of 2: 1 and 1.5 ug, respectively, in a 1.5: 1 ratio. Transformation was performed for 48 hours.
  • Flow cytometer was used to analyze the transformed cell population. As shown in Figure 1 chimeric antibody expression was confirmed by A549 non-small cell lung cancer cell line. Figure 1 shows the results of cloning the antibody gene from the mouse 8F5 antibody and expressed as chimeric recombinant antibody, binding to the surface of the A549 cell positive CD66c antigen.
  • the human antibody gene is encoded Based on sequence 1 31116 room yo ::! Recombinant humanized antibody sequence was selected by the 1 ⁇ 0 method.
  • the human antibody 61 " 1111 ⁇ 1 ⁇ 2 gene used as the 3rd 0116 of the humanized recombinant antibody sequence was most similar to the heavy and light chain sequences of the mouse 00660 antibody 8 5 as shown in Table 5.
  • the heavy chain variable region of the mouse 00660 antibody Amino acid and nucleic acid sequences of the light chain variable region, and the heavy and light chain variable regions The sequence is shown in Table 6 below.
  • humanized 8F5 antibody sequence selected using the above human antibody Germline gene sequence, 12 heavy chain variable regions and 8 light chain variable regions were selected. The sequences are shown in Table 3 below, and the heavy chain variable of the selected humanized antibody was shown. The amino acid sequences, CDR sequences, and framework sequences of the region and the light chain variable region are shown in Tables 6 to 8, and the amino acid sequences of the heavy and light chain variable regions of the chimeric and humanized antibodies are shown in Table 1.
  • Mouse and humanized antibodies preferably have the same heavy chain —R3 and light chain CDR2 sequences. In Table 6 below, the underlined portions are CDR antibody sequences. Darkly underlined sections in Table 7 indicate modified amino acids.
  • Purified antibody was quantified by 0D280 nm measurement, and SDS-PAGE was performed. In addition, the purity and aggregation of the antibody were analyzed by HPLC using a Sepax Zenix-C SEC-300 size exclusion column (Sepax technologies) and analyzed at 280 nm and 220 nm (FIGS. 2A to 2C).
  • Table 9 and Table 10 show the results of the flow cytometer analysis as a result of analysis of the first selected anti-CD66c humanized antibody and chimeric 8F5.
  • the selected eight kinds of antibodies are shown in Table 9 the expression level, the presence or absence of aggregation, the degree of cell binding, specifically summarizes the expression level and molecular weight of the first selected eight antibodies.
  • Table 9 the expression level, the presence or absence of aggregation, the degree of cell binding, specifically summarizes the expression level and molecular weight of the first selected eight antibodies.
  • the eight humanized recombinant antibodies showed a cell binding capacity of% 20% compared to the chimeric antibody, showing a very similar cell binding capacity with chimeric antibodies.
  • expression was normally performed, and aggregation was formed due to instability of the protein itself, and eight humanized antibodies similar to chimeric antibodies were selected as the primary selection. .
  • the cell line binding ability may be different in numerical value, but as shown in FIG. 3, the actual cell binding pattern is similar to that of the chimeric antibody, and is multiplied by the antibody positive rate (% gated) and the mean fluorescence (mean), and compared with the chimeric antibody. Eight species included in the range of 20% were selected, and it was considered that the antibody binding ability is rapidly decreased due to the change of the original protein structure when the mouse antibody CDR region sequence is inserted into the framework region of the humanized antibody. When a very good humanized antibody was selected can do.
  • humanized recombinant antibodies Of the eight humanized recombinant antibodies selected as above, five kinds of humanized recombinant antibodies exhibiting a high binding capacity based on cell binding capacity compared to chimeric antibodies were selected, and these were selected by the method for the 00660 antigen and similar 0066 antigens. Adhesion analysis was performed.
  • CD66c CEACAM6; SinoBiological
  • FIGS. 4A, 12, 4B, and 13 show CECACAM6 ( CD66c) results for antigen
  • Figures 4b and Table 13 show results for CEACAM1 (CD66a) antigen.
  • Stability immediate is 8-k 1 10-1-11 ⁇ 41) urine 1: 113161163111 group 011 301 (1 (less than; It was confirmed through the coupling experiment using the X).
  • Humanized recombinant antibodies were all adjusted to 0.2 mg / ml concentration using PBS (phosphate buf fered sal ine), and left for 4 hours at 50 ° C. as harsh conditions.
  • a 0.2 mg / ml ANS solution was mixed in an amount of 20 ul per 500 ul of the antibody dilution to measure, and after 5 minutes was analyzed under fluorescent conditions at 360 nm exci tat ion and 460 nm emi ssion. In addition, the ANS reagent response was measured even under the condition of 30 minutes at 70 ° C.
  • 5A and 5B show the results of confirming antibody stability under harsh temperature conditions for the five humanized recombinant antibodies shown in Table 11 above.
  • ANS reagent reactivity is measured by fluorescence
  • ANS reagent reaction is also measured under conditions left for another 30 minutes at 70 ° C temperature.
  • most of the five antibodies were insignificant in the ANS reaction under the conditions of 4 hours at 50 ° C., but the fluorescence values of the antibodies were large at the additional 30 minutes at 70 ° C. Increased.
  • protein ID: 3058 recombinant antibody showed the lowest increase in fluorescence value, showing stable results against temperature change among the five recombinant humanized antibodies.
  • ANS reagent reactivity was analyzed by a fluorescent reader in the same manner.
  • fluorescence value variation rate of the antibody with respect to the ANS reagent is measured under low temperature conditions (e.g., 4 ° C) and high temperature conditions (e.g.,
  • the difference of the fluorescence value measured at 62 ° C) is divided by the fluorescence value measured at low temperature.
  • Fluorescence value variation ratio (fluorescence value measured at high temperature condition-fluorescence value measured at low temperature condition) / (fluorescence value measured at low temperature condition)
  • the ANS reagent reactivity was confirmed after being left at 62 ° C for 4 hours at a temperature of 50 ° C under severe conditions. All five recombinant humanized antibodies and chimeric antibodies showed insignificant ANS response values under refrigerated conditions, but the ANS fluorescence values increased with increasing temperature. However, chimeric 8F5 has been tested for ANS reagent reactivity by 62 ° C temperature storage.
  • the humanized antibody according to the present invention is an antibody having a stable ANS reactivity variation of less than 200%, and less than 200% is considered to be very insignificant, and more than that can be interpreted as a significant change in protein structure resulting in ANS reactivity observed. Can be.
  • the humanized antibody according to the present invention has similar antigen binding and cell binding capacity as compared to the chimeric antibody, and the physical stability of the antibody protein itself is increased, which is a very excellent feature in terms of druggabi li ty of therapeutic antibodies. .
  • the five humanized recombinant antibodies selected in Example 2.3 were expressed and analyzed in ⁇ 0 cells used to express the actual most therapeutic antibodies.
  • the light and heavy chain variable region DNA sequences for constituting the five selected humanized recombinant antibodies were subjected to codon optimization, and then synthesized and linked with human IgGl constant region genes by over lay PCR method and the pcDNA3. 4 vectors (Li fe Technology) were cloned. Table 11 above shows the light and heavy chain combinations of humanized antibodies selected for CH0 cell expression.
  • DNA primer sequences used for variable-variable region PCR are shown in Table 14 below.
  • Figs. 6A to 6C Five humanized recombinant antibodies were transfected with ExpiCHO (trademark) Expression System Kit (ThermoFi sher; Cat.No:A29133), and the expressed antibodies were CD66c positive cells as shown in Figs. 6A to 6C. Binding to A549 non-small cell lung cancer cell line was performed by flow cytometer. All five humanized recombinant antibodies showed similar binding capacity to chimeric antibodies. The measured flow cytometer fluorescence value was divided by the amount of antibody expression in the CH0 culture, and the cell surface binding force (relative cel l binding) to the relative amount of antibody expression was plotted. Therefore, it was confirmed that the humanized recombinant antibody is properly expressed in CH0 cells.
  • Fig. 6D shows the relative change of the cell surface binding force of the antibody and its 100%.
  • DNP002 a humanized antibody against CD66c, was expressed and analyzed.
  • antibodies of IgGl type and IgG2 type were prepared.
  • the light and heavy chain variable region DNA sequences for constructing the humanized recombinant antibody were subjected to codon optimization, and then synthesized and linked with human IgGl or IgG2 constant region genes by over lay PCR method, and pcDNA3 using Xhol and EcoRI gene fragments. It was cloned into 4 vectors (Li fe Technology).
  • 2F-PF (2F-Peracetyl-Fucose; Merck, Cat #: 344827) was incubated at 50 uM in the culture medium when the DNP002 IgGl type antibody was expressed. Purification was performed using a Mabselect sure Protein A column (GE Healthcare Li fescience, Cat #: 11003494). Purified antibody was dialyzed with phosphate buffered saline, and the 280 nm absorbance was divided by the absorption coefficient of 1.4 and converted into a concentration unit of "mg / mL", which was then used for the test.
  • Defucosification was evaluated by comparing the reactivity of biotinylated lens culinaris agglutinin (Vector laboratories, Cat #: B-1045) with binding properties to fucose. Fucose bound IgGl DNP002 reacted with Biotinylated Lens culinaris agglutinin and induced TMB coloration by SA_HRP (Jack immunoresearch, Cat #: 016-030-084), but defucose DNP002 showed little relative color development (Table 16).
  • DNP002 with APC bound together with antibodies against anti-HLA-DR-FITC, CDllb-PE, and CD33-PE antibodies against the labeled antigens of MDSC with different fluorescence was added to 100 uL of whole blood and reacted at 4 ° C for 20 minutes.
  • MDSC forward scatter, variable indicating the size of the cell to be analyzed, SSC: side scatter, variable indicating the granularity of the cell to be analyzed, variable indicating the degree of granule in the cell
  • HLA-DR Low variable indicating the degree of granule in the cell
  • MDSC positive DNP002 is 90.9% (upper right region in the right dot plot of Figure 7)
  • MDSC negative DNP002 is 4.4% (upper left region in the right dot plot of Figure 7).
  • MDSCs are divided into subtypes as mononuclear MDCS and granulocytic MDSC. Multinucleated MDSCs express CD66b but are mononuclear.
  • FIG. 7 shows the results of demonstrating that DNP002 (anti-CD66c) reacts in a method of specifying MDSC (except lymphocytes, HLA-DR low / ( ⁇ ), CDllb +, ⁇ 33+) and a specific MDSC. Since DNP002 binds to MDSC, DNP002 can be used as a method for specifying MDSC (FIG. 7), and DNP002 can remove MDSC by inducing ADCC effect (FIG. 8). In the test results below, MDNP002 bound was 90.9%.
  • FIG. 8 shows DNP002 treatment of CD66b positive multinucleated MDSC Killed and reduced in proportion.
  • the viscosity table at the top of the control is a sample before the treatment of DNP002, and the bottom (DNP002) is the result of the decrease of the MDSC after the treatment of DNP002.
  • CD66b is monocytic MDSC and granulocytic
  • the average positive rate of DNP002 on MDSC in total PBMC was 34.3-76.7%, and the average positive rate was 55.1%.
  • Table 17 below analyzes the MDSC reactivity of the DNP002 antibody with 19 gastric cancer patient samples.
  • FIG. 8 shows a representative result of the DNP002 antibody effectively inducing apoptosis of MDSC in the blood, resulting in a significantly reduced MDSC ratio compared to the DNP002 antibody treatment.
  • FIG. 9 is the same test as in FIG. 8, and the test results of five gastric cancer patient blood samples, respectively. Open bar means MDSC ratio before DNP002 treatment and closed bar means relative ratio of MDSC after DNP002 treatment. After treatment with DNP002, the MDSC ratio was significantly reduced in all five patient samples.
  • the blood of two gastric cancer patients was separated from the PBMC layer containing MDSC using Ficol l-Paque PLUS (Ge heal thcare, Cat #: 17-1440-02) solution.
  • Ficol l-Paque PLUS Ge heal thcare, Cat #: 17-1440-02
  • Specific gravity separation of blood cells through the Ficol 1 solution effectively excludes mature neutrophils and enables more accurate MDSC killing.
  • the prepared PBMC was dispensed in a 12 wel l plate at 1 X 10 5 per wel, and DNP002 antibody was added to each wel l to 10 ug / mL and incubated in a 37 ° C incubator for 48 hours. At this time, MDSC killing ability was compared using Nivolumab (Bri stol-Myers Squibb), an antibody against PD-1.
  • Example 5.1 whole blood
  • PBMC peripheral blood mononuclear cells
  • Example 5.2 peripheral blood mononuclear cells
  • DNP002 can mediate ACAM6-positive cells with ADCC.
  • whole blood contains a mixture of neutrophils positive for CEACAM6 target antigen and MDSC, and it is hard to say that only MDSC was selectively dissolved.
  • further experiments were performed on PBMCs that had been neutrophils separated by centrifugation (Example 5.2). This confirmed that the MDSC dissolution by DNP002 was clear.
  • DNP002 antibody preparations were prepared to confirm the MDSC target killing ability of the DNP002 antibody.
  • Antibodies differ in the affinity of FcrRI I I (CD16) expressed in NK cells according to the i sotype, and antibody dependent cell-mediated cytotoxicity (ADCC) increases in view of affinity.
  • FcrRI I I CD16
  • ADCC antibody dependent cell-mediated cytotoxicity
  • ADCC potency of antibodies has been reported to depend not only on the i sotype but also on the sugar chain structure linked to the 297th asparagine amino acid, particularly in the absence of fucose in the sugar chain (Shi tara K., et al, J Immunol). Mehtods. 2005 Nov 30; 306 (1-2) IgG subclass-independent improvement of ant i body-dependent cel lular cytotoxi ci ty by fucose removal from Asn297-1 inked ol igosacchar ides).
  • Red blood cell lysis buffer IX RBC Lysi s Buf f er (ThermoF i sher, Cat #: 00-4333-57) was added to the blood of five gastric cancer patients, and then RBC was dissolved and dispensed 1 X 105 per wel l into a 12 wel l plate. It was prepared by.
  • DNP002 IgGl type Three kinds of antibodies, DNP002 IgGl type, IgG2 type, and af lucosylated IgGl type, were added to the wells to 10 ug / mL, respectively, and incubated in a 37 ° C incubator for one day. After incubation, the cells were washed with and reacted with antibodies to MDSC labeled antigens (anti-HLA-DR, CDllb, ⁇ 33 antibodies) to which each fluorescence was differently bound at 4 ° C. for 20 minutes. After washing with PBS, flow cytometry was implemented. Dyeing intensity was measured in logarithm to fluorescence intensity and expressed in 10 power units.
  • MDSC killing effect was observed in all five gastric cancer patients in order of IgG2, IgGl, afucosylated IgGl type (Fig. 11a, lib).
  • the difference in MDSC killing effect according to isotype and afucosylated (fucose content below 10%) is considered to be due to the affinity between the antibody and FcrRI II. It can be understood as broken.
  • DNP002 IgG2 has a large amount of MDSC remaining, DNP002 IgGl significantly reduced the MDSC ratio.
  • the MDSC killing effect by DNP002 IgGl was significant, but the killing effect by IgG2 was insignificant, and the IgG2 i sotype had no / very low ADCC potency compared with IgGl i sotype. The effect can be inferred by ADCC.
  • Gastric cancer cell line A549 and pancreatic cancer cell line AsPC-1 positive for DNP002, which is the target antigen of DNP002, were prepared by dispensing lxlO 4 per wel l, and the previously separated natural killer cells were dispensed by 2xl0 5 per wel l.
  • the antibody was treated to 10 ug / mL and incubated at 37 ° C for 6 hours.
  • Example 5.2 the result of selective lysis of MDSC by DNP002 and the combined effect of ⁇ cell or NK cell therapy of Example 7 revealed that both CEACAM6-positive cancer cells and CEACAM6-positive MDSC can be targeted and eliminated. have.
  • Example 5.2 and Example 7 showed ADCC against different target cells as MDSC and cancer cells, respectively, in the case of cancer patients in which two types of cells are increased together, DNP002 can simultaneously remove both types of targets. It also indicates that combinations with cell therapies may function to double the efficacy of simultaneously eliminating cancer cells and MDSC targets.
  • CEACAM6 antigen is expressed not only in cancer cells but also in MDSC
  • the DNP002 antibody can be used to detect not only cancer cells but also MDSCs.
  • immunohistochemical staining Immunohi stochemi stry
  • Immunohistochemical staining was performed in the following manner. 3 times for 10 minutes in xylene, 2 times for 10 minutes in 100% alcoho, 5 minutes for 80% and 70% alcohol (al cohol)
  • IX citrate buffer Caitrate buf fer, pH 6.0
  • MDSC tends to be increased in cancer patients, which can detect and confirm MDSC infiltrated into the microtumor environment as shown in Example 8. This indicates that MDSC can be used as a target for diagnosis and treatment regardless of the degree of CEACAM6 positivity on cancer cells when considered in conjunction with the results of Example 5.2 showing that MDSC can be selectively dissolved.
  • the expression of CEACAM6 on the surface of cancer cells may be different.
  • MDSC is increased in most carcinomas regardless of cancers. It can be used for diagnostic and therapeutic purposes.

Abstract

The present invention relates to anti-CD66c antibodies which bind to CD66c expressed on an MDSC, and the use thereof, and relates to anti-CD66c antibodies which bind to an MDSC, and a pharmaceutical composition and a diagnostic composition comprising same. The anti-CD66c antibodies of the present invention target an MDSC inducing immunosuppression and thus can be utilized for treating various diseases due to same.

Description

【명세서】  【Specification】
【발명의 명칭】 [Name of invention]
골수유래억제세포 관련 질환의 예방 및 치료 용도 【기술분야】  Prevention and treatment of bone marrow-derived suppressor cell-related diseases
본 발명은 골수유래억제세포 (myeloi d-der ived suppressor ce l l , MDSC)에서 발현되는 CD66c에 대한 항체 또는 이의 항원 결합 단편을 포함하는 면역 활성 증강제， 및 상기 면역 활성 증강제를 이용한 MDSC 관련 질환의 예방， 치료 또는 개선에 관한 용도에 관한 것이다. 구체적으로， 본 발명은 CD66c에 특이적으로 결합하는 단일클론항체를 이용하여 MDSC의 생성， 사멸 또는 활성을 조절하여 MDSC의 면역 억제능을 감소시키는 효과를 유도함으로써， MDSC 관련 질환의 예방， 치료 또는 개선 용도 또는 진단 용도를 제공한다. 【배경기술】  The present invention provides an immune activity enhancer comprising an antibody against CD66c or an antigen-binding fragment thereof expressed in myeloi d-derived suppressor cells (MDSC), and prevention of MDSC-related diseases using the immune activity enhancer. Or to a use relating to treatment or amelioration. Specifically, the present invention uses a monoclonal antibody that specifically binds CD66c to induce the effect of reducing the immunosuppressive ability of MDSC by controlling the production, death or activity of MDSC, thereby preventing, treating or ameliorating MDSC-related diseases. Provides a use or diagnostic use. Background Art
최근 암의 치료에 있어 항체나 면역 세포 백신을 이용한 면역 요법 치료에 대한 연구가 활발히 진행되고 있지만, 암세포의 면역 회피 및 억제 작용은 이러한 치료 효과를 저해시킨다. 암세포는 자신에 대한 면역 반응을 막기 위해 각종 면역 세포들의 활동을 저하시키고, 비활성 수지상세포， regul atory T 세포 (Treg) , Tumor-associ ated macrophage (TAM)와 같은 면역 억제 기능을 가진 세포들을 유도하는데， 이러한 면역억제 세포의 하나로 최근 골수유래 면역억제 세포 (mye loi d-der ived suppressor ce l l , MDSC)의 역할이 알려지면서 큰 주목을 받고 있다.  Recently, studies on immunotherapy treatment using antibodies or immune cell vaccines have been actively conducted in the treatment of cancer, but immune evasion and suppression of cancer cells inhibits these therapeutic effects. Cancer cells reduce the activity of various immune cells to prevent their immune response and induce immune-suppressing cells such as inactive dendritic cells, regulatory T cells (Treg), and Tumor-associated macrophage (TAM). ， As one of these immunosuppressive cells, the role of bone marrow-derived immunosuppressive cells (mye loi d-der ived suppressor ce ll, MDSC) has recently been known and attracts great attention.
MDSC는 면역억제 기능을 가지는 골수 유래 미성숙 골수 세포의 집합으로 정의되며, 건강한 개체에서는 수가 제한적이나, 만성/급성 감염, 암 등의 병적인 상태에서 말초혈액， 림프기관， 비장， 암 조직 등에 축적되는 것으로 보고되고 있다.  MDSC is defined as a collection of bone marrow-derived immature bone marrow cells with immunosuppressive functions. In healthy individuals, MDSC is limited, but accumulates in peripheral blood, lymphoid organs, spleen, and cancer tissues in pathological conditions such as chronic / acute infections and cancer. It is reported.
MDSC는 T세포 및 ■ 세포의 면역반응을 억제하고， 면역억제 세포 ( immunosuppress ive cel l )인 Treg 세포의 생성을 유도함으로써, 암세포의 성장을 촉진시키며， 또한 암세포의 원격 전이를 유도할 수도 있는 것으로도 확인되었다.  MDSCs can inhibit the immune response of T cells and cells and induce the production of Treg cells, which are immunosuppressive cells, thereby promoting cancer cell growth and also inducing distant metastasis of cancer cells. Was also confirmed.
지금까지 알려진 MDSC의 면역억제 기전은 크게 네 가지 형태로 나눌 수 있는데， 첫 번째는 림프구가 필요로 하는 영양소를 결핍시키는 방법이다. 두 번째 형태는 산화스트레스를 형성하는 것으로, 활성산소나 활성질소를 만들어 T 세포의 증식에서 기능까지 다양한 과정을 저해한다. 세 번째는 림프구의 이동 (traf f icking)과 생존에 영향을 주는 방식이다. T 세포가 림프절로 reci rculat ion 하는 과정을 저해하거나， 종양의 중심으로 T 세포가 이동하는 것을 방해하기도 하고, T 세포 사멸을 유도하는 등의 기전이 알려져 있다. 네 번째는 항원 특이 natural Treg 세포를 증식시키고， 나이브 (naive) CD4+ T세포를 Treg 으로 전환하는 과정을 촉진하는 것으로 알려져 있다. The immunosuppressive mechanisms of MDSC known to date are largely divided into four types. The first is how to deplete the nutrients lymphocytes need. The second form forms oxidative stress, which produces free radicals or nitrogen, which inhibits a variety of processes from the proliferation of T cells to function. The third is how it affects traf f icking and survival. Mechanisms such as inhibiting the reciprocal ion of T cells into lymph nodes, preventing T cells from moving to the center of the tumor, and inducing T cell death are known. Fourth, it is known to proliferate antigen-specific natural Treg cells and to accelerate the process of converting naïve CD4 + T cells to Tregs.
MDSC의 가장 큰 특징 중의 하나는 형태， 표현형, 및 기능에 있어 다양성을 가진다는 것이다. MDSC의 표지 마커로는 Lineage(-) , HLA- DRL0W/(-) , CDllb(+) , CD33C+) 가 알려져 있다. 이러한 표지 마커들은 수지상 세포， 대식 세포， 과립성백혈구의 전구체 세포들과 같은 여러가지 다른 종류의 골수성 세포에서 공통적으로 발현되고 있어서, MDSC는 면역 억제 기능을 가진 골수성 유래 세포의 집합군으로 정의되어 왔다. 이러한 One of the greatest features of MDSC is its diversity in form, phenotype, and function. Lineage (-), HLA-DRL0W / (-), CDllb (+), and CD33C + are known as marker markers of MDSC. These marker markers are commonly expressed in several different types of myeloid cells, such as dendritic cells, macrophages, granulocytes and precursor cells, so that MDSC has been defined as a group of myeloid-derived cells with immunosuppressive functions. Such
MDSC 의 다양성은 MDSC의 기원 및 특성을 연구하는데 있어 서로 다른 분석으로 이어져 큰 혼란을 주어 왔다. 이에 MDSC의 세부군을 밝히려는 연구가 진행되어 현재 MDSC는 80% 의 다핵구성 (granulocyt ic) MDSC와 20% 의 단핵구성 (monocyt i c) MDSC로 이루어져 있음이 밝혀겼다. 이 두 세포는 모양과 표현형에서만 차이를 보이는 것이 아니라, 면역을 억제하는 기전도 다른 것으로 밝혀졌는데 다핵구성 MDSC는 활성산소를 매개로 T세포 사이의 접촉을 통해 항원특이적인 면역억제를 유도하는 반면， 단핵구성 MDSC는 주로 arginase의 고발현 및 다양한 면역억제 사이토카인을 매개로 하여 면역 억제 기능을 나타낸다. The diversity of MDSCs has led to significant confusion in the analysis of the origins and characteristics of MDSCs. Research into the subgroups of MDSC has been underway, and it has now been found that MDSC is composed of 80% multinucleated (IC) and 20% mononuclear (IC) MDSCs. The two cells differ not only in shape and phenotype, but also in immunosuppressive mechanisms. Multinucleated MDSCs induce antigen-specific immunosuppression through contact with T cells via free radicals. Mononuclear MDSCs exhibit immunosuppressive functions mainly through high expression of arginase and the mediation of various immunosuppressive cytokines.
최근 연구를 통해 암환자에서 조성되는 면역억제 환경에 MDSC의 축적이 관여한다는 것이 보고되고 있으며, 이는 거의 모든 암종에서 공통적으로 나타났다. MDSC가 증가하는 정도는， 암의 병기가 진행될수록 커진다는 것이 많은 연구를 통해 뒷받침되고 있다. 이에 MDSC의 증가 정도를， 암환자의 낮은 생존율과 치료 반응률에 대한 예후 지표로 활용하고자 하는 연구가 활발하게 진행 중이다. 암의 병태생리에서 MDSC가 중요한 역할을 하고 있음은 분명해 보인다  Recent studies have reported the involvement of MDSCs in the immunosuppressive environment in cancer patients, which has been common in almost all carcinomas. Many studies have supported the increase in MDSC as the stage of cancer increases. Therefore, studies are actively underway to use the increase of MDSC as a prognostic indicator for the low survival rate and treatment response rate of cancer patients. It seems clear that MDSCs play an important role in the pathophysiology of cancer.
【발명의 상세한 설명】 2019/221574 1»（：1^1{2019/006007 [Detailed Description of the Invention] 2019/221574 1 »（： 1 ^ 1 {2019/006007
【기술적 과제】 [Technical problem]
본 발명의 일 예는 ¾©就에서 발현되는 0예6（：에 결합하는 항체 및 이의 항원결합단편을 포함하는 면역 증강제, 면역 활성화제, 또는 ¾10就에 의한면역 억제능을 감소또는 제거하기 위한조성물에 관한 것이다.  One embodiment of the present invention is an immune enhancer, an immune activator, or a composition for reducing or eliminating immune suppression by ¾10 을 comprising an antibody binding to 0 Example 6 (:) and antigen-binding fragment thereof expressed in ¾ © 就It is about.
본 발명의 일 예는， ^）엤에서 발현되는 0066（：에 결합하는 항체 및 이의 항원결합단편을 포함하는 ¾©洗 관련 질환의 예방, 치료， 또는 개선용 약학적 조성물또는 이의 용도에 관한 것이다.  One embodiment of the present invention relates to a pharmaceutical composition or the use thereof for the prevention, treatment or amelioration of ¾ © 洗 related diseases, including an antibody binding to 0066 (:) and an antigen-binding fragment thereof expressed in ^) 엤. .
본 발명의 일 예는， 光（：에서 발현되는 0예6（；에 결합하는 항체 및 이의 항원결합단편을 이를 필요로 하는 대상에게 투여하는 단계를 포함하는 대상의 면역 반응을증가시키거나활성화시키는 방법을 제공한다.  One embodiment of the present invention is to increase or activate an immune response of a subject comprising administering to a subject in need thereof an antibody and antigen-binding fragment thereof that binds to Example 6 (; Provide a method.
본 발명의 추가 일예는， 犯）%에서 발현되는 0066（：에 결합하는 항체 및 이의 항원결합단
Figure imgf000005_0001
접촉하는 단계를 포함하는,
Figure imgf000005_0002
활성을 억제하는 방법에 관한 것이다. An additional embodiment of the present invention is an antibody that binds to 0066 (:) expressed in i)% and an antigen-binding group thereof
Figure imgf000005_0001
Contacting,
Figure imgf000005_0002
It relates to a method of inhibiting activity.
또한, 본 발명의 일 예는 상기 면역 증강제 또는 면역 활성화제를, ¾©洗 관련 질환을 갖는 대상에게 투여하는 단계를 포함하는， 볘就 관련 질환의 예방， 치료， 또는 개선방법에 관한 것이다.  In addition, one embodiment of the present invention relates to a method for preventing, treating, or ameliorating a related disease, comprising administering the immune enhancing agent or immune activator to a subject having a ¾ © related disease.
본 발명의 일 예는， 볘엤에서 발현되는 0066（：에 결합하는 항체 및 이의 항원결합단편을 포함하는 면역 증강제 또는 면역 활성화제를, ¾0）況 관련 질환을 갖는 대상에게 투여하는 단계를 포함하는， 1©엤 관련 질환의 예방， 치료， 또는 개선방법에 관한 것이다.  One embodiment of the present invention, comprising administering an immune enhancer or immune activator comprising an antibody that binds to 0066 (:) and an antigen-binding fragment thereof expressed in VII to a subject having a disease related to ¾0 況, A method for preventing, treating, or ameliorating a disease.
본 발명에 따른 ¾0此（：에서 발현되는 0^6（：에 결합하는 항체 및 이의 항원결합단편은， 볘엤의 면역 억제능을 제거 또는 감소시키거나， 1«湖（：의 세포수를 감소시키며， 구체적으로 ¾©엤의 활성, 생성 또는 사멸을 조절하거나， 사멸을유도하여 달성할수 있다.  Antibodies and antigen-binding fragments thereof, which bind to 0 ^ 6 (:) expressed in ¾0 此 （此） in accordance with the present invention, eliminate or reduce the immunosuppressive ability of ，, or reduce the number of cells of 1 «湖 （： Specifically, it can be achieved by controlling the activity, production or killing of ¾ ©, or by inducing killing.
【기술적 해결방법】 Technical Solution
본 발명은
Figure imgf000005_0003
발현되는 0^6（：에 결합하는 항체 및 이의 항원결합단편를 포함하는 면역 증강용, 면역 활성화용， 또는 볘洗에 의한 면역 억제능을 감소또는 제거하기 위한용도에 관한 것이다. The present invention
Figure imgf000005_0003
The present invention relates to an immune enhancing agent, an immune activating agent, or an agent for reducing or eliminating the immune suppression ability of the antibody, including an antibody that binds to 0 ^ 6 (expressed) and an antigen-binding fragment thereof.
또한, 본 발명의 추가 일 예는， ¾0就에서 발현되는 0066（：에 결합하는 항체 및 이의 항원결합단편를 포함하는 엤 관련 질환， 예를 들면, 암， 감염성 질환의 예방， 치료또는 경감용도에 관한 것이다.  In addition, a further embodiment of the present invention relates to the prevention, treatment or alleviation of 엤 -associated diseases, including cancer, infectious diseases, including antibodies that bind 0066 (:) and antigen-binding fragments thereof expressed at ¾0 就. will be.
구체적으로， 쌔就에서 발현하는 0^6（：에 특이적으로 결합하는 항체를 2019/221574 1»（：1^1{2019/006007 Specifically, antibodies that bind specifically to 0 ^ 6 （： 2019/221574 1 »（： 1 ^ 1 {2019/006007
이용하여 光(：에 의한 면역억제반응을 감소시키는 효과를 유도함으로써 , 질환 예방 또는 치료 및 진단 용도에 관한 것이다. 상기 항체는 다클론 항체 또는 단클론 항체일 수 있으며 , 마우스 항체 , 키메릭 항체 또는 인간화 항체일 수 있다.The present invention relates to a disease prevention or treatment and diagnostic use by inducing an effect of reducing an immunosuppressive reaction by phototherapy. The antibody may be a polyclonal antibody or a monoclonal antibody, and may be a mouse antibody, a chimeric antibody, or a humanized antibody. It may be an antibody.
Figure imgf000006_0001
항원결합단편을 암호화하는 핵산 분자를 제공한다.
Figure imgf000006_0001
Provided are nucleic acid molecules encoding antigen binding fragments.
다른 예는 상기 핵산 분자를 포함하는 재조합 벡터를 제공한다. 상기 재조합 벡터는 상기 핵산 분자를 숙주세포에서 발현시키기 위한 발현 벡터로 사용될 수 있다.  Another example provides a recombinant vector comprising the nucleic acid molecule. The recombinant vector may be used as an expression vector for expressing the nucleic acid molecule in a host cell.
다른 예는 상기 핵산 분자 또는 상기 재조합 벡터를 포함하는 재조합 세포를 제공한다. 상기 재조합 세포는 상기 핵산 분자 또는 상기 재조합 벡터를 숙주세포에 형질전환시켜서 얻어진 것일 수 있다.  Another example provides a recombinant cell comprising said nucleic acid molecule or said recombinant vector. The recombinant cell may be obtained by transforming the nucleic acid molecule or the recombinant vector into a host cell.
다른 예는 상기
Figure imgf000006_0002
항체 또는 이의 항원결합단편의 제조 방법을 제공한다. 상기 제조 방법은 상기 핵산 분자를 숙주세포에서 발현시키는 단계를 포함할 수 있다. 상기 발현시키는 단계는 상기 재조합 세포를 배양하는 단계를 포함할 수 있으며, 임의로， 상기 얻어진 세포 배양물로부터 항체를 분리 및/또는 정제하는 단계를 추가로 포함할 수 있다. Another example above
Figure imgf000006_0002
Provided are methods for preparing an antibody or antigen-binding fragment thereof. The preparation method may include expressing the nucleic acid molecule in a host cell. The expressing step may include culturing the recombinant cell, and optionally, may further include separating and / or purifying the antibody from the obtained cell culture.
(3) 상기 핵산 분자 또는 상기 재조합 벡터로 형질전환된 재조합 세포를 준비하는 단계;  (3) preparing recombinant cells transformed with the nucleic acid molecule or the recombinant vector;
0)) 상기 핵산 분자의 충분한 발현을 위한 조건 및/또는 기간에서 상기 재조합 세포를 배양하는 단계; 및  0)) culturing said recombinant cell under conditions and / or duration for sufficient expression of said nucleic acid molecule; And
(0) 상기 (ø) 단계에서 얻어진 배양물로부터
Figure imgf000006_0003
항체 또는 이의 항원결합단편을 분리 및/또는 정제하는 단계. 이하， 본 발명을 더욱 자세히 설명하고자 한다. (0) from the culture obtained in the step (ø)
Figure imgf000006_0003
Isolating and / or purifying the antibody or antigen-binding fragment thereof. Hereinafter, the present invention will be described in more detail.
일 구체예에서, 상기 제조 방법은， ¾0 에서 발현되는 0066(：에 결합하는 항체 및 이의 항원결합단편을 포함하는 면역 증강제， 면역 활성화제， 또는 關就에 의한 면역 억제능을 감소 또는 제거하기 위한 조성물에 관한 것이다. In one embodiment, the production method is a composition for reducing or eliminating immune suppression by an immune enhancer, immune activator, or 關 就 comprising an antibody binding to 0066 (:) and antigen-binding fragment thereof expressed at ¾0. It is about.
Figure imgf000006_0004
면역억제 기능을 가지는 골수 유래 미성숙 골수 세포의 집합으로 정의되며， 건강한 개체에서는 수가 제한적이나, 만성/급성 감염， 암 등의 병적인 상태에서 말초혈액, 림프기관， 비장, 암 조직 등에 축적되는 것으로 보고되고 있다. MDSC는 T세포 및 ■ 세포의 면역반응을 억제하고, 면역억제 세포 (immunosuppressive cell)인 Treg 세포의 생성을 유도함으로써, 암세포의 성장을 촉진시키며, 또한 암세포의 원격 전이를 유도할 수도 있는 것으로도 확인되었다. 지금까지 알려진 MDSC의 면역억제 기전은, 림프구가 필요로 하는 영양소를 결핍시키는 방법, 림프구의 이동 (trafficking)과 생존에 영향을 주는 방법, 활성산소나 활성질소를 만들어 T 세포의 증식에서 기능까지 다양한 과정을 저해하는 산화스트레스를 형성하는 방법， T세포가 림프절로 recirculation하는 과정을 저해하거나 종양의 중심으로 T 세포가 이동하는 것을 방해하기도 하고, T 세포 사멸을 유도하는 등의 기전이 알려져 있다. 또한， 항원 특이적 natural Treg 세포를 증식시키고, na_ve 抑 4+ T세포를 Treg 으로 전환하는 과정을 촉진하는 것으로 알려져 있다.
Figure imgf000006_0004
It is defined as a collection of immature bone marrow-derived immature bone marrow cells that have immunosuppressive functions, and is limited in healthy individuals, but accumulates in peripheral blood, lymphoid organs, spleen, and cancer tissues in pathological conditions such as chronic / acute infection and cancer. It is becoming. MDSCs have been shown to promote cancer cell growth by inhibiting T-cell and cell immune responses and inducing the production of Treg cells, which are immunosuppressive cells, and may also induce distant metastasis of cancer cells. It became. The immunosuppressive mechanisms of MDSCs known to date range from how the lymphocytes deplete the nutrients they need, how they affect the trafficking and survival of lymphocytes, and which produce free radicals or free radicals, from the proliferation of T cells to their functions. Methods of forming oxidative stress that inhibits the process, such as inhibiting the process of recirculation of T cells into lymph nodes, or preventing T cells from moving to the center of the tumor, inducing T cell death are known. It is also known to promote antigen-specific natural Treg cells and to promote the process of converting na_ve 抑 4+ T cells into Tregs.
MDSC는 면역억제 기능을 가지는 골수 유래 미성숙 골수 세포의 집합으로 정의되며， 건강한 개체에서는 수가 제한적이나, 만성/급성 감염， 암 등의 병적인 상태에서 말초혈액, 림프기관, 비장, 암 조직 등에 축적되는 것으로 보고되고 있다. 암종에 있어서 MDSC 축적과 면역억제 기능은 대장암， 섬유육종, 흉선종， 폐암， 중피종， 림프종, 전립선암， 두경부암, 흑색종등에서 보고 되었다 (Gabri lovich DI, et al . , Coordinated regulation of myeloid cells by tumors , Nat Rev Immunol . 12(4):253-68 (2012)) . MDSC는 암뿐만 아니라 Trypanosoma cruzi , Listeria monocytogenes, Leishmania major, helminths, Candida albicans, MDSC is defined as a set of bone marrow-derived immature bone marrow cells that have immunosuppressive functions. In healthy individuals, MDSC is limited, but accumulates in peripheral blood, lymphoid organs, spleen, and cancer tissues in pathological conditions such as chronic / acute infection and cancer. It is reported. MDSC accumulation and immunosuppressive functions in carcinoma have been reported in colorectal cancer, fibrosarcoma, thymoma, lung cancer, mesothelioma, lymphoma, prostate cancer, head and neck cancer, and melanoma (Gabri lovich DI, et al., Coordinated regulation of myeloid cells by tumors, Nat Rev Immunol. 12 (4): 253-68 (2012)). In addition to cancers, MDSCs can be found in Trypanosoma cruzi, Listeria monocytogenes, Leishmania major, helminths, Candida albicans,
Porphyromonas gingival is 등의 감염 또는 톡소플라즈마증， 다균성 패혈증의 질환에서도 축적되어 면역억제를 유발하는 것으로 알려져 있다 (Garbri lovich DI , et al . , Myeloid-derived suppressor cells as regulators of the immune systems. Nat Rev Immunol . 9(3):162-74 (2009)) . 본 명세서에서 MDSC는 비림프구성 HLA-DRLow/(-) , CDllb+, 및 CD33+ 표현형을 보이는 것이고， 상기 표현형을 나타내면서 CD66c발현하는 MDSC가 본원 발명에 따른 항- CD66c 항체 또는 이의 항원결합단편의 표적이 될 수 있다. 구체적으로, 본 발명에 따른 조성물은 비림프구성 HLA-DRLow/(-), CDllb+, CD33+ 표현형을 보이는 MDSC 중에서 CD66c 양성 MDSC가 축적된 상기 질환을 타깃하여 MDSC에 의한 면역결핍， 면역저하 또는 면역 손상을 개선 또는 치료하기 위한 방안을 제시한다. 예를 들면， 본 발명은 점도표 (dot plot)에서 세포의 크기에 따라 림프구를 제외하고 단핵구와 2019/221574 1»（：1^1{2019/006007 It has been known to accumulate in infections such as Porphyromonas gingival is or in diseases of toxoplasmosis and multiseptic sepsis (Garbri lovich DI, et al., Myeloid-derived suppressor cells as regulators of the immune systems.Nat Rev Immunol. 9 (3): 162-74 (2009)). In the present specification, MDSC shows a nonlymphogenic HLA-DRLow / (-), CDllb +, and CD33 + phenotype, and the MDSC expressing CD66c expressing the phenotype may be a target of an anti-CD66c antibody or antigen-binding fragment thereof according to the present invention. Can be. Specifically, the composition according to the present invention targets the disease in which CD66c-positive MDSCs accumulate among MDSCs exhibiting nonlymphogenic HLA-DRLow / (-), CDllb +, CD33 + phenotypes. Suggest ways to improve or treat the problem. For example, according to the present invention, the present invention relates to monocytes, except lymphocytes, according to the size of cells in a dot plot. 2019/221574 1 »（： 1 ^ 1 {2019/006007
과립구 영역만을 지정한 뒤， - 의 발현이 없거나 낮은 군을 선택하고， 그군에서 0）1113와抑 33에 양성인 그룹을關就로 지정할수 있다. After specifying only the granulocyte region, a group with no or low expression of-can be selected, and the group positive in 0) 1113 and 33 can be designated as 關 就.
본 발명 따른 ¾©就에서 발현되는 0^6（：에 결합하는 항체 및 이의 항원결합단편 또는 상기 항체 또는 항원결합단편을 포함하는 면역 증강제를 이용하여 ¾： 관련 질환의 예방， 치료 또는 개선용 약학적 조성물 또는 용도를 제공할수 있다.  Pharmacy for the prevention, treatment or amelioration of ¾: related diseases by using an antibody that binds to 0 ^ 6 (:) expressed in ¾ © and the antigen-binding fragment thereof or an immune enhancer comprising the antibody or antigen-binding fragment according to the present invention. Suitable compositions or uses.
본 발명에 따른
Figure imgf000008_0002
항체에 의한
Figure imgf000008_0001
의 용해 효과를 전혈（ ^ 13100（1）과 쌔（：에서 모두 0묘 能6 양성인 세포를 뺘洗 세포수 감소 또는 세포 사멸을 유도할 수 있으며， 바람직하게는 새 방식으로 溫）엤의 세포 수 감소 또는 세포 사멸을 유도할 수 있다. 전혈에는 期此 6 타깃 항원이 양성인 호중구와 犯）엤 가 혼합되어 있어,
Figure imgf000008_0003
만을 선택적으로 용해하였다고 보기 어려우나, 호중구를 제거하고 얻어진 말초혈액 단핵세포作요版：）에 대해서
Figure imgf000008_0004
항체에 의한 ¾0）엤 의 선택적인 용해가가능하다. According to the invention
Figure imgf000008_0002
By antibody
Figure imgf000008_0001
The effect of lysing on the whole blood (^ 13100 (1) and Sah) can be reduced or reduced the number of cells in cells which are positive for both seedlings and positive cells, preferably in a new way. Reduction or cell death. Whole blood contains a mixture of neutrophils positive for 期 此 6 target antigen and 犯） 엤,
Figure imgf000008_0003
Although it is difficult to selectively dissolve the bay, the peripheral blood mononuclear cells produced by removing neutrophils are produced.
Figure imgf000008_0004
Selective dissolution of ¾0 엤엤 by the antibody is possible.
상기
Figure imgf000008_0005
관련 질환은
Figure imgf000008_0006
의한 면역 억제 활성을 나타내는 질환으로서， 00660 양성인 ¾©엤가 증가된 질환의 기준인 정상 세포 대비 증가한 질환이며, 예를 들면, 특정 질환을 갖는 대상에서 00660 양성인 光（：의 수 또는 활성이 , 대응하는 정상 대상의 시료의 단위 부피당 ¾10 의 수 또는 활성을 100% 기준으로 할 때, 약 200%이상, 약 300%이상， 약 500%이상, 약 700%이상, 약 1,000%이상, 약 1,500%이상 일 수 있으며， 예를 들면 약 200 내지 5,000%, 또는 200% 내지 3,000%, 200 내지 1,500% 등일 수 있다. 예를 들면, ¾0洗 관련 질환을 갖는 것으로 의심되는 대상 및 정상인의 시료, 혈액을 취하여, 유세포 분석기로 !）（:의 시료 내 세포 수를 분석하여， 쌔엤 관련 질환을 갖는 것으로 의심되는 대상의 쌔엤의 세포 수가 정상인 세포수와 비교하여 증가여부를 결정한다. 구체적으로, 시료 （예， 혈액）의 단위 부피당 汉： 세포의 수가 정상인에 비해서 증가한 경우 일 수 있으며 정상 대상의 시료의 단위
Figure imgf000008_0007
수 또는 활성을 100% 기준으로 할 때， 약 200%이상， 약 300%이상, 약 500%이상, 약 700%이상， 약 1,000%이상， 약 1,500%이상 일 수 있으며, 예를 들면 약 200 내지 5,000%, 또는 200%내지 3,000%, 200내지 1,500%등일 수 있다. remind
Figure imgf000008_0005
Related diseases
Figure imgf000008_0006
Disease exhibiting immunosuppressive activity, which is an increased disease relative to normal cells, which is a standard for increased disease of 00660 positive ¾ ©, and, for example, a number or activity of 00660 positive light (： Based on 100% of the number or activity of ¾10 per unit volume of the sample of normal subject, about 200%, about 300%, about 500%, about 700%, about 1,000%, about 1,500% or more And, for example, about 200 to 5,000%, or 200% to 3,000%, 200 to 1,500%, and the like. For example, a sample of a subject suspected of having a ¾0 洗 disease and a sample of a normal person, blood may be taken and analyzed by the flow cytometer for the number of cells in the sample of! Determine whether the number of cells increases compared to the number of normal cells. Specifically, 당 per unit volume of a sample (eg blood) may be the case where the number of cells has increased compared to a normal person,
Figure imgf000008_0007
When based on 100% of water or activity, about 200% or more, about 300% or more, about 500% or more, about 700% or more, about 1,000% or more, or about 1,500% or more, for example, about 200 to 5,000%, or 200% to 3,000%, 200 to 1,500%, and so on.
구체적인 볘엤는, 예를 들면 비림프구성 此 1^（ /（-）， 0）1比+, 抑 33+표현형을 보이는볘就 중에서 00660 양성 «（：가 증가된 또는 축적된 질환또는 상기 ¾© 세포수가 정상 세포 수에 비해 증가된 질환일 수 있다. 상기 MDSC 관련 질환의 예는 만성/급성 감염 및 암 등을 포함하며， 구체적으로 MDSC에 의한 면역 억제능을 나타내는 만성/급성 감염 및 암일 수 있으며， 예를 들면 비림프구성 HLA-DRLow/(-) , CDllb+ , CD33 + 표현형을 보이는 MDSC 중에서 CD66c 양성 MDSC가 축적된 질환， 또는 MDSC 중에서 CD66c 양성 MDSC가축적된 감염질환 및 암등을포함한다. Specifically, for example, non-lymphoid 구성 1 ^ (/ (-), 0) 1 ratio +, 抑 33+ phenotype among the phenomena showing increased or accumulated 00660 positive «(： or above ¾ © The disease may be an increase in cell number compared to normal cell number. Examples of the MDSC-related diseases include chronic / acute infections and cancers, and specifically, may be chronic / acute infections and cancers that exhibit immunosuppressive effects by MDSC, for example, non-lymphogenic HLA-DRLow / (-), CDllb +, CD33 + phenotype, MD66 accumulation of CD66c-positive MDSC in the disease, or MD66 CD66c-positive MDSC accumulation in the disease and cancer and the like.
상기 MDSC 관련 감염질환은 Trypanosoma cruzi , Li ster ia monocytogenes , Lei shmania major , helminths , Candida albicans , Porphyromonas gingival i s 등의 감염 또는 톡소플라즈마증， 다균성 패혈증의 질환일 수 있다.  The MDSC-related infectious diseases may be infections of Trypanosoma cruzi, Lister ia monocytogenes, Lei shmania major, helminths, Candida albicans, Porphyromonas gingival i s, or toxoplasmosis, multi-septic sepsis.
예를 들면, 상기 MDSC 관련 암은 CD66c 양성인 MDSC가 증가된 암일 수 있으며 , 고형암 및 혈액암을 포함하며 , 상기 고형암의 예는 대장암, 섬유육종, 흉선종, 폐암, 중피종, 림프종, 전립선암, 두경부암， 흑색종， 위암， 간암, 또는 유방암일 수 있으며, 바람직하게는 대장암, 위암 또는 간암일 수 있다. 상기 암 및 암전이의 예방, 억제， 또는 치료 용도는 예를 들면， 암 세포 성장을 억제 할 수 있다. 상기 혈액암 (hematopoiet ic mal ignancy)의 예는 급성골수구성백혈병 (acute myeloid leukemi a) , 급성림프구성 백혈병 (acute lymphoblast i c leukemia) , 급성단구성백혈병 (acute monocyt i c leukemi a) , 또는 호지킨림프종 (Hodgkin1 s lymphoma) , 비호지킨 림프종 (non-Hodgkin 1 s lymphoma)일 수 있다. For example, the MDSC-related cancer may be a CD66c positive MDSC-increased cancer, including solid cancer and hematologic cancer, and examples of the solid cancer include colorectal cancer, fibrosarcoma, thymoma, lung cancer, mesothelioma, lymphoma, prostate cancer, head Cervical cancer, melanoma, gastric cancer, liver cancer, or breast cancer, preferably colon cancer, stomach cancer or liver cancer. The use of the prophylaxis, inhibition, or treatment of cancer and cancer metastasis can inhibit cancer cell growth, for example. Examples of the hematopoiet ic mal ignancy are acute myeloid leukemi a, acute lymphoblast ic leukemia, acute monocyt ic leukemi a, or Hodgkin's lymphoma. Hodgkin 1 s lymphoma and non-Hodgkin 1 s lymphoma.
본 발명은 MDSC에서 발현되는 CD66c에 결합하는 항체 및 이의 항원결합단편에 관한 것으로서, CD66c(Cluster of Di f ferent i at ion 66c)는 CEACAM 6 (carcinoembryonic ant igen-related cel l adhesion molecule 6) 또는 NCA(non-speci f ic cross-react ing glycoprotein ant i gen) -90으로도 알려진 단백질로, 세포 접착 (cel l adhesion)과 연관된 중요한 단백질로 알려져 있으며 이에 한정되지 않지만 바람직하게는 서열번호 1의 아미노산 서열 (Genbank Protein No . AAH05008)로 표시될 수 있다.  The present invention relates to an antibody that binds to CD66c expressed in MDSC and antigen-binding fragment thereof, wherein CD66c (Cluster of Diferent i at ion 66c) is CEACAM 6 (carcinoembryonic ant igen-related cel l adhesion molecule 6) or NCA. (non-speci f ic cross-reacting glycoprotein ant i gen) -90, also known as an important protein associated with cell adhesion (cel l adhesion), but is not limited to this preferably the amino acid sequence of SEQ ID NO: 1 (Genbank Protein No. AAH05008).
본 명세서에서 "항체’’라 함은， 면역계 내에서 항원의 자극에 의하여 만들어지는 물질을 의미하는 것으로서 그 종류는 특별히 제한되지 않는다. 상가 항체는 비자연적으로 생성된 것, 예컨대， 재조합적 또는 합성적으로 생성된 것일 수 있다. 상기 항체는 동물 항체 (예컨대， 마우스 항체 등) , 키메릭 항체, 인간화 항체 또는 인간 항체일 수 있다. 상기 항체는 단클론 항체 또는 다클론 항체일 수 있다. 상기 항- CD66c항체 또는 항원결합단편은 앞서 설명한 CD66c 의 특정 항원결정부위에 특이적으로 결합하는 것으로, 동물 항체 (예컨대, 마우스 항체) , 키메릭 항체， 인간화 항체， 및 이들의 항원결합단편들로 이루어진 군에서 선택된 것일 수 있다. 상기 동물 항체는 인간 이외의 동물 종으로부터 유래된 것일 수 있으며， 예컨대, 쥐, 생쥐, 염소， 기니피그， 당나귀， 토끼， 말， 라마, 낙타， 조류 (예컨대， 닭， 오리 등) 등으로부터 유래된 것일 수 있으나 이에 한정되는 것은 아니다. 이와 같은 동물 항체로부터 키메릭 항체 및/또는 인간화 항체를 제작하는 기술은 관련 기술 분야에 잘 알려져 있다. 상기 인간화 항체는 IgG ( IgGl , IgG2, IgG3 , IgG4) , IgM, IgA, IgD, IgE 또는 임의의 subc l ass 같은 임의의 적합한 아이소타입일 수 있으며， 바람작하게는 IgGl 또는 IgG2 아이소타입일 수 있으며, 더욱 바람직하게는 탈푸코오스화된 IgGl 또는 IgG2 아이소타입일 수 있다. As used herein, the term "antibody" refers to a substance produced by stimulation of an antigen in the immune system, and the kind thereof is not particularly limited. The antibody may be an animal antibody (eg, a mouse antibody, etc.), a chimeric antibody, a humanized antibody, or a human antibody, and the antibody may be a monoclonal antibody or a polyclonal antibody. The anti-CD66c antibody or antigen-binding fragment specifically binds to a specific epitope of CD66c as described above, and is composed of animal antibodies (eg, mouse antibodies), chimeric antibodies, humanized antibodies, and antigen-binding fragments thereof. It may be selected from the group consisting of. The animal antibody may be derived from an animal species other than human. For example, the animal antibody may be derived from a rat, a mouse, a goat, a guinea pig, a donkey, a rabbit, a horse, a llama, a camel, a bird (eg, a chicken, a duck, etc.). But it is not limited thereto. Techniques for making chimeric and / or humanized antibodies from such animal antibodies are well known in the art. The humanized antibody may be any suitable isotype, such as IgG (IgGl, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE or any subc ass, and may preferably be an IgGl or IgG2 isotype. , More preferably afucoseized IgGl or IgG2 isotype.
또한 본 명세서에서 항체는， 특별한 언급이 없는 한， 항원 결합능을 보유한 항체의 항원 결합 단편도 포함하는 것으로 이해될 수 있다. 본 명세서에서 11상보성 결정부위 (Complementar i ty-determining regions , CDR) 1'라 함은, 항체의 가변 부위 중에서 항원과의 결합 특이성을 부여하는 부위를 의미한다. 앞서 설명한 항체의 항원 결합 단편은 상기 상보성 결정부위를 하나 이상 포함하는 항체 단편일 수 있다. 용어， ”CDR( complementar i ty determining region)"은 면역글로불린의 중쇄 및 경쇄의 고가변 영역 (hypervar i able region)의 아미노산 서열을 의미한다. 중쇄 및 경쇄는 각각 3개의 CDR을 포함할 수 있다 (CDRH1 , 抑 RH2, CDRH3 및 抑 RL1 , CDRL2, CDRL3) . 상기 CDR은 항체가 항원 또는 에피토프에 결합하는 데 있어서 주요한 접촉 잔기를 제공할 수 있다. 한편， 본 명세서에 있어서， 용어， "특이적으로 결합1' 또는 "특이적으로 인식”은 당업자에게 통상적으로 공지되어 있는 의미와 동일한 것으로서, 항원 및 항체가 특이적으로 상호작용하여 면역학적 반응을 하는 것을 의미한다. In addition, the antibody herein may be understood to include antigen-binding fragments of an antibody having antigen-binding ability, unless otherwise specified. In the present specification, 11 complementarity determining regions (Complementar i ty-determining regions (CDRs)) 1 'means a site that provides binding specificity with the antigen among the variable sites of the antibody. The antigen-binding fragment of the antibody described above may be an antibody fragment comprising one or more complementarity determining regions. The term “complement i ty determining region (CDR)” refers to the amino acid sequence of the hypervar i able region of the heavy and light chains of an immunoglobulin. The heavy and light chains may each comprise three CDRs ( CDRH1, 抑 RH2, CDRH3, and 1 RL1, CDRL2, CDRL3) The CDRs may provide major contact residues for the antibody to bind antigen or epitope. Meanwhile, in the present specification, the term “specifically Binding 1 'or "specifically recognized" is the same as commonly known to those skilled in the art and means that the antigen and the antibody specifically interact to produce an immunological response.
용어, "항원 결합 단편"은 면역글로불린 전체 구조에 대한 그의 단편으로， 항원이 결합할 수 있는 부분을 포함하는 폴리펩타이드의 일부를 의미한다. 예를 들어 , scFv, (scFv)2, scFv-Fc , Fab, Fab' 또는 F(ab’)2일 수 있으나, 이에 한정되지 않는다.  The term “antigen binding fragment” refers to a portion of a polypeptide that includes a portion to which an antigen can bind, as a fragment thereof for the entire structure of an immunoglobulin. For example, it may be, but is not limited to, scFv, (scFv) 2, scFv-Fc, Fab, Fab ', or F (ab') 2.
본 발명에 따른 항- CD66c는 CD66c를 특이적으로 인식 및/또는 결합하고 항체는 마우스 항체, 키메릭 항체 또는 인간화 항체를 포함한다. 본 발명에서 키메릭 항체는 가변 영역 서열들이 하나의 종으로부터 유래되고 불변 영역 서열들이 다른 종으로부터 유래된 항체들， 예를 들어， 가변 영역 서열들이 마우스 항체로부터 유래되고 불변 영역 서열들이 인간 항체로부터 유래된 항체를 의미한다. 본 발명에서 인간화 항체란 인간에서 면역성이 적으면서 비인간 항체의 활성을 보유하는 항체를 의미한다. 이는， 예를 들어， 비인간 CDR 영역을 유지시키고, 항체의 나머지 부분을 인간 대응부 (counter parts)로 치환함으로써 제조될 수 있다. 예를 들어， 하기 문헌들이 참조된다: Morr i son et al， Proc . Nat l . Acad. ScL USA, 81 : 6851- 6855(1984)； Morr i son and Oi , Adv. Immunol . , 44 : 65-92 (1988)； Verhoeyen et al , Science , 239 : 1534-1536 ( 1988)； Pad lan, Molec . Immun. , 28 :489-Anti-CD66c according to the present invention specifically recognizes and / or binds to CD66c and the antibody comprises a mouse antibody, chimeric antibody or humanized antibody. In the present invention, chimeric antibodies are antibodies in which variable region sequences are derived from one species and constant region sequences are derived from another species, eg, variable region sequences are derived from mouse antibodies and constant region sequences are derived from human antibodies. Mean antibody. In the present invention, the humanized antibody refers to an antibody that retains the activity of a non-human antibody while having low immunity in humans. It can be prepared, for example, by maintaining non-human CDR regions and replacing the rest of the antibody with human counter parts. For example, reference is made to Morr i son et al, Proc. Nat l. Acad. ScL USA, 81: 6851-6855 (1984); Morr i son and Oi, Adv. Immunol. , 44: 65-92 (1988); Verhoeyen et al, Science, 239: 1534-1536 (1988); Pad lan, Molec. Immun. , 28: 489-
498 ( 1991)； Pad lan, Molec . Immun. , 31(3) : 169-217 ( 1994) . 498 (1991); Pad lan, Molec. Immun. , 31 (3): 169-217 (1994).
본 발명에서 항체 절편은 항체 경쇄 가변 영역 (VL) 및 항체 중쇄 가변 영역 (VH)를 포함하여 CD66c 에피토프를 선택적으로 인식할 수는 것이면 이에 제한이 없으나， Fab , Fab ' , F(ab ' )2 , scFv, dsFv 및 CDR로 이루어진 군에서 선택된 것일 수 있다. 특히， 상기 scFv는 상기 중쇄 가변부위 (VH) 및 경쇄 가변부위 (VL)를 링커 폴리펩타이드로 연결하여 단쇄 (single chain)로 만든항체 절편이다.  In the present invention, the antibody fragment may include, but is not limited to, an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), as long as it can selectively recognize a CD66c epitope, Fab, Fab ', F (ab') 2. , scFv, dsFv and CDR may be selected from the group consisting of. In particular, the scFv is an antibody fragment made of a single chain by connecting the heavy chain variable region (VH) and light chain variable region (VL) with a linker polypeptide.
용어 "힌지 영역 (hinge region)”은 항체의 중쇄에 포함되어 있는 영역으로서， CH1 및 CH2 영역 사이에 존재하며， 항체 내 항원 결합 부위의 유연성 (f lexibi l i ty)를 제공하는 기능을 하는 영역을 의미한다. 예컨대, 상기 힌지는 인간 항체로부터 유래한 것일 수 있으며， 구체적으로， IgA, IgE, 또는 IgG, 예컨대, IgGl， IgG2, IgG3, 또는 IgG4로부터 유래한 것일 수 있다.  The term “hinge region” refers to a region that is included in the heavy chain of an antibody, which exists between CH1 and CH2 regions, and which functions to provide f lexibi li ty of antigen binding sites in the antibody. For example, the hinge may be derived from a human antibody, specifically, may be derived from IgA, IgE, or IgG such as IgGl, IgG2, IgG3, or IgG4.
항-抑 66c 항체는 다클론 항체 또는 단클론 항체일 수 있으며， 예컨대, 단클론 항체일 수 있다. 단클론 항체는 당 업계에 널리 알려진 방법대로 제조될 수 있다. 예컨대, phage di splay기법을 이용해서 제조될 수 있다. 상기 인간화 항체는 마우스항체나 키메릭 항체와 달리 인체 투여 시， 면역원성의 원인을 대폭 줄이는 차별성 외에도 안정성 측면에서 키메릭 8F5 항체보다 10배 이상의 높은 안정성을 보였다. 구체적으로는， 높은 온도， 예를 들면 온도 62°C에서 ANS 결합에 의한 형광값 변이율이 200% 미만으로 안정성이 높다. The anti-VIII 66c antibody may be a polyclonal antibody or a monoclonal antibody, for example a monoclonal antibody. Monoclonal antibodies can be prepared according to methods well known in the art. For example, it may be prepared using a phage di splay technique. Unlike the mouse antibody or chimeric antibody, the humanized antibody showed more than 10 times higher stability than chimeric 8F5 antibody in terms of stability in addition to the difference in greatly reducing the cause of immunogenicity in human administration. Specifically, the fluorescence value variation rate due to ANS binding at high temperature, for example, 62 ° C. is less than 200% stability is high.
구체적인 가혹조건에서 항체 물성의 변이로 인해 키메릭 8F5 항체는 1 ,406 % 의 ANS 반응성 변화를 보인 반면， 인간화 항체는 114%， 133% 정도의 상대적으로 미미한 변화를 보여 상당히 단백질 측면에서 안정화되었음을 알수 있다. Under specific harsh conditions, chimeric 8F5 antibody showed 1,406% ANS reactivity change due to variation of antibody properties, while humanized antibody 114%, 133% Relatively minor changes in degree suggest that they have been significantly stabilized in terms of protein.
상기 키메릭 8F5 및 인간화 항체는 T 세포의 활성화를 증가시키며， 이는 T 세포 활성인자에 의한 활성화 증대 및 서로 다른 사람의 동종 수지상 세포와 T 세포 혼합으로 인한 T 세포 활성 조건에서도 활성 증대를 보이는 경우를 들 수 있다. 이러한 T 세포 활성화 유도는 암세포와의 공동 배양 시 암세포의 사멸을 유도하였으며， 다양한 암세포와 공동 배양 조건에서 T세포 활성화유도하는 경우를들 수 있다.  The chimeric 8F5 and humanized antibodies increase the activation of T cells, which indicates that activation is increased by T cell activators and even when T cell activity conditions are due to the mixing of T cells and homologous dendritic cells of different humans. Can be mentioned. Such induction of T cell activation induced death of cancer cells in co-culture with cancer cells, and may induce T cell activation in various cancer cells and co-culture conditions.
본 발명에 따른 항체 또는 이의 단편은， 종양 퇴화 (tumor regression) 활성 및 종양 세포주에 대해 직접적인 억제 효과를 갖는다. 본 명세서에서 종양의 퇴화는 종양 크기의 감소 및/또는 종양 세포의 성장 저해， 중단 또는 감소 등을 유도 또는 촉진하는 것을 포함한다. 종양의 크기 감소는 예를 들면， 본 발명의 항체 또는 이의 단편을 포함하는 조성물을 처리하기 전을 100%를 기준으로, 상기 항체 또는 이의 단편을 포함하는 조성물을 투여한 경우에 얻어진 종양의 크기가 97%이하， 95%이하， 90%이하， 85%이하, 80%이하， 75% 이하의 크기를 갖는 경우 등을 들 수 있다. 본 발명에 따른 항체는 항체-의존성 세포매개성 세포독성 (ADCC, Ant i body-dependent cel 1 -mediated cytotoxici ty)과 보체계 의존 세포독성 (CDC, complement -dependent cytotoxi ci ty)을 가지며, 바람직하게는 ADCC특성을 갖는다.  Antibodies or fragments thereof according to the invention have a tumor regression activity and a direct inhibitory effect on tumor cell lines. Degeneration of a tumor herein includes inducing or facilitating a decrease in tumor size and / or inhibition, interruption or reduction of growth of tumor cells. Tumor size reduction is, for example, based on 100% prior to treatment of a composition comprising an antibody or fragment thereof, the size of the tumor obtained when the composition comprising the antibody or fragment thereof is administered. Or less than 97%, 95% or less, 90% or less, 85% or less, 80% or less, or 75% or less. Antibodies according to the present invention have antibody-dependent cell-mediated cytotoxicity (ADCC, Ant i body-dependent cel 1 -mediated cytotoxici ty) and complement-dependent cytotoxi ci ty (CDC), preferably It has ADCC characteristics.
본 발명에 따른 항체 또는 이의 항원결합단편은 자연살상 (natural ki l ler) 세포 또는■세포 유래 세포 치료제를 병용하여 MDSC 관련 질환을 개선 또는 치료할수 있다.  The antibody or antigen-binding fragment thereof according to the present invention can ameliorate or treat MDSC-related diseases by using a natural cell or cell-derived cell therapeutic agent.
구체적으로， 본 발명에 따른 항- CD66c 항체는 암세포 살해능이 자연 살해 세포와의 병용에 의해 증가하며， 이를 통해, CEACAM6 양성인 암세포뿐만 아니라， 역시 CEACAM6 양성인 MDSC 의 효과적인 제거를 위해 ■ 세포또는■세포치료제와의 병용 처리의 효과가우수하다.  Specifically, the anti-CD66c antibody according to the present invention increases the cancer cell killing ability by the use with natural killer cells, and through this, for the effective removal of not only CEACAM6-positive cancer cells, but also CEACAM6-positive MDSC cells ■ or cell therapy The effect of combined treatment with is excellent.
구체적인 실험으로서, EZ-cytox enhanced cel l vi abi l i ty ki t (Daei l Lab) 를 이용하여 세포 생존율을 즉정한 결과， 두 종의 암세포주 모두에서 자연 살해 세포의 병용에 의한 세포 사멸 효과가 단독처리 시 대비 증가하는 것으로 확인되었다 (도 12a 및 도 12b) . 본 발명에 따른 항- CD66c 항체에 의한 MDSC 의 선택적 용해 결과와 ■ 세포 또는 ■ 세포치료제의 병용효과를 통해， CEACAM6 양성인 암세포 및 CEACAM6 양성인 MDSC 를 모두 표적으로 하여 제거할 수 있다. 본 발명에 따른 항-CD66C 항체는 각각 MDSC 와 암세포로 다른 표적 세포에 대해 ADCC 를 보이지만， 실제 두 종류의 세포가 함께 증가되어 있는 암환자의 경우 본 발명에 따른 항-CD66c항체로 두 종류의 표적을 동시에 제거할 수 있으며, 또한 ■ 세포치료제와의 병용으로 암세포 및 MDSC표적을동시 제거하는 효능이 더욱 향상된다. 본 발명에 따른 항체는 항체에 결합된 당잔기인 FUC0湖를 일부 또는 전부 제거한 것일 수 있다. 본 발명에 따른 푸코오스 제거 항체는 MDSC의 세포 사멸효과를 가지며, 일예에서 본 발명에 따른 항체는 저 푸코오스 형태 또는 탈푸코오스 (defucose)형태의 항체가 fucose형태의 항체에 비해 MDSC 살상에 대한 효능이 우수하여 면역 활성 증강 효능이 크다. 본 명세서에 사용되는 바와 같이， "정상 푸코스’’ 또는 ’’정상 푸코스 함량’’은 푸코스 함량이 전형적으로 90% 이상인 항체를 지칭한다. 본 발명에 따른 저 푸코오스 또는 탈 푸코오스 형태의 항체는， 푸코스 함량이 약 10이하%인 항체， 약 7%이하， 또는 약 5%이하일 수 있으며， 예를 들면 0 내지 약 10%, 0내지 약 7%, 또는 0내지 약 5%범위일 수 있다. As a concrete experiment, EZ-cytox enhanced cel l vi abi li ty kt (Daei l Lab) was used to immediately determine the cell survival rate, resulting in a combination of natural killer cells in both cancer cell lines. It was found to increase relative to treatment (FIGS. 12A and 12B). Through the selective lysis of MDSC by the anti-CD66c antibody according to the present invention and the combined effect of cell or cell therapy, both CEACAM6 positive cancer cells and CEACAM6 positive MDSC Can be removed by targeting. Anti-CD66 C antibody according to the present invention, respectively, MDSC and cancer cells show ADCC against other target cells, but in the case of cancer patients in which the two types of cells are actually increased together two kinds of anti-CD66c antibody according to the present invention Targets can be removed at the same time, and ■ the combination with cell therapy improves the efficacy of simultaneously removing cancer cells and MDSC targets. The antibody according to the present invention may be a part or all of the FUC0 ′ which is a sugar residue bound to the antibody. The fucose-removing antibody according to the present invention has a cell killing effect of MDSC, and in one embodiment, the antibody according to the present invention is characterized by low fucose or defucose type antibodies against MDSC killing compared to fucose type antibodies. It is excellent in efficacy and has a great effect on enhancing immune activity. As used herein, "normal fucose" or `` normal fucose content '' refers to an antibody that typically has a fucose content of at least 90%, low fucose or tal fucose form according to the present invention. The antibody of may be an antibody having a fucose content of about 10% or less, about 7% or less, or about 5% or less, for example, in the range of 0 to about 10%, 0 to about 7%, or 0 to about 5%. Can be.
구체적으로， 본 발명에 적용되는 항체는 다음의 상보성 결정부위 (complementarity determining region； CDRs)를 포함하는, 항-CD66c 항체 또는 이의 항원 결합단편일 수 있다:  Specifically, the antibody applied to the present invention may be an anti-CD66c antibody or antigen binding fragment thereof, comprising the following complementarity determining regions (CDRs):
서열번호 1또는서열번호 9의 아미노산서열을포함하는 CDR-H1 , 서열번호 2또는서열번호 10의 아미노산서열을포함하는 CDR-H2, 서열번호 3의 아미노산서열을포함하는 CDR-H3,  CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3,
서열번호 4, 서열번호 11 또는 서열번호 12의 아미노산 서열을 포함하는 CDR-L1 ,  CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 11 or SEQ ID NO: 12,
서열번호 5의 아미노산서열을포함하는 CDR-L2, 및  CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and
서열번호 6또는서열번호 13의 아미노산서열을포함하는抑R-L3임 . 상기 항체의 중쇄 가변영역은 서열번호 22 , 23, 24, 25, 26 또는 27의 아미노산 서열을 포함하는 프레임워크 (V-FR1) , 서열번호 32, 33， 34, 35, 36 또는 37의 아미노산 서열을 포함하는 프레임워크 (V-FR2) , 서열번호 42, 43, 44, 45, 46 또는 47의 아미노산 서열을 포함하는 프레임워크 (V- FR3) , 및 서열번호 52, 53, 54, 55, 56 또는 57의 아미노산 서열을 포함하는 프레임워크 (V-FR4)로 이루어진 군에서 선택된 적어도 하나의 프레임 워크를 포함하는 것인 항-CD66c 항체 또는 이의 항원 결합 단편일 수 있다. 2019/221574 1»（：1^1{2019/006007 抑 R-L3 comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 13. The heavy chain variable region of the antibody comprises a framework comprising the amino acid sequence of SEQ ID NO: 22, 23, 24, 25, 26 or 27 (V-FR1), amino acid sequence of SEQ ID NO: 32, 33, 34, 35, 36 or 37 Framework comprising the amino acid sequence of SEQ ID NO: 42, 43, 44, 45, 46 or 47 (V-FR3), and SEQ ID NO: 52, 53, 54, 55, 56 Or an anti-CD66c antibody or antigen binding fragment thereof comprising at least one framework selected from the group consisting of a framework comprising the amino acid sequence of 57 (V-FR4). 2019/221574 1 »（： 1 ^ 1 {2019/006007
상기 항체의 경쇄 가변영역은， 서열번호 28， 29, 30 또는 31의 아미노산 서열을 포함하는 프레임워크（丄 犯）, 서열번호 38, 39， 40 또는 41의 아미노산 서열을 포함하는 프레임워크
Figure imgf000014_0001
서열번호 48, 49, 50, 또는 51의 아미노산 서열을 포함하는 프레임워크（丄-요3）, 및 서열번호 58, 59, 60 또는 61의 아미노산 서열을 포함하는 프레임워크（丄-요4）로 이루어지는 군에서 선택된 적어도 하나의 프레임 워크를 포함하는 것인 항- 00660항체 또는 이의 항원 결합단편일 수 있다. The light chain variable region of the antibody comprises a framework comprising the amino acid sequence of SEQ ID NO: 28, 29, 30 or 31, a framework comprising the amino acid sequence of SEQ ID NO: 38, 39, 40 or 41
Figure imgf000014_0001
A framework comprising the amino acid sequence of SEQ ID NO: 48, 49, 50, or 51 (XIII) and a framework comprising the amino acid sequence of SEQ ID NO: 58, 59, 60 or 61 (XIII) It may be an anti-00660 antibody or antigen-binding fragment thereof comprising at least one framework selected from the group consisting of.
상기 항체는서열번호 7， 14, 15， 16， 17또는 18의 아미노산서열을 포함하는 중쇄 가변영역; 및 서열번호 8， 19， 20, 또는 21의 아미노산 서열을 포함하는 경쇄 가변영역을 포함하는
Figure imgf000014_0002
또는 이의 항원 결합단편일 수 있다. The antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, 14, 15, 16, 17 or 18; And a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8, 19, 20, or 21
Figure imgf000014_0002
Or an antigen binding fragment thereof.
본 발명에 따른 마우스 항체 또는 키메릭 항체의 일예는 서열번호 1 내지 3의 아미노산 서열을 포함하는 ■ 영역의 001?를 결정하는 아미노산 서열 및 서열번호 4 내지 6의 아미노산 서열을 포함하는 1영역의
Figure imgf000014_0003
결정하는 아미노산 서열로 이루어지는 군에서 선택된 1종 이상의 아미노산 서열을 포함하는 항체 또는 이의 항원 결합 단편일 수 있다. 상기 마우스 항체 또는 키메릭 항체의 일예에
Figure imgf000014_0004
서열과 가변영역 서열을 하기 표 1에 정리한다. One example of a mouse antibody or chimeric antibody according to the present invention is an amino acid sequence that determines 001? Of the region comprising the amino acid sequence of SEQ ID NOs: 1 to 3 and an amino acid sequence of the amino acid sequence of SEQ ID NOs: 4 to 6
Figure imgf000014_0003
It may be an antibody or antigen-binding fragment thereof comprising at least one amino acid sequence selected from the group consisting of amino acid sequences to be determined. In one example of the mouse antibody or chimeric antibody
Figure imgf000014_0004
Sequences and variable region sequences are summarized in Table 1 below.
구체적으로， 본 발명의 항체의 일예는 m 영역의
Figure imgf000014_0005
결정하는 아미노산 서열로서 서열번호 1（00^）, 서열번호 2犯에2） 및 서열번호 301«3） 및/또는 I 영역의
Figure imgf000014_0006
결정하는 아미노산 서열인 서열번호 4犯 1）， 서열번호 501«2） 및 서열번호 6此에3）의 아미노산 서열을 포함한다. Specifically, one example of the antibody of the present invention is the
Figure imgf000014_0005
The amino acid sequence to be determined is SEQ ID NO: 1 (00 ^), SEQ ID NO: 2 犯 2) and SEQ ID NO: 301 «3) and / or the region of I
Figure imgf000014_0006
Amino acid sequences of SEQ ID NO: 4 ′ 1), SEQ ID NO: 501 «2) and SEQ ID NO: 6 ′ 3), which are amino acid sequences to be determined.
상기 마우스 항체 또는 키메릭 항체는서열번호 7의 아미노산서열을 포함하는 영역 및 서열번호 8의 아미노산 서열을 포함하는 1영역을 포함하는 것일 수 있다.  The mouse antibody or chimeric antibody may include a region including the amino acid sequence of SEQ ID NO: 7 and one region including the amino acid sequence of SEQ ID NO: 8.
본 발명에 따른 마우스 항체 또는 키메릭 항체를 유효성분으로 포함하는
Figure imgf000014_0007
질병 또는 이의 증상의 개선， 예방 또는 치료용 약학 조성물, 키트또는 치료방법에 관한 것이다. Mouse antibody or chimeric antibody according to the invention as an active ingredient
Figure imgf000014_0007
A pharmaceutical composition, kit or method for treating, preventing or treating a disease or symptom thereof.
본 발명에 따른 마우스 항체 또는 키메릭 를 들면 기탁번호 Accession No. for the mouse antibody or chimeric according to the present invention
［（比卵내 - 요예의 하이브리도마로부터 생산된
Figure imgf000014_0008
항체의 。 - , ◦예내2, 抑묘내3， ◦예_1그， ◦예- ， 및 포함하는 항-◦예此 항체 또는 이의 항원 결합 단편을 포함하는 ^）엤 관련 질병 또는 이의 증상의 예방 또는 치료용 약학 조성물에 관한 것이다. 상기 하이브리도마 세포는 한국세포주은행 (Korean Cel l Line Research Foundat ion, KCLRF)에 ’8F5’로 2010년 2월 22일자로 기탁번호 KCLRF-BP-00230로 기탁된 것이며， KR 10- 1214177등록공보에 자세히 기재되어 있다. （（比 卵 nae- Produced from the Hybridoma of Yo-Ye
Figure imgf000014_0008
O) of an antibody, including, eg, Example 2, Myocardium 3, ◦Example _1 Example, ◦Example-, and containing an anti-◦prescription antibody or antigen-binding fragment thereof. It relates to a pharmaceutical composition for prophylaxis or treatment. The hybridoma cells were deposited with the deposit number KCLRF-BP-00230 dated February 22, 2010 as '8F5' to the Korean Cel Line Research Foundat ion (KCLRF), KR 10-1214177. It is described in detail in.
본 발명은 상기 마우스 항체 또는 키메릭 항체를 이용하여 항- CD66c 항체 8F5의 아미노산 서열과 사람 항체 서열을 프레임 워크 서열에 기초하여 제조한다. 인간화 재조합 항체 후보 중에서 발현 정도와 aggregat ion 유무, 세포결합 정도를 기준으로 인간화 후보항체 중에서 발현이 정상적으로 이뤄지며， 단백질 자체의 불안정성으로 형성되는 aggregat ion 이 적으며, 또한 타겟 항원 양성의 세포에 결합하는 능력이 키메릭 항체와 유사한 인간화 항체 후보를 선별한다. 구체적으로 세포결합 양상은 키메릭 항체와 비슷한 수준으로서 항체 양성률 (% gated)과 형광 평균값 (mean)을 곱하고 이를 키메릭 항체와 상대 비교하여 土 20% 범위 내 포함되는 후보 항체를 선별한다. 따라서, 본 발명에서 선정된 항체군은 통상 인간화 항체 생성 시， 인간화 항체의 framework region 에 마우스 항체 CDR 부위 서열을 삽입하였을 때 원래 단백질 구조의 변경으로 항체 결합력이 급격히 저하된다는 결과를 고려하였을 때， 매우 우수한 인간화 항체를 선별하였다할수 있다.  The present invention uses the mouse antibody or chimeric antibody to prepare the amino acid sequence and the human antibody sequence of anti-CD66c antibody 8F5 based on the framework sequence. The expression of humanized candidate antibodies is normally expressed on the basis of the expression level, the presence or absence of aggregat ion, and the degree of cell binding among the candidate humanized recombinant antibodies, the aggregat ion formed by the instability of the protein itself is small, and the ability to bind to the target antigen-positive cells. Humanized antibody candidates similar to this chimeric antibody are selected. Specifically, the cell binding pattern is similar to that of the chimeric antibody, multiplying the antibody positive rate (% gated) and the mean fluorescence (mean), and comparing it with the chimeric antibody to select candidate antibodies included in the range of 土 20%. Therefore, the antibody group selected in the present invention is very considerably in consideration of the result that the antibody binding ability is drastically reduced due to the change in the original protein structure when the mouse antibody CDR region sequence is inserted into the framework region of the humanized antibody. Good humanized antibodies can be selected.
바람직하게는, 키메릭 항체 대비하여 세포결합력을 기준으로 높은 결합력을 나타내는 다섯 종류의 인간화 재조합 항체를 선택하고， 이들을 ELISA 방법으로 CD66c 항원 및 유사 CD66 항원들에 대한 결합력 분석을 실시한다.  Preferably, five kinds of humanized recombinant antibodies exhibiting high avidity based on cell binding capacity as compared to chimeric antibodies are selected, and these are subjected to avidity analysis for CD66c antigen and similar CD66 antigens by ELISA method.
또한， 본원 발명에 따른 인간화 항체는 키메릭 항체에 비해 우수한 안정성을 나타내며, 예를 들면 ANS 반응성 변이%가 200% 미만으로 안정한 항체이며， 200% 미만은· 변화가 매우 미미한 것으로 간주되고,. 그 이상은 의미 있는 단백질 구조'변경이 이뤄져 ANS 반응성이 관찰된 것으로 해석될 수 있다. 따라서， 본 발명에 따른 인간화항체는 키메릭 항체 대비 유사한 항원 결합혁 및 세포 결합력을 가지며， 항체 단백질 자체의 물리적 안정성이 증가하였으며， 이러한 사실은 치료용 항체의 druggabi l i ty 측면에서 매우우수한특정이라할수 있다. In addition, the humanized antibody according to the present invention exhibits excellent stability compared to chimeric antibodies, for example, an antibody having a stable ANS reactivity variation of less than 200%, and less than 200% is considered to be very insignificant. Beyond that yirwojyeo the protein structure, which change can be interpreted as meaning the ANS reactivity observed. Therefore, the humanized antibody according to the present invention has similar antigen binding properties and cell binding capacity as compared to chimeric antibodies, and the physical stability of the antibody protein itself is increased, which is very excellent in terms of druggabi li ty of therapeutic antibodies. have.
ANS 시약에 대한 항체의 형광값 변이율은 저온 조건 (예， 4°C )에서 측정한 형광값과， 고온 조건 (예， 62°C )에서 측정한 형광값의 차 (di f ference)를， 저온조건에서 측정한 형광값으로 나눈 값을 의미한다. 2019/221574 1»（：1^1{2019/006007 The fluorescence value variation rate of the antibody against the ANS reagent is determined by the difference between the fluorescence value measured at low temperature conditions (eg, 4 ° C) and the fluorescence value measured at high temperature conditions (eg, 62 ° C), Means the value divided by the fluorescence value measured at low temperature conditions. 2019/221574 1 »（： 1 ^ 1 {2019/006007
［수학식］ [Mathematical formula]
형광값 변이율 = （고온 조건에서 측정한 형광값- 저온 조건에서 측정한 형광값）/（저온조건에서 측정한 형광값）  Fluorescence value variation rate = (fluorescence value measured at high temperature condition-fluorescence value measured at low temperature condition) / (fluorescence value measured at low temperature condition)
구체적인 항체 형광값 변이율을 얻는 방법으로서， 냉장조건（4 °0 및 온도에서 각각 4시간 방치한 후에 요 시약 반응성을 형광 리더기로 분석하여 형광값으로 표시하고， 상기 수학식을 이용하여 형광값 변이율을 측정할수 있다. 본 발명에 따른 인간화 항체의 일예는 서열번호 9 내지 13의 아미노산 서열을 포함하는 중쇄 또는 경쇄 가변영역의
Figure imgf000016_0001
결정하는 아미노산 서열로 이루어지는 군에서 선택된 1종 이상의 아미노산 서열을 포함할 수 있으며， 추가로 마우스 항체 또는 키메릭 항체의 일예는 서열번호 1 내지 3의 아미노산 서열을 포함하는 영역의
Figure imgf000016_0002
결정하는 아미노산서열 및 서열번호 4내지 6의 아미노산 서열을 포함하는 영역의 결정하는 아미노산 서열로 이루어지는 군에서 선택된 1종 이상의 아미노산서열을포함하는 항체일 수 있다. As a method of obtaining a specific antibody fluorescence variation rate, after cooling for 4 hours at 4 ° 0 and temperature, the reagent reactivity is analyzed by a fluorescence reader and displayed as a fluorescence value. The interest rate can be measured. One example of a humanized antibody according to the present invention comprises a heavy or light chain variable region comprising the amino acid sequence of SEQ ID NOs.
Figure imgf000016_0001
It may include one or more amino acid sequence selected from the group consisting of the amino acid sequence to be determined, and further one example of a mouse antibody or chimeric antibody of the region comprising the amino acid sequence of SEQ ID NO: 1 to 3
Figure imgf000016_0002
It may be an antibody comprising at least one amino acid sequence selected from the group consisting of the amino acid sequence to be determined and the amino acid sequence to determine the region comprising the amino acid sequence of SEQ ID NO: 4 to 6.
구체적으로, 인간화 항체의 일예는 서열번호 1 또는 9의 아미노산 서열을 포함하는 쌔 영역의 0 1을 결정하는 아미노산 서열， 서열번호 2 또는 10의 아미노산 서열을 포함하는 영역의 0 2를 결정하는 아미노산 서열， 서열번호 3의 아미노산 서열을 포함하는 영역의 0 3을 결정하는 아미노산서열을포함하는 중쇄 가변영역일 수 있다.  Specifically, one example of a humanized antibody is an amino acid sequence that determines 0 1 of an ass region comprising an amino acid sequence of SEQ ID NO: 1 or 9, and an amino acid sequence that determines 0 2 of a region comprising an amino acid sequence of SEQ ID NO: 2 or 10 It may be a heavy chain variable region comprising an amino acid sequence that determines 0 3 of the region comprising the amino acid sequence of SEQ ID NO: 3.
또한 인간화 항체의 일예는 서열번호 4 , 11 또는 12의 아미노산 서열을 포함하는 영역의 0예1을 결정하는 아미노산 서열, 서열번호 5의 아미노산 서열을 포함하는 I 영역의
Figure imgf000016_0003
결정하는 아미노산 서열， 서열번호 6 또는 서열번호 13의 아미노산 서열을 포함하는 VI 영역의 0 3을 결정하는 아미노산서열을포함하는중쇄 가변영역일 수 있다. In addition, one example of a humanized antibody is an amino acid sequence determining 0 example 1 of a region comprising the amino acid sequence of SEQ ID NO: 4, 11 or 12, a region of I region comprising the amino acid sequence of SEQ ID NO: 5
Figure imgf000016_0003
It may be a heavy chain variable region comprising an amino acid sequence that determines 0 3 of the VI region including the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 13 to determine.
인간화 항체의 일예는 서열번호 7 및 서열번호 14 내지 18의 아미노산 서열을 포함하는 중쇄 가변영역로 이루어지는 군에 선택된 중쇄 가변영역과， 서열번호 8 및 서열번호 19 내지 21의 아미노산 서열을 포함하는 경쇄 가변영역로 이루어지는 군에서 선택된 경쇄 가변영역을 포함할 수 있으며, 단 서열번호 7 및 서열번호 8을 포함하는 항체는 제외된다.  One example of a humanized antibody is a heavy chain variable region selected from the group consisting of a heavy chain variable region comprising SEQ ID NO: 7 and amino acid sequences of SEQ ID NOs: 14-18, and a light chain variable comprising the amino acid sequence of SEQ ID NO: 8 and SEQ ID NOs: 19-21 It may include a light chain variable region selected from the group consisting of regions, except that the antibody comprising SEQ ID NO: 7 and SEQ ID NO: 8.
상기 인간화 항체의 일예에 서열과 가변영역 서열을 하기 2019/221574 1»(：1/10公019/006007 In one example of the humanized antibody sequence and variable region sequence 2019/221574 1 »(： 1/10 公 019/006007
표 1에 정리한다. It summarizes in Table 1.
【표 1]  [Table 1]
Figure imgf000017_0001
2019/221574 1»(：1^1{2019/006007
Figure imgf000018_0001
본 발명에 따른 인간화 항체의 일예의 프레임 워크 서열을 하기 표 2 및 3에 나타내며， 상기 항체는 중쇄 가변영역의 프레임 워크 1 내지 4 , 및 경쇄 가변영역의 프레임 워크 1 내지 4로 이루어지는 군에서 선택된 1종 이상의 프레임 워크를 포함하는 항체일 수 있다.
Figure imgf000017_0001
2019/221574 1 »(： 1 ^ 1 {2019/006007
Figure imgf000018_0001
An example framework sequence of the humanized antibody according to the present invention is shown in Tables 2 and 3, wherein the antibody is selected from the group consisting of frameworks 1 to 4 of the heavy chain variable region, and frameworks 1 to 4 of the light chain variable region. It may be an antibody comprising at least a framework.
구체적으로， 중쇄 가변영역에서 프레임 워크 1의 아미노산 서열은 서열번호 23 내지 27을 포함할 수 있으며， 프레임 워크 2의 아미노산 서열은 서열번호 32 내지 37을 포함할 수 있으며, 프레임 워크 3의 아미노산 서열은 서열번호 43 내지 47을 포함할 수 있으며, 및 프레임 워크 4의 아미노산 서열은 서열번호 53 내지 57을 포함할 수 있다.  Specifically, the amino acid sequence of the framework 1 in the heavy chain variable region may include SEQ ID NO: 23 to 27, the amino acid sequence of the framework 2 may include SEQ ID NO: 32 to 37, the amino acid sequence of the framework 3 SEQ ID NOs: 43-47, and the amino acid sequence of Framework 4 may include SEQ ID NOs: 53-57.
경쇄 가변영역에서 프레임 워크 1의 아미노산 서열은 서열번호 29 내지 31을 포함할 수 있으며， 프레임 워크 2의 아미노산 서열은 서열번호 39 내지 41을 포함할 수 있으며， 프레임 워크 3의 아미노산 서열은 서열번호 49 내지 51을 포함할 수 있으며， 및 프레임 워크 4의 아미노산 서열은 서열번호 59 내지 61을 포함할 수 있다. 상기 인간화 항체의 일예에 따른 프레임워크 서열을 하기 표에 나타낸다.  In the light chain variable region, the amino acid sequence of Framework 1 may include SEQ ID NOs: 29 to 31, the amino acid sequence of Framework 2 may include SEQ ID NOs: 39 to 41, and the amino acid sequence of framework 3 may be SEQ ID NO: 49 To 51, and the amino acid sequence of Framework 4 may include SEQ ID NOs: 59-61. Framework sequences according to one example of such humanized antibodies are shown in the table below.
【표 2】  Table 2
Figure imgf000018_0002
Figure imgf000018_0002
Figure imgf000019_0001
Figure imgf000019_0001
【표 3】 Table 3
Figure imgf000019_0002
상기 인간화 항체는 서열번호 14 내지 18의 아미노산 서열로 이루어지는 군에서 선택되는 ■영역 및 서열번호 19 내지 21의 아미노산 서열로 이루어지는 군에서 선택된 1영역을 포함하는 것일 수 있다. 구체적으로， 상기 인간화 항체의 예는, 서열번호 15의 아미노산 서열을 포함하는 ■영역 및 서열번호 21의 아미노산 서열로 이루어지는 군에서 선택된 영역을 포함하는 항체 0¾8+\¾6) , 서열번호 18의 아미노산 서열을 포함하는 ■영역 및 서열번호 21의 아미노산 서열로 이루어지는 군에서 선택된 1영역을 포함하는 항체 (\¾8+\¾11) , 서열번호 16의 아미노산 서열을 포함하는 VH영역 및 서열번호 19의 아미노산 서열로 이루어지는 군에서 선택된 영역을 포함하는 항체 (Vk5+VH7)， 서열번호 17의 아미노산 서열을 포함하는 VH영역 및 서열번호 20의 아미노산 서열로 이루어지는 군에서 선택된 VL영역을 포함하는 항체 (Vk7+VH6) , 서열번호 15의 아미노산 서열을 포함하는 VH영역 및 서열번호 20의 아미노산 서열로 이루어지는 군에서 선택된 VL영역을 포함하는 항체 (Vk7+VH10)， 서열번호 16의 아미노산서열을 포함하는 VH영역 및 서열번호 20의 아미노산 서열로 이루어지는 군에서 선택된 VL영역을 포함하는 항체 (Vk7+VH7)， 서열번호 14의 아미노산 서열을 포함하는 VH영역 및 서열번호 20의 아미노산 서열로 이루어지는 군에서 선택된 VL영역을 포함하는 항체 (Vk7+VH5)， 및 서열번호 16의 아미노산 서열을 포함하는 VH영역 및 서열번호 21의 아미노산 서열로 이루어지는 군에서 선택된 VL영역을 포함하는 항체 (Vk8+VH7)일 수 있다. 상기 항체의 구체적인 조합 및 아미노산 서열은 하기 표 6에 나타낸다. 상기 항체의 바람직한 일예는， 서열번호 15의 아미노산 서열을 포함하는 VH영역 및 서열번호 21의 아미노산 서열을 포함하는 VL영역을 포함하는 항체 (Vk8+VH6) , 서열번호 18의 아미노산 서열을 포함하는 개영역 및 서열번호 21의 아미노산 서열로 이루어지는 군에서 선택된 VL영역을 포함하는 항체 (Vk8+VH11) , 서열번호 16의 아미노산 서열을 포함하는 VH영역 및 서열번호 19의 아미노산 서열로 이루어지는 군에서 선택된 VL영역을 포함하는 항체 (Vk5+VH7) , 서열번호 17의 아미노산 서열을 포함하는 영역 및 서열번호 20의 아미노산 서열로 이루어지는 군에서 선택된 VL영역을 포함하는 항체 (Vk7+VH6) , 서열번호 15의 아미노산 서열을 포함하는 VH영역 및 서열번호 20의 아미노산 서열로 이루어지는 군에서 선택된 VL영역을 포함하는항체 (Vk7+VH10)일 수 있다.
Figure imgf000019_0002
The humanized antibody may include a region selected from the group consisting of amino acid sequences of SEQ ID NOs: 14 to 18 and one region selected from the group consisting of amino acid sequences of SEQ ID NOs: 19 to 21. Specifically, examples of the humanized antibody include antibody 0¾8 + \ ¾6) comprising an amino acid sequence of SEQ ID NO: 15 and a region selected from the group consisting of the amino acid sequence of SEQ ID NO: 21, and an amino acid sequence of SEQ ID NO: 18. An antibody comprising one region selected from the group consisting of a region comprising an amino acid sequence of SEQ ID NO: 21 (\ ¾8 + \ ¾11), and an amino acid sequence of SEQ ID NO: 16 An antibody comprising a region selected from the group consisting of a VH region comprising and an amino acid sequence of SEQ ID NO: 19 (Vk5 + VH7), a VH region comprising an amino acid sequence of SEQ ID NO: 17 and an amino acid sequence of SEQ ID NO: 20 An antibody comprising a VL region (Vk7 + VH6), a VH region comprising an amino acid sequence of SEQ ID NO: 15, and a VL region selected from the group consisting of the amino acid sequence of SEQ ID NO: 20 (Vk7 + VH10), SEQ ID NO: 16 An antibody (Vk7 + VH7) comprising a VH region comprising an amino acid sequence of SEQ ID NO: 20 and an amino acid sequence of SEQ ID NO: 20, a VH region comprising the amino acid sequence of SEQ ID NO: 14, and an amino acid sequence of SEQ ID NO: 20 An antibody (Vk7 + VH5) comprising a VL region selected from the group consisting of: a VH region comprising the amino acid sequence of SEQ ID NO: 16 and a SEQ ID NO: 21 It may be an antibody (Vk8 + VH7) comprising a VL region selected from the group consisting of amino acid sequences. Specific combinations and amino acid sequences of the antibodies are shown in Table 6 below. Preferred examples of the antibody include an antibody comprising a VH region comprising an amino acid sequence of SEQ ID NO: 15 and a VL region comprising an amino acid sequence of SEQ ID NO: 21 (Vk8 + VH6), a open region comprising an amino acid sequence of SEQ ID NO: 18 And an antibody (Vk8 + VH11) comprising a VL region selected from the group consisting of the amino acid sequence of SEQ ID NO: 21, a VH region comprising the amino acid sequence of SEQ ID NO: 16, and a VL region selected from the group consisting of the amino acid sequence of SEQ ID NO: 19; An antibody (Vk7 + VH6) comprising an antibody (Vk5 + VH7), a region comprising the amino acid sequence of SEQ ID NO: 17, and a VL region selected from the group consisting of the amino acid sequence of SEQ ID NO: 20, an amino acid sequence of SEQ ID NO: 15; It may be an antibody (Vk7 + VH10) comprising a VH region comprising and a VL region selected from the group consisting of the amino acid sequence of SEQ ID NO: 20.
CD66C항체 또는 항체 절편은 다양한 표지기 (labeling agent) , 독성 물질 또는 항종양제와 결합할 수 있다. 당해 기술 분야에 있어서 주지 방법에 의해 본 발명의 항체는 상기 표지기， 독소， 또는 항종양제와 결합될 수 있는 것은 당업자에게 분명하다. 이러한 결합은 항체 또는 항원의 발현 후， 부착부위에 화학적으로 수행하는 것도 가능하고， 혹은 DNA 레벨로 본 발명의 항체 또는 항원 내에 결합산물을 조작해 넣어도 괜찮다. 이어서 본 명세서에 있어서 이하로 기재하는 적절한 숙주계에 있어서 DNA를 발현시키고 그리고 발현시킨 단백질을 회수해, 필요에 따라 재생시킨다. 결합은 당해 기술 수준에 있어서 알려진 링커를 통해 달성해도 괜찮다. 특히 이 기술과 함께 산성 조건 혹은 환원 조건 하에서 또는 특정 프로테아제에 대한 노출시에 독소 또는 항종양제를 방출하는, 다양한 링커를 이용할 수 있다. 특정 양태에 있어서는 표지기， 독소, 또는 항종양제를 다양한 길이의 스페이서 암에 의해 결합시키고 잠재적인 입체 장애를 감소시키는 것이 바람직한경우도 있다. CD66C antibody or antibody fragments can be combined with various labeling agents, toxic substances or antitumor agents. It is apparent to those skilled in the art that antibodies of the present invention can be combined with the labeling group, toxin, or antitumor agent by methods well known in the art. Such binding may be performed chemically at the attachment site after the expression of the antibody or antigen, or the binding product may be manipulated into the antibody or antigen of the present invention at the DNA level. Next, in the appropriate host system described below in the present specification, DNA is expressed and the expressed protein is recovered and regenerated as necessary. The linkage may be accomplished through a linker known in the art. In particular with this technique various linkers are available which release toxins or anti-tumor agents under acidic or reducing conditions or upon exposure to certain proteases. In certain embodiments it may be desirable to bind a label, toxin, or antitumor agent by spacer arms of varying lengths and to reduce potential steric hindrance.
CD66c의 항원결정부위에 대한 항체 또는 항체 절편은 CD66c 단백질， CD66c의 항원결정부위, CD66c의 항원결정부위를 포함하는 CD66c 단백질 일부， 또는 CD66c의 항원결정부위를 발현하는 세포를 항원으로 이용하여 통상의 방법으로 제조가능 하다. 일례로, CD66c 항체 제조방법은， (a) CD66c 단백질, CD66c의 항원결정부위， CD66c의 항원결정부위를 포함하는 Antibodies or antibody fragments against the antigenic region of CD66c are conventionally used as antigens using cells expressing the CD66c protein, the antigenic region of CD66c, a portion of the CD66c protein including the antigenic region of CD66c, or the antigenic region of CD66c. It can be manufactured by the method. In one embodiment, the method for producing a CD66c antibody includes (a) a CD66c protein, an antigenic determining site of CD66c, and an antigenic determining site of CD66c.
CD66c 단백질 일부, 또는 CD66c의 항원결정부위를 발현하는 세포를 동물에 주입하여 면역화 시키는 단계, (b) CD66c에 특이적인 항체를 생산하는 비장세포를 수득하는 단계 및 (c) 상기 비장세포를 골수종세포와 융합시켜, CD66c에 대한 항체를 생산하는 하이브리도마 세포를 선별하는 단계를 포함하는 CD66c 항체를 생산하는 생산주 제조방법으로 통하여 실시가능 하다. 상기 생산주는 생체외 ( in vi tro)에서 배양하거나, 생체내 주입하여 항체를 분리할 수 있다. 일례로， 마우스의 복강내에 삽입하고 복수로부터 분리， 정제한다. 단클론 항체의 분리 및 정제는 배양 상층액 또는 복수를 이온교환 크로마토그래피 (DEAE 또는 DE52 등)， 항 면역글로불린 칼럼 또는 프로테인 A 컬럼 등의 친화성 크로마토그래피를 이용하여 실시할 수가 있다. 본 발명에 따른 항체가 결합하는 항원결정부위는 MDSC 특이적인 발현을 나타낸다. 따라서 , 항- CD66c 항체는 MDSC의 검출에 유용하게 사용할 수 있을 뿐만 아니라 독성물질을 포함시켜 MDSC만을 특이적으로 세포살상 (cytotoxici ty) 시킬 수 있다. Immunizing an animal by injecting a portion of the CD66c protein or a cell expressing the antigenic determinant site of CD66c into the animal, (b) obtaining splenocytes producing an antibody specific for CD66c, and (c) the splenocytes into myeloma cells. It is possible to carry out through the production method of the production line for producing a CD66c antibody comprising a step of fusion with, hybridoma cells for producing an antibody against CD66c. The producer may isolate the antibody by culturing in vitro or in vivo. For example, the mouse is inserted into the abdominal cavity and separated from the ascites and purified. Isolation and purification of monoclonal antibodies can be carried out using affinity chromatography, such as ion exchange chromatography (DEAE or DE52), anti-immunoglobulin column or protein A column, in culture supernatant or ascites. The epitope to which the antibody according to the present invention binds exhibits MDSC-specific expression. Therefore, anti-CD66c antibodies can be usefully used for detection of MDSCs, and can also specifically poison MDSCs by including toxic substances.
다른 예는 본 발명에 따른 항체의 CD66c을 포함하는 MDSC의 검출을 위한 마커로서의 용도를 제공하며， 이에 CD66c에 대한 항체 또는 이의 항원결합단편을 이용하여 MDSC 검출 및 MDSC 관련 질환의 진단용도 또는 진단의 정보를 제공하는용도사용될 수 있다.  Another example provides the use of an antibody according to the invention as a marker for the detection of MDSCs comprising CD66c, wherein the antibody or antigen-binding fragment thereof for CD66c is used to detect MDSC and to diagnose or diagnose MDSC-related diseases. It can also be used for informational purposes.
예를 들면， CD66c에 대한 항체 또는 이의 항원결합단편을 이용하여 상기 항체의 항원결정부위와 상호작용하는 물질을 포함하는 MDSC의 검출용 조성물을 제공한다. 상기 상호작용하는 물질은 CD66c에 위치하는 항원결정부위와 상호작용 가능한 모든 물질, 예컨대， 화학물질 (smal l molecular chemi cal ) , 항체， 항체의 항원결합단편， 앱타머 등으로 이루어진 군에서 선택된 1종 이상일 수 있다. For example, the present invention provides a composition for detecting MDSC, including a substance interacting with an antigen-determining site of the antibody by using an antibody against CD66c or an antigen-binding fragment thereof. The interacting substance is composed of all substances capable of interacting with the epitope located at CD66c, such as a chemical molecular molecule, an antibody, an antigen-binding fragment of the antibody, and an aptamer. It may be one or more selected from the group.
본 발명 진단 조성물은 다양한 세포， 조직 또는 다른 적절한 시료에 있어서의, CD66c의 바람직하지 않은 발현 또는 과잉 발현의 검출로서， 시료를 본 발명의 항체와 접촉시키는 것 및 시료에 있어서 CD66c의 존재를 검출하는 것을 포함한 검출에 유용하다. 따라서， 본 발명 진단 조성물을 이하로 정의하는 발병 또는 질환상태를 평가하기 위해 이용해도 무방하다. 특히 CD66c를 발현하는， MDSC를 본 발명의 항체, 항체 단편 또는 유도체로 타겟팅해도 괜찮다. 본 발명의 항체가 결합한 세포는 그러므로 보체계 등의 면역계 기능에 의해 또는 세포 매개성 세포 독성에 의해 공격되고 따라서 , CD66C의 바람직하지 않은 발현 또는 과잉 발현을 나타내는 세포의 수가 감소하거나또는 이러한세포가사멸된다.  The diagnostic composition of the present invention detects undesirable or overexpression of CD66c in various cells, tissues or other suitable samples, comprising contacting the sample with the antibody of the invention and detecting the presence of CD66c in the sample. It is useful for detection including. Therefore, the diagnostic composition of the present invention may be used for evaluating the onset or disease state defined below. In particular, MDSC expressing CD66c may be targeted to the antibody, antibody fragment or derivative of the present invention. The cells bound by the antibodies of the present invention are therefore attacked by immune system functions such as the complement system or by cell mediated cytotoxicity, thus reducing the number of cells exhibiting undesirable or overexpression of CD66C or killing such cells. .
구체적인 예로서， 본 발명에 따른 CD66c에 대한 항체 또는 이의 항원결합단편을 이용한 MDSC 관련 질환의 진단 방법 또는 진단용 조성물을 제공한다.  As a specific example, the present invention provides a method or diagnostic composition for diagnosing MDSC-related diseases using an antibody against CD66c or an antigen-binding fragment thereof.
MDSC 관련 질환, 예를 들면 암을 진단하기 위하여， 본 발명에 따른 In order to diagnose MDSC related diseases, for example cancer,
CD66c에 대한 항체 또는 이의 항원결합단편을 이용하는 경우, 암 조직 또는 암세포의 자체에서의 CEACAM6 항원 발현과 상관없이 암조직 주변의 침윤된 MDSC를 표적으로 진단 및 치료에 활용할 있다. 본 발명에 따른 CD66c에 대한 항체는 고형암 세포에서 발현되는 CD66c에 결합할 뿐만 아니라, 고형암에서 CD66c를 발현하지 않는 암에서도， 암에 의한 MDSC 증가 상태를 MDSC에서 발현되는 CD66c를 표적으로 하여 탐지하여 암을 탐지할 수 있다. 구체적으로, 암세포에서 CEACAM6 가 양성인 폐 선암 ( lung adenocarcinoma)과 암세포 자체에서는 CEACAM6가 음성인 편평상피암 ( lung squamous cel l carcinoma) , 비뇨기계 암 (Ur inary bladder cancer) 및 피부 암 (Melanoma mal ignancy) 조직에 CEACAM6 면역 염색한 결과 암 조직의 비 종양부위에 CEACAM6 양성인 MDSC가 있는 것을 확인하였다 (도 13) . When using antibodies to CD66c or antigen-binding fragments thereof, infiltrated MDSCs around cancer tissues can be used for diagnosis and treatment as targets, regardless of the expression of CEACAM6 antigen in cancer tissues or cancer cells themselves. The antibody against CD66c according to the present invention not only binds to CD66c expressed in solid cancer cells, but also in cancers that do not express CD66c in solid cancer, and detects the increased state of MDSC caused by cancer by targeting CD66c expressed in MDSC to cancer. Can be detected. Specifically, lung adenocarcinoma positive for CEACAM6 in cancer cells and lung squamous cell carcinoma (Ur inary bladder cancer) and skin cancer (Melanoma mal ignancy) tissues for which CEACAM6 is negative in cancer cells themselves CEACAM6 immunostaining confirmed that there was a CEACAM6 positive MDSC at the non-tumor site of cancer tissue (FIG. 13).
따라서， 암세포 세포표면의 CEACAM6 양성도와 상관없이， 암환자에서는 MDSC 가 증가되는 경향을 보이며， 이는 실시예 8의 결과와 같이 미세종양환경에 침윤되어 있는 MDSC 를 검출하고 확인할 수 있다. 이는 MDSC를 선택적으로 용해할 수 있음을 보인 실시예 5.2의 결과와 함께 고려하였을 때， 암세포 상의 CEACAM6 양성도와 상관없이 MDSC 를 표적으로 진단 및 치료목적에 사용할 수 있음을 나타낸다. 암 종에 따라 암세포 세포표면의 CEACAM6 발현 유무는 다를 수 있으나， 이와 상관없이 대부분의 암 종에서 MDSC는 증가되기 때문에， 본원 발명에 따른 CD66c 항체를 이용하여 MDSC를 표적 치료를 통해 대부분의 암 종을 대상으로 한 범용의 진단 및 치료적 목적으로 이용할수 있다. Therefore, regardless of the CEACAM6 positivity of the cancer cell surface, MDSC tends to increase in cancer patients, which can detect and confirm the MDSC infiltrating into the microtumor environment as shown in Example 8. This, in combination with the results of Example 5.2 showing that MDSCs can be selectively dissolved, indicates that MDSCs can be used for diagnostic and therapeutic purposes, regardless of the degree of CEACAM6 positivity on cancer cells. Depending on the type of cancer, the presence or absence of CEACAM6 expression on the surface of cancer cells may vary. Since MDSC is increased in carcinomas, MDSC can be used for general purpose diagnostic and therapeutic purposes for most carcinomas through targeted therapy using the CD66c antibody according to the present invention.
본 발명의 일 양태에 있어서는 본 발명의 항체， 항체 단편 또는 유도체를 표지기에 결합시킨다. 이러한 항체는 진단 애플리케이션에 특히 적합하다.  In one aspect of the present invention, the antibody, antibody fragment or derivative of the present invention is bound to a labeling group. Such antibodies are particularly suitable for diagnostic applications.
본 발명에 따른 조성물은 단독 활성 약제로서 투여할 수 있고, 또는 다른 약제와조합하여 투여될 수 있다.  The composition according to the invention can be administered as the sole active agent or in combination with other agents.
MDSC 검사방법은 (a) CD66c 항체를 MDSC를 포함하는 시료에 반응시키고, (b) 상기 항체에 대하여 양성반응을 나타내는 시료를 MDSC로 판단하는 것이다. 상기 시료는 림프액， 골수， 혈액 또는 혈구일 수 있으나 이에 한정되는 것은 아니다. 상기 CD66c 항체를 이용하여 MDSC 관련 질환을 검사하는 경우, 상기 CD66c 항체는 항원-항체 반응성을 확인할 수 있는 물질로 표지된 것일 수 있다. 사용 가능한상기 물질로는 방사성동위원소， 형광물질， 발광물질, 크로모젠 (chromogen) , 또는 기타 염색 물질 등이 있다. 또한본 발명의 CD66c 항체는 MDSC 질환을 진단하기 위한진단키트로 제공될 수 있다. 상기 진단키트는 CD66c 항체 이외에, 항원-항체반응 검출수단을 포함할 수 있다. 상기 검출수단은 유세포측정, 면역조직화학염색, 효소결합 면역톱착 분석 (enzyme l inked immunosorbent assay： ELISA) , 방사선 면역측정법 (radioimmunoassay: RIA) , 효소 면역분석 (enzyme immunoassay: EIA) , 형광면역분석 (Floresence immunoassay: FIA) 및 발광면역분석 ( luminescence immunoassay： LIA)으로 이루어진 군으로부터 선택된 방법을실시하기 위한통상의 물질 일 수 있다.  In the MDSC test method, (a) a CD66c antibody is reacted with a sample containing MDSC, and (b) a sample having a positive reaction with the antibody is determined as MDSC. The sample may be, but is not limited to, lymph, bone marrow, blood or blood cells. When the MDSC-related disease is tested using the CD66c antibody, the CD66c antibody may be labeled with a substance capable of confirming antigen-antibody reactivity. The materials that can be used include radioisotopes, fluorescent materials, luminescent materials, chromogens, or other dyeing materials. In addition, the CD66c antibody of the present invention can be provided as a diagnostic kit for diagnosing MDSC disease. The diagnostic kit may include antigen-antibody reaction detection means in addition to the CD66c antibody. The detection means may be flow cytometry, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay ( Floresion immunoassay (FIA) and luminescence immunoassay (LIA) may be a conventional material for carrying out the method selected from the group consisting of.
상기 고형암의 치료 효과는 암세포 (특히， 암줄기세포) 또는 이를 포함하는 암조직의 성장 억제 (양적 감소)， 사멸 (apoptosi s) 효과뿐 아니라, 이동 (migrat ion) , 침습 ( invasion) , 전이 (metastasi s) 등을 억제하여 이로 인한 암의 악화를 억제하는 효과를 포함한다. 본 발명에 따른 항체는 STING agoni st 또는 5_Fu 와 병용 처리하여 효과를 극대화 하고자， 본 발명의 항체와 병용 처리 시 보다 높은 효과를 얻을 것으로 기대할수 있다.  The therapeutic effect of the solid cancer is not only growth inhibition (quantitative reduction), apoptosi s effect of cancer cells (especially cancer stem cells) or cancer tissues including the same, but also migration (migrat ion), invasion, metastasis (metastasi). s) and the like to inhibit the exacerbation of cancer thereby. The antibody according to the present invention may be expected to obtain a higher effect when combined with the antibody of the present invention in order to maximize the effect by co-treatment with STING agoni st or 5_Fu.
본 명세서에서, "대상” 또는 ’’환자’’는 MDSC 관련 질환의 경감, 예방 및/또는 치료를 필요로 하는 환자를 의미하는 것으로, 모든 포유류， 예컨대 인간， 원숭이 등의 영장류, 마우스, 래트 등의 설치류일 수 있고， MDSC 관련 질환을 앓고 있거나 MDSC 관련 질환의 증상을 갖거나， MDSC 관련 질환 발병의 위험이 있는 환자일 수 있다. As used herein, "subject" or `` patient '' refers to a patient in need of alleviation, prevention and / or treatment of MDSC-related diseases and includes all mammals such as primates such as humans, monkeys, mice, rats, and the like. May be a rodent of MDSC The patient may be suffering from, having symptoms of, or at risk of developing a MDSC-related disease.
본 발명에 따른 항체 혹은 항체 절편의 투여는 허용된 모든 약물 투여방법에 의해 시행될 수 있다. 구체적으로 예를 들면， CD66c 항체를 유효성분으로 포함하는 치료제를 MDSC 관련 질환을 갖는 대상， 즉 인간 또는 동물에 경구 또는 비경구， 바람직하기로는 비경구로 투여하는 것이다. 상기 치료제는 약리학적으로 허용 가능한 부형제를 포함할 수 있으며， 이의 투여량은 환자의 상태에 따라 적절히 조절하는 것이 바람직하나， 일예로 1일 3 mg 내지 6 , 000 mg 일 수 있다. 치료제의 제형은 액제， 산제， 유제， 현탁제 또는 주사제일 수 있으나, 이에 한정되는 것은 아니다.  Administration of the antibody or antibody fragment according to the invention can be carried out by any method of drug administration permitted. Specifically, for example, a therapeutic agent containing a CD66c antibody as an active ingredient is administered orally or parenterally, preferably parenterally, to a subject having an MDSC-related disease, that is, a human or an animal. The therapeutic agent may include a pharmacologically acceptable excipient, and the dosage thereof may be appropriately adjusted according to the condition of the patient, but may be, for example, 3 mg to 6, 000 mg per day. The formulation of the therapeutic agent may be, but is not limited to, a liquid, powder, emulsion, suspension or injection.
본 발병은 CD66c 항원결정부위에 대한 항체, 항체 절편 (F( ab’)2 , Fab 및 Fv 등)， 및 리간드 이뤄진 군에서 선택된 항체를 이용하여 MDSC 관련 질환을 치료하는 방법을 제공한다. 항체 혹은 항체 절편은 바람직하기는 단클론 또는 다클론 항체로 이루어진 군에서 선택되며， 바람직하기로는 사람과 동물에서 기원한 것이다. 상기 CD66c 항체 또는 항체 절편은 상기에서 언급한 독소물질이 더욱 포함된 것일 수 있다. 독소물질은 항체에 융합， 접합, 결합, 또는 링크될 수 있으며， 이는 공지의 기술로 실시가능 하다.  The onset provides a method for treating MDSC related diseases using antibodies selected from the group consisting of antibodies against CD66c epitopes, antibody fragments (F (ab ′) 2, Fab and Fv, etc.), and ligands. The antibody or antibody fragment is preferably selected from the group consisting of monoclonal or polyclonal antibodies, preferably from humans and animals. The CD66c antibody or antibody fragment may further contain the above-mentioned toxin. Toxins can be fused, conjugated, bound, or linked to an antibody, which can be implemented by known techniques.
본 발명의 의약 조성물은 단독 활성 약제로서 투여해도 괜찮고 또는 기타 약제와 조합하고， 바람직하게는 당해 기술 분야에 있어서 문제의 질환의 치료에 적합한 것이 알려지는 것과 조합해 투여해도 괜찮다. 또한， 본 발명의 항체를 투여하는 방법은， 다른 항암치료, 예를 들면 화학적 요법, 방사선 요법， 세포치료제와 병행하여 수행할 수 있다. 상기 화학적 요법 또는 세포 치료제에 사용되는 다양한 MDSC 관련 질환을 치료로 알려진 치료제를 사용할 수 있다.  The pharmaceutical composition of the present invention may be administered as the sole active agent or in combination with other agents, preferably in combination with those known to be suitable for the treatment of the disease in question. In addition, the method of administering the antibody of the present invention can be carried out in parallel with other anticancer therapies, such as chemotherapy, radiation therapy, and cell therapy. Therapeutic agents known to treat various MDSC related diseases used in the chemotherapy or cell therapy may be used.
【발명의 효과】 【Effects of the Invention】
본 발명은 골수유래억제세포 (mye l o i d-der i ved suppressor ce l l , MDSC)에서 발현되는 CD66c에 대한 항체 또는 이의 항원 결합 단편을 포함하는 면역 활성 증강제 , 및 상기 면역 활성 증강제를 이용한 MDSC 관련 질환의 예방, 치료 또는 개선에 관한 용도를 제공한다.  The present invention provides an immune activity enhancer comprising an antibody against CD66c or an antigen-binding fragment thereof expressed in myeloid-derived suppressor cells (MDSC), and MDSC-related diseases using the immune activity enhancer. To provide for the prevention, treatment or amelioration of
【도면의 간단한 설명】 도 1은 마우스 8F5 항체로부터 항체 유전자를 클로닝하여 키메릭 재조합 항체로 발현시켜, CD66c 항원 양성인 A549 세포 표면에 결합함을 보여주는 결과이다. [Brief Description of Drawings] 1 shows the result of cloning the antibody gene from mouse 8F5 antibody and expressing it with chimeric recombinant antibody, binding to A549 cell surface that is CD66c antigen positive.
도 2a 내지 도 2c는 96종의 인간화 재조합 항체 중에서 1차 선정된 8종의 인간화 재조합 항체에 대한 HPLC 분석 결과로서, 각 항체 별로 왼쪽 2A to 2C show HPLC analysis results of eight humanized recombinant antibodies selected first from 96 humanized recombinant antibodies.
0D 220 nm, 오른쪽 0D 280 nm 분석한 결과를 나타내고 있다. 항체의 aggregat ion 및 절편 등의 항체 유래 불순물 포함 여부를 나타내는 결과이다. The results of the analysis of 0D 220 nm and the right 0D 280 nm are shown. It is a result which shows whether the antibody-containing impurities, such as an aggregat ion and a fragment, of an antibody are included.
도 3a 내지 도 3e는 96종의 인간화 재조합 항체 중에서 1차 선정된 8종의 인간화 재조합 항체에 대한 CD66c 항원 양성 세포 표면 결합을 확인한 결과로서, 키메릭 항체와 비교하여 유사한 정도의 세포 표면 결합을 나타낸다.  3A to 3E show CD66c antigen positive cell surface binding to eight humanized recombinant antibodies selected first from 96 humanized recombinant antibodies, and show similar levels of cell surface binding compared to chimeric antibodies. .
도 4a 및 도 4b는 96종의 인간화 재조합 항체 중에서 선정된 5종의 인간화 재조합 항체에 대한 CD66c 항원에 대한 결합력을 ELISA통해 확인한 결과로서， 도 4a는 CECACAM6 （抑 66c） 항원에 대한 결과이며, 도 4b는 CEACAM1 （抑 66a） 항원에 대한결과이다.  Figures 4a and 4b is a result confirmed by ELISA binding to CD66c antigen to five humanized recombinant antibodies selected from 96 humanized recombinant antibodies, Figure 4a is a result for the CECACAM6 (66c) antigen, 4b is the result for CEACAM1 (66a) antigen.
도 5a 및 도 5b는 96종의 인간화 재조합 항체 중에서 선정된 5종의 인간화 재조합 항체에 대한 가혹 온도 조건에서의 항체 안정성을 확인한 결과이다.  5A and 5B show the results of antibody stability under harsh temperature conditions for five humanized recombinant antibodies selected from 96 humanized recombinant antibodies.
도 6a 내지 도 6d는 CH0에서 발현시킨 인간화 재조합 항체의 CD66c 항원 양성 세포 A549의 세포표면 결합을나타낸 결과이다.  6A to 6D show the cell surface binding of CD66c antigen positive cells A549 of the humanized recombinant antibody expressed at CH0.
도 7의 상단은 MDSC 분석 방법에 대한 이해를 돕기 위한 모식도로, 점도표（dot plot）에서 세포의 크기에 따라 림프구를 제외하고 단핵구와 과립구 영역만을 지정한후에， HLA-DR의 발현이 없거나 낮은 군을 선택하고， 그 군에서 CDllb와 CD33에 양성인 그룹을 MDSC로 특정한 점도표 모식도이며， 하단은 지정된 MDSC군에서 DNP002의 양성률을 확인한결과이다.  7 is a schematic diagram to help understand the MDSC analysis method. After specifying only monocytes and granulocyte regions except lymphocytes according to the size of cells in a dot plot, the group having no or low expression of HLA-DR is shown. The viscosity table is a schematic diagram of MDSC selected from the group that is positive for CDllb and CD33 in the group, and the bottom is the result of confirming the positive rate of DNP002 in the designated MDSC group.
도 8는 DNP002 처리 후 MDSC 사멸효과를 분석한 대표적인 결과로, 탈푸코오스 DNP002에 의하여 MDSC가 현격하게 감소하였음을 보여준다. CD66b는 granulocyt i c MDSC에서 발현하나 monocyt i c MDSC에서 발현하지 않으므로 MDSC 아형구분에 사용한다. 도 8에서 특정한 MDSC 대부분은 CD66b 양성이므로 granulocyt i c MDSC라 구분할 수 있으며， DNP002 처리에 의하여 granulocyt ic MDSC가현격하게 감소하였다.  8 is a representative result of analyzing the MDSC killing effect after DNP002 treatment, showing that the MDSC was significantly reduced by the fucose DNP002. CD66b is expressed in granulocyt i c MDSC but not in monocyt i c MDSC. In FIG. 8, since most of the specific MDSCs are CD66b-positive, they can be classified as granulocyt i c MDSCs, and granulocytic MDSCs are significantly reduced by DNP002 treatment.
도 9는 5명의 환자에서 탈푸코오스 DNP002 처리 후 MDSC사멸효과를 분석한 것으로, 5명의 환자 모두에서 DNP002로 인해 MDSC군의 수가 대조군 대비 감소함을 백분율로 비교하여 나타낸 결과로서， 그래프에서 가로축에 기재된 P#1는 pat ient ' s whole blood #1를 의미하며 세로축은 상대적인 MDSC viabi l i ty %변화를 의미한다. Figure 9 shows the effect of MDSC killing after treatment with fucose DNP002 in 5 patients In the analysis, the percentage of the MDSC group was decreased by DNP002 compared to the control group in all 5 patients. P # 1 on the horizontal axis in the graph means pat ient 's whole blood # 1. Represents the relative MDSC viabi li ty% change.
도 10은 위암 환자의 혈액에서 분리된 PBMC에 DNP002 항체를 처리한 후 MDSC사멸효과를유세포분석기를 이용하여 분석한 결과를 나타낸다. 도 11a는 DNP002의 i sotype 에 따른 MDSC 사멸 효과를 비교한 결과로서， 탈푸코오스 (afucosylated) IgGl 타입의 DNP002 가 혈액 내 MDSC 사멸을 가장효과적으로유발하는 현상을 확인한결과이다.  Figure 10 shows the results of analyzing the effect of MDSC killing by flow cytometry after treatment with DNP002 antibody to PBMC isolated from the blood of gastric cancer patients. Figure 11a is a result of comparing the MDSC killing effect according to the I sotype of DNP002, a fucosylated IgGl type of DNP002 is the result confirming the phenomenon that most effectively induces MDSC killing in the blood.
도 lib는 5명의 위암 환자에서 DNP002의 i sotype 에 따른 MDSC사멸 효과를 비교한 결과로서， 환자 모두에사 탈푸코오스 (afucosyl ated) IgGl 타입의 DNP002 가 혈액 내 MDSC 사멸을 가장 높게 나타냈음을 대조군 대비 백분율로 비교하여 나타낸 결과로서, 그래프에서 가로축에 기재된 P#1 는 pat ient ' s whole blood #1를 의미하며 세로축은 상대적인 MDSC vi abi l i ty % 변화를 의미한다.  Lib compares MDSC killing effect according to i sotype of DNP002 in 5 patients with gastric cancer. In all patients, afucosyl ated IgGl type DNP002 showed the highest MDSC killing in blood. As a result of comparison as a percentage, in the graph, P # 1 in the horizontal axis means patient 's whole blood # 1 and the vertical axis indicates relative MDSC vi a bi li ty% change.
도 12a 및 도 12b는 DNP002 의 타깃 항원인 CEACAM6 가 양성인 위암 세포주 A549 와 췌장암 세포주 AsPC-1에 DNP002 항체 단독, NK 세포 단독， 및 DNP002 항체와■ 세포의 병용 조건에서 분석한 세포 사멸 효과에 대한 결과이다.  12A and 12B show the results of apoptosis effects analyzed in combination with DNP002 antibody alone, NK cells alone, and DNP002 antibody in gastric cancer cell line A549 and pancreatic cancer cell line AsPC-1 positive for DNP002, which is CEACAM6 positive. to be.
도 13은 암세포에서 CEACAM6 가 양성인 폐 선암 ( lung adenocarcinoma)과 암세포 자체에서는 CEACAM6가 음성인 편평상피암 ( lung squamous cel l carcinoma) , 비뇨기계 (Ur inary bladder cancer) 및 피부 암 (Melanoma mal ignancy) 조직에 CEACAM6 면역 염색한 결과 암 조직의 비 종양부위에 CEACAM6 양성인 MDSC가 있는 것을 확인한사진이다.  FIG. 13 shows lung adenocarcinoma positive for CEACAM6 in cancer cells and lung squamous cancer (Ur inary bladder cancer), urinary tract (Melanoma mal ignancy) tissue for CEACAM6 negative in cancer cells themselves. The result of CEACAM6 immunostaining showed that there was a CEACAM6 positive MDSC at the non-tumor site of cancer tissue.
【발명의 실시를위한 형태】 [Form for implementation of the invention]
하기의 실시예를들어 본 발명을 더욱 자세히 설명할 것이나, 발명의 권리범위가하기 실시예로 한정되는 것은 아니다. <실시예 1>항- CD66c키메릭 항체의 제조  The present invention will be described in more detail with reference to the following examples, but the scope of the invention is not limited to the following examples. Example 1 Preparation of Anti-CD66c Chimeric Antibody
1.1. 항- CD66c항체 유전자서열 클로닝  1.1. Anti-CD66c Antibody Gene Cloning
8F5 항체 유전자는 Mouse Ig-Pr imer Set (Mi l l ipore , Cat . #: 69831)을 이용하여 클로닝하였다. 8F5 하이브리도마로부터 분리한 RNA로부터 Mouse Ig-Primer Set을 이용하여 PCR을 수행하고， 이를 pGem-T 벡터 (Promega, Cat. #： A3600)에 삽입한후， 시퀀싱을 통해 DNA염기서열을 확인하였으며 , IMGT site (www. imgt .org)를 통해 마우스 항체 유전자를 확인하였다. 분석된 8F5 항체의 중쇄 및 경쇄 가변영역 서열은 다음과 같다. The 8F5 antibody gene was cloned using Mouse Ig-Pr imer Set (Mill ipore, Cat. #: 69831). Isolated from 8F5 hybridomas PCR was performed using a Mouse Ig-Primer Set from RNA, inserted into a pGem-T vector (Promega, Cat. #: A3600), and DNA sequencing was confirmed by sequencing. The IMGT site (www.imgt) .org) to identify mouse antibody genes. The heavy and light chain variable region sequences of the analyzed 8F5 antibody are as follows.
【표 4]  [Table 4]
Figure imgf000027_0003
Figure imgf000027_0003
1-2. 키메릭 항체 제조 1-2. Chimeric Antibody Manufacturing
상기 제작된
Figure imgf000027_0001
마우스 항체 85의 아미노산 서열을 기초로， 항체를 제작하였다. 라스미드 제작Produced above
Figure imgf000027_0001
Antibodies were prepared based on the amino acid sequence of mouse antibody 85. Lasmid production
Figure imgf000027_0002
키메릭 항체의 발현을 위하여， 중쇄 발현용 플라스미드와 경쇄 발현용 플라스미드를 각각 제작하였다 . 경쇄 발현용 플라스미드는 pOptiVEC (Invitrogen 사) 벡터를 사용하였고, 중쇄 발현용 플라스미드는 pcDNA3.3 (Invitrogen사) 벡터를사용하였다.
Figure imgf000027_0002
For expression of chimeric antibodies, heavy chain expression plasmids and Light chain expression plasmids were prepared, respectively. The light chain expression plasmid was used as a pOptiVEC (Invitrogen) vector, and the heavy chain expression plasmid was used as a pcDNA3.3 (Invitrogen) vector.
추가적인 아미노산 삽입 없이 항체 각각의 가변영역 코딩 cDNA와 불변영역 코딩 cDNA를 연속적인 아미노산 서열로서 발현되도록 하기 위하여， 상기 클로닝한 가변영역의 코딩 염기서열과 알려진 human IgGl 불변영역 (중쇄) 및 kappa 불변영역 (경쇄) 코딩 염기서열을 연결한 유전자 조각 각각을 합성 (Bioneer 사)하였다. 이와 같이 합성한 중쇄 및 경쇄 발현 유전자는 제한 효소 Xho I 과 Sal I으로 자른 후， 경쇄 유전자조각은 pOptiVec 벡터에， 중쇄 유전자 조각은 pcDNA3.3 벡터에 각각 ligation하여 완전한 항체 발현용 플라스미드를 제작하였다 ( pcDNA3.3-ant i-CD66c 중쇄 발현 플라스미드 및 p0ptiVEC-anti-CD66c경쇄 발현 플라스미드).  In order to express the variable region coding cDNA and the constant region coding cDNA of each antibody as a continuous amino acid sequence without additional amino acid insertion, the coding sequence of the cloned variable region and the known human IgGl constant region (heavy chain) and kappa constant region ( Light chain) each of the gene fragments linked to the coding sequence was synthesized (Bioneer). The heavy and light chain expression genes thus synthesized were cut with restriction enzymes Xho I and Sal I, and the light chain fragments were ligation to the pOptiVec vector and the heavy chain fragments to the pcDNA3.3 vector to prepare a complete antibody expression plasmid ( pcDNA3.3-ant i-CD66c heavy chain expression plasmid and p0ptiVEC-anti-CD66c light chain expression plasmid).
1-2-2. 형질전환 1-2-2. Transformation
상기 제작된 pcDNA3.3-ant i-CD66c중쇄 발현 플라스미드와 pOptiVEC- anti-CD66c 경쇄 발현 플라스미드를 抑0세포 유래인 The above-described pcDNA3.3-ant i-CD66c heavy chain expression plasmid and pOptiVEC-anti-CD66c light chain expression plasmid were derived from 抑 0 cells.
DG44세포 (Invitrogen)에 transfect ion시켜 형질전환과정을수행하였다. The transformation process was performed by transfecting DG44 cells (Invitrogen).
우선 transfection 3일전에 부유상태의 DG44 세포를 5%FBS가 포함된 MEMa 배지에 적응시킴으로서 부착상태 세포로 변환시켜 형질전환 효율을 높일 수 있게 적응시켰다. 형질전환은 ViaFect transfection regent (Pr omega, Cat.#: E4981)를 사용하여 6wel 1 plate에서 수행하였다. 형질전환 하루 전날 1 X 105 cells/well의 농도로 계대 배양하여 부착상태로 적응된 DG44세포를 준비하였고, 형질전환에 사용된 DNA의 양은 pcDNA3.3-ant i-CD66c 중쇄 발현 플라스미드와 p0ptiVEC-anti_CD66c 경쇄 발현 플라스미드 각각 2ug, 1.5ug씩 1.5 : 1비율의 조합으로 사용하였다. 형질전환은 48시간 동안 수행하였다. 형질전환된 세포군을 분석하기 위하여 유세포분석기 (flow cytometer) 을 이용하였다. 도 1과 같이 키메릭 항체의 발현은 A549비소세포폐암 세포주로 확인하였다. 도 1은 마우스 8F5 항체로부터 항체 유전자를 클로닝하여 키메릭 재조합 항체로 발현시켜, CD66c항원 양성인 A549세포 표면에 결합함을보여주는 결과이다.  First, three days prior to transfection, suspended DG44 cells were transformed into adherent cells by adapting to MEMa medium containing 5% FBS to increase transformation efficiency. Transformation was performed on 6wel 1 plate using ViaFect transfection regent (Pr omega, Cat. #: E4981). One day prior to transformation, DG44 cells were prepared by attachment at a concentration of 1 X 105 cells / well, and the amount of DNA used for transformation was pcDNA3.3-ant i-CD66c heavy chain expression plasmid and p0ptiVEC-anti_CD66c. Light chain expression plasmids were used in a combination of 2: 1 and 1.5 ug, respectively, in a 1.5: 1 ratio. Transformation was performed for 48 hours. Flow cytometer was used to analyze the transformed cell population. As shown in Figure 1 chimeric antibody expression was confirmed by A549 non-small cell lung cancer cell line. Figure 1 shows the results of cloning the antibody gene from the mouse 8F5 antibody and expressed as chimeric recombinant antibody, binding to the surface of the A549 cell positive CD66c antigen.
<실시예 2>인간화항- CD66c단클론항체의 제작 Example 2 Preparation of Humanized Antibody-CD66c Monoclonal Antibody
2.1 In silico Humanization0!] 의한재조합항체 서열 선정 마우스 00660 항체, 85 （중쇄 아미노산서열: 7 중쇄 코딩 : 묘요 10 0: 62； 경쇄 아미노산서열: 況요 犯 0: 8； 경쇄 코딩 0 : 況 0 犯炯: 2.1 In silico Humanization 0 !] Mouse 00660 antibody, 85 (heavy chain amino acid sequence: 7 heavy chain coding: summary 10 0: 62; light chain amino acid sequence: needed 犯 0: 8; light chain coding 0: 況 0 犯 炯:
Figure imgf000029_0001
Figure imgf000029_0001
유지하여 항원 결합력이 동등하거나 우수하면, 사람 항체 유전자를 코딩하고
Figure imgf000029_0002
서열을 기반으로 1 31116방0 요 ）：! 에 대한 부위 서열을 재조합한 인간화 항체 서열을 1^0 방법으로 선별하였다. 마우스 00660 항체 8 5 의 중쇄， 경쇄 각각 서열과 가장 유사성이 높아 인간화 재조합 항체 서열의 3신 0116으로 사용한 사람 항체 61"1111^½ 유전자는 아래 표 5와 같다. 상기 마우스 00660 항체의 중쇄 가변영역과 경쇄 가변영역의 아미노산 서열과 핵산 서열, 그리고 중쇄 가변영역과 경쇄 가변영역의
Figure imgf000029_0003
서열을 하기 표 6에 나타낸다. If the antigen binding ability is equal or superior, the human antibody gene is encoded
Figure imgf000029_0002
Based on sequence 1 31116 room yo ：：! Recombinant humanized antibody sequence was selected by the 1 ^ 0 method. The human antibody 61 " 1111 ^ ½ gene used as the 3rd 0116 of the humanized recombinant antibody sequence was most similar to the heavy and light chain sequences of the mouse 00660 antibody 8 5 as shown in Table 5. The heavy chain variable region of the mouse 00660 antibody Amino acid and nucleic acid sequences of the light chain variable region, and the heavy and light chain variable regions
Figure imgf000029_0003
The sequence is shown in Table 6 below.
【표 5]  [Table 5]
Figure imgf000029_0004
위의 사람 항체 Germline유전자 서열을 이용하여 선정한 인간화 8F5 항체 서열로서， 중쇄 가변영역 12종， 경쇄 가변영역 8종을 선별하였으며， 해당 서열은 아래 표 3과 같으며, 상기 선별된 인간화 항체의 중쇄 가변영역과 경쇄 가변영역의 아미노산 서열， CDR 서열 및 프레임워크 서열을 하기 표 6 내지 표 8에 나타내고 chimeric 항체와 인간화 항체의 중쇄 및 경쇄 가변영역의 아미노산 서열을 표 1에 나타냈다. 마우스 항체와 인간화 항체는 중쇄 抑R3와 경쇄 CDR2 서열이 동일한 것이 바람직하다. 하기 표 6에서 진하게 밑줄을 그어 표시한 부분은 CDR 항체서열이다. 표 7에서 진하게 밑줄을 그어 표시한 부분은 변경된 아미노산을 나타낸다.
Figure imgf000029_0004
As a humanized 8F5 antibody sequence selected using the above human antibody Germline gene sequence, 12 heavy chain variable regions and 8 light chain variable regions were selected. The sequences are shown in Table 3 below, and the heavy chain variable of the selected humanized antibody was shown. The amino acid sequences, CDR sequences, and framework sequences of the region and the light chain variable region are shown in Tables 6 to 8, and the amino acid sequences of the heavy and light chain variable regions of the chimeric and humanized antibodies are shown in Table 1. Mouse and humanized antibodies preferably have the same heavy chain —R3 and light chain CDR2 sequences. In Table 6 below, the underlined portions are CDR antibody sequences. Darkly underlined sections in Table 7 indicate modified amino acids.
【표 6】
Figure imgf000029_0005
2019/221574 1»(：1/10公019/006007 Table 6
Figure imgf000029_0005
2019/221574 1 »(： 1/10 公 019/006007
Figure imgf000030_0001
Figure imgf000030_0001
【표 7】 Table 7
Figure imgf000030_0002
2019/221574 1»（：1/10公019/006007
Figure imgf000031_0001
Figure imgf000030_0002
2019/221574 1 »（： 1/10 公 019/006007
Figure imgf000031_0001
【표 8］ [Table 8]
Figure imgf000031_0002
2.2 인간화 재조합 항체의 발현 및 분석
Figure imgf000031_0002
2.2 Expression and Analysis of Humanized Recombinant Antibodies
선별된 항체 서열들은 각각 사람 IgGl 중쇄 불변영역과 kappa 경쇄 불변영역과 연결하여 사람 IgGl 형태로 293T 세포에서 발현시켰다. Transfection 한 후, 7일 뒤에 배양액은 KanCap A resin (Kaneca 사)을 이용하여 인간화 재조합 항체를 정제하였다.  Selected antibody sequences were expressed in human IgGl form in 293T cells in connection with human IgGl heavy chain constant region and kappa light chain constant region, respectively. After 7 days, the culture medium was purified with humanized recombinant antibody using KanCap A resin (Kaneca).
정제한 항체는 0D280 nm 측정으로 정량하였으며， SDS-PAGE 를 수행하였다. 또한， Sepax Zenix-C SEC-300 size exclusion column (Sepax technologies 사)으로 HPLC를 수행하여 280nm 와 220nm로 분석함으로써， 항체의 순도 및 aggregation여부를 분석하였다 (도 2a내지 도 2c)  Purified antibody was quantified by 0D280 nm measurement, and SDS-PAGE was performed. In addition, the purity and aggregation of the antibody were analyzed by HPLC using a Sepax Zenix-C SEC-300 size exclusion column (Sepax technologies) and analyzed at 280 nm and 220 nm (FIGS. 2A to 2C).
2.3 인간화 재조합 항체의 세포 결합 및 항원 결합분석 2.3 Cell Binding and Antigen Binding Assay of Humanized Recombinant Antibodies
2-3-1 세포결합 분석  2-3-1 Cell Binding Assay
발현시킨 96종의 인간화 재조합 항체를 각각 동일 양 (lug)을 CD66c 양성 세포인 A549 비소세포폐암 세포주가 담긴 시험관에 넣어 4°C에서 30분간 반응시킨 후 PBS로 수세하고, FITC-conjugated goat ant i-Human IgGEach of the 96 humanized recombinant antibodies expressed in the same amount (lug) in a test tube containing A549 non-small cell lung cancer cell line, which is a CD66c positive cell, was reacted at 4 ° C for 30 minutes, washed with PBS, and washed with PBS, FITC-conjugated goat ant i Human IgG
(DiNona Inc, Korea)를 넣어 4°C에서 15분간 반응하였다. 다시 PBS로 수세한 후 유세포분석기 (Stratedigm， SlOOOEXi)로 분석하여 그 결과를 아래에 기재하였다. (DiNona Inc, Korea) was added and reacted at 4 ° C for 15 minutes. After washing with PBS again, a flow cytometer (Stratedigm, SlOOOOi) was analyzed and the results are described below.
96종의 인간화 재조합 항체 후보 중에서 발현 정도와 aggregation 유무, 세포결합 정도를 기준으로 8종을 1차로 선정하였다 (표 9 및 표 10 , 도 3a 내지 도 3e) . 하기 표 9 및 표 10은 1차 선정된 항 CD66c 인간화 항체 및 키메릭 8F5 의 분석 결과로서 표 10은 Flow cytometer 분석 결과를 나타낸다.  Among the 96 humanized recombinant antibody candidates, eight were selected based on the expression level, the presence of aggregation, and the degree of cell binding (Table 9 and Table 10, FIGS. 3A to 3E). Table 9 and Table 10 show the results of the flow cytometer analysis as a result of analysis of the first selected anti-CD66c humanized antibody and chimeric 8F5.
【표 9]  [Table 9]
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000032_0001
Figure imgf000033_0001
【표 10] Table 10
Figure imgf000033_0002
상기 선정된 8종의 항체는 발현 정도와 aggregation유무， 세포결합 정도를 표 9에 나타냈으며， 구체적으로 1차 선정된 8종 항체에 대한 발현 정도와 분자량을 정리한 것이다. 또한， 표 10의 유세포 분석 결과를 살펴보면， 8종의 인간화 재조합 항체가 키메릭 항체 대비 土 20%의 세포 결합력을 나타내어 키메릭 항체와 매우 유사한 세포 결합력을 나타냄을 확인하였다. 이로써 96종의 인간화 후보항체 중에서 발현이 정상적으로 이뤄지며， 단백질 자체의 불안정성으로 형성되는 aggregation 이 적으며， 또한 타겟 항원 양성의 세포에 결합하는 능력이 키메릭 항체와 유사한 인간화 항체 8종을 1차 선별하였다.
Figure imgf000033_0002
The selected eight kinds of antibodies are shown in Table 9 the expression level, the presence or absence of aggregation, the degree of cell binding, specifically summarizes the expression level and molecular weight of the first selected eight antibodies. In addition, looking at the flow cytometry results of Table 10, it was confirmed that the eight humanized recombinant antibodies showed a cell binding capacity of% 20% compared to the chimeric antibody, showing a very similar cell binding capacity with chimeric antibodies. As a result, among the 96 humanized candidate antibodies, expression was normally performed, and aggregation was formed due to instability of the protein itself, and eight humanized antibodies similar to chimeric antibodies were selected as the primary selection. .
특히 세포주 결합력은 수치상으로는 차이가 있어 보이나， 도 3과 같이 실제 세포결합 양상은 키메릭 항체와 비슷한 수준으로서 항체 양성률 (% gated)과 형광 평균값 (mean)을 곱하고 이를 키메릭 항체와 상대 비교하여 士 20% 범위 내 포함되는 8종을 선별한 것으로서, 통상 인간화 항체 생성 시， 인간화 항체의 framework region 에 마우스 항체 CDR 부위 서열을 삽입하였을 때 원래 단백질 구조의 변경으로 항체 결합력이 급격히 저하된다는 결과를 고려하였을 때， 매우 우수한 인간화 항체를 선별하였다 할 수 있다. In particular, the cell line binding ability may be different in numerical value, but as shown in FIG. 3, the actual cell binding pattern is similar to that of the chimeric antibody, and is multiplied by the antibody positive rate (% gated) and the mean fluorescence (mean), and compared with the chimeric antibody. Eight species included in the range of 20% were selected, and it was considered that the antibody binding ability is rapidly decreased due to the change of the original protein structure when the mouse antibody CDR region sequence is inserted into the framework region of the humanized antibody. When a very good humanized antibody was selected can do.
2-3-2 항원 결합 분석 2-3-2 Antigen Binding Assay
상기와 같이 선정한 8종 인간화 재조합 항체 중에서, 키메릭 항체 대비하여 세포결합력을 기준으로 높은 결합력을 나타내는 다섯 종류의 인간화 재조합 항체를 선택하고， 이들을 此比쇼 방법으로 00660 항원 및 유사 0066 항원들에 대한 결합력 분석을 실시하였다.  Of the eight humanized recombinant antibodies selected as above, five kinds of humanized recombinant antibodies exhibiting a high binding capacity based on cell binding capacity compared to chimeric antibodies were selected, and these were selected by the method for the 00660 antigen and similar 0066 antigens. Adhesion analysis was performed.
【표 11】  Table 11
Figure imgf000034_0001
CD66c (CEACAM6； SinoBiological 사) 와 CEACAM1 항원
Figure imgf000034_0001
CD66c (CEACAM6; SinoBiological) and CEACAM1 Antigen
(SinoBiological 사)을 96 well plate에 웰 당 100 씩 코팅한 후， blocking하였다. 일차항체 양은 lOug/ml 부터 3 배씩 희석하여 37 °C 온도에서 1시간 결합시켰으며， 이차항체로서 goat anti-Human Ig -HRP 접합체 (Jackson ImmunoResearch 사)를 1 : 10000으로 희석하여 37 °C 온도에서 30분간 결합시켰다. 각 단계 사이는 3번씩 수세과정을 거치고 TMB 반응하였다. IN H2S04 용액으로 TMB 용액과 동량 ( 100ul) 처리하여 반응 중지한 후, 450nm로 0D값을 측정하였다. (SinoBiological Co., Ltd.) was coated on a 96 well plate per 100 wells, and then blocked. The amount of primary antibody was diluted three times from lOug / ml and bound for 1 hour at 37 ° C. As a secondary antibody, goat anti-Human Ig -HRP conjugate (Jackson ImmunoResearch) was diluted 1: 10000 at 37 ° C Bound for 30 minutes. Each step was washed three times and reacted with TMB. After the reaction was stopped by treating the same amount (100ul) with TMB solution with IN H2S04 solution, 0D value was measured at 450nm.
상기 실험결과로서 96종의 인간화 재조합 항체 중에서 선정된 5종의 인간화 재조합 항체에 대한 CD66c항원에 대한 결합력을 도 4a, 표 12 및 도 4b, 표 13 에 나타냈으며， 도 4a와 표 12 는 CECACAM6 (CD66c) 항원에 대한 결과이며， 도 4b와 표 13 은 CEACAM1 (CD66a) 항원에 대한 결과이다.  As a result of the experiment, the binding capacity of the CD66c antigen to five humanized recombinant antibodies selected from 96 humanized recombinant antibodies is shown in FIGS. 4A, 12, 4B, and 13, and FIGS. 4A and 12 show CECACAM6 ( CD66c) results for antigen, Figures 4b and Table 13 show results for CEACAM1 (CD66a) antigen.
도 4a, 표 12 및 도 4b, 표 13의 항원에 대한 결합력 결과로부터， CEACAM6에 대해서는 모든 항체들이 키메릭 항체와 유사한 결합 양상을 보였으며, 期ACAM1에 대해서는 약한 결합과 결합하지 않은 그룹으로 양분되었다.  From the results of binding to the antigens of FIGS. 4A, 12, and 4B, Table 13, all antibodies showed a similar binding pattern to chimeric antibodies for CEACAM6, and were divided into groups that did not bind weak binding to 期 ACAM1. .
【표 12]
Figure imgf000034_0002
2019/221574 1»（：1^1{2019/006007
Figure imgf000035_0001
Table 12
Figure imgf000034_0002
2019/221574 1 »（： 1 ^ 1 {2019/006007
Figure imgf000035_0001
【표 13】 Table 13
Figure imgf000035_0002
Figure imgf000035_0002
2 .4 인간화 재조합 항체의 안정성 분석 2 .4 Stability Analysis of Humanized Recombinant Antibodies
상기 항원 및 세포결합 양상을 통해 선정한， 상기 실시예 3.3의 Example 3.3 of the above selected by the antigen and cell binding pattern
5종의 인간화 재조합 항체를 높은 온도 조건에 방치하여 항체의 안정성 여부를 분석하는 실험을 수행하였다. Five humanized recombinant antibodies were left at high temperature to perform experiments to analyze the stability of the antibodies.
안정성 즉정은 8 -크 1 10-1-1¼1）뇨1:113161163111군011 301（1 （이하 ; 크 사）를 이용한 결합 실험을 통해 확인하였다. 는 단백질 변성 시 노출되는 소수성 부위에 결합할 때와 그렇지 않을 때의 형광 파장이 달라 이러한 파장 변화를 측정함으로써 단백질의 변성 유무를 확인할 수 있는 화합물이다. 인간화 재조합 항체를 PBS (phosphate buf fered sal ine)를 이용해 0.2 mg/ml 농도로 모두 맞춘 후， 가혹 조건으로서 50 °C 온도에서 4시간 동안 방치하였다. 0.2 mg/ml ANS 용액을， 측정하기 위한 항체 희석액 500 ul 당 20 ul 넣어 섞어준 후, 5 분 후에 형광리더기로 360 nm exci tat ion, 460 nm emi ssion 조건에서 분석하였다. 추가적으로 70 °C 온도에서 30분간 더 방치한조건에서도 ANS시약반응을측정하였다. Stability immediate is 8-k 1 10-1-1¼1) urine 1: 113161163111 group 011 301 (1 (less than; It was confirmed through the coupling experiment using the X). Is a compound that can determine whether the protein is denatured by measuring the wavelength change because the fluorescence wavelength is different when binding to the hydrophobic region exposed during protein denaturation. Humanized recombinant antibodies were all adjusted to 0.2 mg / ml concentration using PBS (phosphate buf fered sal ine), and left for 4 hours at 50 ° C. as harsh conditions. A 0.2 mg / ml ANS solution was mixed in an amount of 20 ul per 500 ul of the antibody dilution to measure, and after 5 minutes was analyzed under fluorescent conditions at 360 nm exci tat ion and 460 nm emi ssion. In addition, the ANS reagent response was measured even under the condition of 30 minutes at 70 ° C.
상기 표 11에 나타낸 5종의 인간화 재조합 항체에 대한 가혹 온도 조건에서의 항체 안정성을 확인한 결과를 도 5a 및 도 5b에 나타냈다. 즉 가혹 조건으로서 50 °C 온도에서 4시간 동안 항체를 방치한 후 ANS 시약 반응성을 형광으로 측정하고, 추가로 70 °C 온도에서 30분간 더 방치한 조건에서도 ANS 시약 반응을 측정한 결과를 나타낸다. 도 5a 의 실험결과에 나타낸 바와 같이, 50 °C 온도에서 4시간 방치 조건에서는 5 종의 항체 대부분이 ANS 반응이 미미했었으나， 70 °C 온도에서 추가 30분 방치 조건에서는 대부분 항체의 형광값이 크게 증가하였다. 그 중에서는 protein ID： 3058 재조합 항체가 가장 낮은 형광값 증가세를 보여， 5 종류의 재조합 인간화항체 중에서 가장온도 변화에 대해 안정적인 결과를보였다. 5A and 5B show the results of confirming antibody stability under harsh temperature conditions for the five humanized recombinant antibodies shown in Table 11 above. In other words, after leaving the antibody at 50 ° C temperature for 4 hours as harsh conditions, ANS reagent reactivity is measured by fluorescence, and ANS reagent reaction is also measured under conditions left for another 30 minutes at 70 ° C temperature. As shown in the experimental results of FIG. 5A, most of the five antibodies were insignificant in the ANS reaction under the conditions of 4 hours at 50 ° C., but the fluorescence values of the antibodies were large at the additional 30 minutes at 70 ° C. Increased. Among them, protein ID: 3058 recombinant antibody showed the lowest increase in fluorescence value, showing stable results against temperature change among the five recombinant humanized antibodies.
또한， ANS 시약 반응성 변화율을 측정하고자, 냉장조건 (4 + 2 °C ) 및 62 °C 온도에서 각각 4시간 방치한 후에 ANS 시약 반응성을 상기 동일한 방법으로 형광 리더기로 분석하였다. 또한， ANS 시약에 대한 항체의 형광값 변이율은 저온 조건 (예, 4°C )에서 측정한 형광값과， 고온 조건 (예,In addition, in order to measure the rate of change of ANS reagent reactivity, after leaving for 4 hours at refrigeration conditions (4 + 2 ° C) and 62 ° C respectively, ANS reagent reactivity was analyzed by a fluorescent reader in the same manner. In addition, the fluorescence value variation rate of the antibody with respect to the ANS reagent is measured under low temperature conditions (e.g., 4 ° C) and high temperature conditions (e.g.,
62°C )에서 측정한 형광값의 차 (di f ference)를， 저온 조건에서 측정한 형광값으로 나눈 값을 의미한다. The difference of the fluorescence value measured at 62 ° C) is divided by the fluorescence value measured at low temperature.
［수학식］  [Mathematical formula]
형광값 변이율 = (고온 조건에서 측정한 형광값- 저온 조건에서 측정한형광값)/ (저온조건에서 측정한 형광값)  Fluorescence value variation ratio = (fluorescence value measured at high temperature condition-fluorescence value measured at low temperature condition) / (fluorescence value measured at low temperature condition)
도加와 같이 가혹조건의 온도 (50 °C )에서 온도를좀 더 올린 62 °C 온도에서 4시간 방치 후， ANS 시약 반응성을 확인하였다. 5종이 재조합 인간화 항체와 키메릭 항체 모두 냉장 조건에서는 ANS 반응 값이 미미했으나， 온도를 상승함에 따라 ANS 형광값이 증가하였다. 그러나， 키메릭 8F5는 62 °C 온도조건 보관실험에 의하여 ANS 시약 반응성이As shown in FIG. 4, the ANS reagent reactivity was confirmed after being left at 62 ° C for 4 hours at a temperature of 50 ° C under severe conditions. All five recombinant humanized antibodies and chimeric antibodies showed insignificant ANS response values under refrigerated conditions, but the ANS fluorescence values increased with increasing temperature. However, chimeric 8F5 has been tested for ANS reagent reactivity by 62 ° C temperature storage.
1 ,406%까지 증가하였으며， 동시에 침전물이 발생하여 불안정한 결과를 보였다. 그러나， 인간화 항체 5종은 키메릭 항체보다 ANS 시약 반응성 변화가 현저히 낮게 측정되었으며， 침전물이 발생되지 않았다. 특히, Protein ID 3019 인간화 항체와 Protein ID 3058 인간화 항체는 각각It increased up to 1,406%, and at the same time, precipitates were generated, resulting in unstable results. However, five humanized antibodies showed significantly lower changes in ANS reagent reactivity than chimeric antibodies, and no precipitates were generated. Especially, Protein ID 3019 Humanized Antibody and Protein ID 3058 Humanized Antibody
114%, 133%로 ANS 반응성의 변화가 측정되어 가장 안정한 항체로 평가되었다. ANS 시약 반응성이 커진다는 의미는 단백질 구조 상 안쪽에 배치되어 있던 소수성 아미노산의 노출이 증가한다는 의미이고, 이는 단백질 구조의 변성과 이로 인한 단백질 aggregate 즉， 침전물 생성의 원인이 된다. 본 발명에 따른 인간화 항체는 ANS 반응성 변이%가 200% 미만으로 안정한 항체이며, 200% 미만은 변화가 매우 미미한 것으로 간주되고， 그 이상은 의미 있는 단백질 구조 변경이 이뤄져 ANS 반응성이 관찰된 것으로 해석될 수 있다. 따라서, 본 발명에 따른 인간화 항체는 키메릭 항체 대비 유사한 항원 결합력 및 세포 결합력을 가지며, 항체 단백질 자체의 물리적 안정성이 증가하였으며， 이러한 사실은 치료용 항체의 druggabi l i ty측면에서 매우우수한특징이라할수 있다. Changes in ANS reactivity were measured at 114% and 133%, and evaluated as the most stable antibody. Increased reactivity of ANS reagents means increased exposure of hydrophobic amino acids that have been placed inward on the protein structure, leading to denaturation of the protein structure and thus protein aggregates, that is, sediment formation. The humanized antibody according to the present invention is an antibody having a stable ANS reactivity variation of less than 200%, and less than 200% is considered to be very insignificant, and more than that can be interpreted as a significant change in protein structure resulting in ANS reactivity observed. Can be. Therefore, the humanized antibody according to the present invention has similar antigen binding and cell binding capacity as compared to the chimeric antibody, and the physical stability of the antibody protein itself is increased, which is a very excellent feature in terms of druggabi li ty of therapeutic antibodies. .
2.5 인간화 재조합항체의 CH0세포 발현 및 분석 2.5 CH0 Cell Expression and Analysis of Humanized Recombinant Antibodies
상기 실시예 2.3에서 선정된 5종의 인간화 재조합 항체를 실제 대부분의 치료용 항체를 발현시키기 위해 사용하는 抑 0 세포에 발현시켜 분석하였다. 선정된 5종의 인간화 재조합 항체를 구성하기 위한 경쇄 및 중쇄 가변영역 DNA 서열을 코돈 최적화 과정을 거친 후, 합성하여 사람 IgGl 불변영역 유전자와 over lay PCR 방법으로 연결하고 Xhol 과 EcoRI 유전자 조각으로 pcDNA3.4 벡터 (Li fe Technology 사)에 클로닝하였다. 상기 표 11은 CH0 세포 발현을 위해 선별된 인간화 항체의 경쇄 및 중쇄 조합을 나타낸다.  The five humanized recombinant antibodies selected in Example 2.3 were expressed and analyzed in 抑 0 cells used to express the actual most therapeutic antibodies. The light and heavy chain variable region DNA sequences for constituting the five selected humanized recombinant antibodies were subjected to codon optimization, and then synthesized and linked with human IgGl constant region genes by over lay PCR method and the pcDNA3. 4 vectors (Li fe Technology) were cloned. Table 11 above shows the light and heavy chain combinations of humanized antibodies selected for CH0 cell expression.
가변-불변영역 유전자 PCR을 위해 사용한 DNA pr imer 서열은 아래 표 14와 같다.  DNA primer sequences used for variable-variable region PCR are shown in Table 14 below.
【표 14】  Table 14
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000037_0001
Figure imgf000038_0001
5 종의 인간화 재조합 항체는 ExpiCHO (trademark) Expression System Ki t (ThermoFi sher 사; Cat . No:A29133)을 이용하여 Transient transfect ion 시켰으며, 발현시킨 항체는 도 6a 내지 도 6c와 같이 CD66c 양성 세포인 A549 비소세포폐암 세포주에 결합시켜 Flow cytometer로 분석하였다. 5종류 인간화 재조합 항체는 모두 키메릭 항체와 유사한 결합력을 보였다. 상기 측정한 Flow cytometer 형광값을 CH0 배양액의 항체 발현 양으로 나누어, 상대적인 항체 발현양에 대한 세포표면 결합력 (relat ive cel l binding)을 표시한 것을 도식화하면 도 6d와 같았다. 따라서， 인간화 재조합 항체는 CH0 세포에서도 적절히 발현되는 것을 확인하였다. 항체의 세포 표면 결합력을 구하고 이를 100% 로 하여 상대적인 변화를도시한 것이 도 6d이다. Five humanized recombinant antibodies were transfected with ExpiCHO (trademark) Expression System Kit (ThermoFi sher; Cat.No:A29133), and the expressed antibodies were CD66c positive cells as shown in Figs. 6A to 6C. Binding to A549 non-small cell lung cancer cell line was performed by flow cytometer. All five humanized recombinant antibodies showed similar binding capacity to chimeric antibodies. The measured flow cytometer fluorescence value was divided by the amount of antibody expression in the CH0 culture, and the cell surface binding force (relative cel l binding) to the relative amount of antibody expression was plotted. Therefore, it was confirmed that the humanized recombinant antibody is properly expressed in CH0 cells. Fig. 6D shows the relative change of the cell surface binding force of the antibody and its 100%.
<실시예 3> DNP002항체의 CH0세포발현 및 분석 Example 3 CH0 Cell Expression and Analysis of DNP002 Antibody
대부분의 치료용 항체를 발현시키기 위해 사용하는 CH0 세포에서 항 Antibodies in CH0 cells used to express most therapeutic antibodies
CD66c에 대한 인간화 항체인 DNP002를 발현시켜 분석하였다. DNP002 항체의 아형에 따른 기능 차이를 확인하기 위해 IgGl 타입과 IgG2 타입의 항체를 제조하였다. DNP002, a humanized antibody against CD66c, was expressed and analyzed. In order to confirm the functional differences according to subtypes of the DNP002 antibody, antibodies of IgGl type and IgG2 type were prepared.
인간화 재조합 항체를 구성하기 위한 경쇄 및 중쇄 가변영역 DNA 서열을 코드 (codon) 최적화 과정을 거친 후， 합성하여 사람 IgGl 또는 IgG2 불변영역 유전자와 over lay PCR 방법으로 연결하고 Xhol 과 EcoRI 유전자 조각으로 pcDNA3.4벡터 (Li fe Technology사)에 클로닝하였다.  The light and heavy chain variable region DNA sequences for constructing the humanized recombinant antibody were subjected to codon optimization, and then synthesized and linked with human IgGl or IgG2 constant region genes by over lay PCR method, and pcDNA3 using Xhol and EcoRI gene fragments. It was cloned into 4 vectors (Li fe Technology).
【표 15】 2019/221574 1»(：1/10公019/006007 Table 15 2019/221574 1 »(： 1/10 公 019/006007
Figure imgf000039_0001
Figure imgf000040_0002
탈푸코오스 (afucosylated) DNP002 인간화 항체를 제조하기 위하여, DNP002 IgGl 타입의 항체 발현 시， 2F-PF(2F-Peracetyl-Fucose； Merck, Cat#： 344827) 를 배양액에 50 uM로 투여하여 배양하였으며, Mabselect sure Protein A column (GE Healthcare Li fescience, Cat#: 11003494) 사용하여 정제하였다. 정제한 항체는 phosphate buffered saline으로 투석하고 280 nm 흡광값을 흡광계수 1.4로 나누어 "mg/mL" 농도 단위로 변환하고 이후 시험에 사용하였다.
Figure imgf000039_0001
Figure imgf000040_0002
In order to prepare afucosylated DNP002 humanized antibody, 2F-PF (2F-Peracetyl-Fucose; Merck, Cat #: 344827) was incubated at 50 uM in the culture medium when the DNP002 IgGl type antibody was expressed. Purification was performed using a Mabselect sure Protein A column (GE Healthcare Li fescience, Cat #: 11003494). Purified antibody was dialyzed with phosphate buffered saline, and the 280 nm absorbance was divided by the absorption coefficient of 1.4 and converted into a concentration unit of "mg / mL", which was then used for the test.
탈푸코오스화는 푸코오스에 결합특성이 있는 Biotinylated Lens culinaris agglutinin (Vector laboratories , Cat#：B-1045) 반응성을 상대적으로 비교하여 평가하였다. 푸코오스가 결합되어 있는 IgGl DNP002는 Biotinylated Lens culinaris agglutinin과 반응하고 SA_HRP( Jackson immunoresearch, Cat#: 016-030-084)에 의하여 TMB 발색을 유도하였으나 탈푸코오스화 DNP002는 상대적인 발색이 미미하였다 (표 16).  Defucosification was evaluated by comparing the reactivity of biotinylated lens culinaris agglutinin (Vector laboratories, Cat #: B-1045) with binding properties to fucose. Fucose bound IgGl DNP002 reacted with Biotinylated Lens culinaris agglutinin and induced TMB coloration by SA_HRP (Jack immunoresearch, Cat #: 016-030-084), but defucose DNP002 showed little relative color development (Table 16).
【표 16】  Table 16
Figure imgf000040_0003
Figure imgf000040_0003
＜실시예
Figure imgf000040_0001
항체의 반응성 조사 MDSC에 대한 DNP002 항체의 반응성을 유세포 분석 (flow cytometric analysis)을통해 확인하였다. <Example
Figure imgf000040_0001
Investigation of Reactivity of Antibodies Reactivity of the DNP002 antibody to MDSC was confirmed by flow cytometric analysis.
구체적으로 위암환자의 혈액을준비한 뒤 , 각 형광이 다르게 결합된 MDSC의 표지 항원들에 대한 항체 (항-HLA-DR-FITC, CDllb-PE, CD33-PE 항체)와 함께, APC가 결합된 DNP002 항체를 전혈 100 uL에 첨가하고 20분간 4°C에서 반응시켰다. 적혈구 용해 버퍼 IX RBC Lysis Buffer (ThermoFisher , Cat#： 00-4333-57) 5 ml을 첨가하여 실온에서 30분간 반응시킨 후 원심분리하여 분해된 RBC를 제거하고， 다시 PBS로 수세한후 유세포 분석을 시행하였다. 염색강도는 형광강도에 대한 log 로 측정하고 10승 단위로 표시하였다. Specifically, after preparing the blood of gastric cancer patients, DNP002 with APC bound together with antibodies against anti-HLA-DR-FITC, CDllb-PE, and CD33-PE antibodies against the labeled antigens of MDSC with different fluorescence. The antibody was added to 100 uL of whole blood and reacted at 4 ° C for 20 minutes. 5 ml of erythrocyte lysis buffer IX RBC Lysis Buffer (ThermoFisher, Cat #: 00-4333-57) was added and allowed to react at room temperature for 30 minutes, followed by centrifugation to remove the degraded RBC, followed by washing with PBS. Was implemented. Dyeing intensity was measured in logarithm to fluorescence intensity and expressed in 10 power units.
결과 분석은， 점도표(dot plot)에서 세포의 크기에 따라 림프구를 제외하고 단핵구와 과립구 영역만을 지정한 뒤, HLA-DR의 발현이 없거나 낮은 군을 선택하고， 그 군에서 CDllb와 CD33에 양성인 그룹을 MDSC로 지정하였다. 지정된 MDSC군에서 DNP002의 양성률을 확인하였다 (도 7) . 구체적으로， 도 7에서 상단에 나타낸 그래프 중에서 왼쪽부터 오른쪽 방향으로, 1) FSC 및 SSC dot plot에서 monocytes와 granulocytes만을 구획화(gating)， 즉， lymphocytes를 제외. (FSC: forward scatter , 분석하는 세포의 사이즈를 나타내는 변수， SSC： side scatter, 분석하는 세포의 granularity를 나타내는 변수로서， 세포 내 granule이 있는 정도를 나타내는 변수)이며， 2) HLA-DR Low혹은 (-) 그룹을 구획화한 것이며, 3) 상기 1번， 2번 구획화 중에서 CDllb와 CD33 이 양성인 그룹이 MDSC이다. DNP002양성인 MDSC는 90.9 % (도 7의 오른쪽 dot plot 내에서 upper right 부위)이고， DNP002 음성인 MDSC는 4.4% (도 7의 오른쪽 dot plot 내에서 upper left 부위 ) 이다. MDSC는 단핵구성 MDCS와 다핵구성 (granulocytic) MDSC로 아형으로 구분되며， 다핵구성 MDSC는 CD66b를 발현하나 단핵구성 The analysis of the results showed that in the dot plot, only the monocytes and granulocyte regions were excluded except for lymphocytes according to the cell size, and the group with no or low expression of HLA-DR was selected, and the groups positive for CDllb and CD33 were selected. It was designated MDSC. Positive rate of DNP002 was confirmed in the designated MDSC group (FIG. 7). Specifically, in the left-to-right direction of the graph shown in the upper part in FIG. 7, 1) gating only monocytes and granulocytes in FSC and SSC dot plots, ie, excluding lymphocytes. (FSC: forward scatter, variable indicating the size of the cell to be analyzed, SSC: side scatter, variable indicating the granularity of the cell to be analyzed, variable indicating the degree of granule in the cell), 2) HLA-DR Low or ( -) The group was partitioned, and 3) the CDllb- and CD33-positive group among the 1 and 2 partitions was MDSC. MDSC positive DNP002 is 90.9% (upper right region in the right dot plot of Figure 7), MDSC negative DNP002 is 4.4% (upper left region in the right dot plot of Figure 7). MDSCs are divided into subtypes as mononuclear MDCS and granulocytic MDSC. Multinucleated MDSCs express CD66b but are mononuclear.
MDCS는 CD66b를 발현하지 않아 MDSC 아형 구분에 CD66b 발현 여부는 유용하게 사용할수 있다. Since MDCS does not express CD66b, the expression of CD66b in the MDSC subtype can be usefully used.
도 7은 MDSC를 특정하는 방법 (림프구 제외， HLA-DR low/(-)， CDllb+, 抑 33+)과 특정한 MDSC에서 DNP002(anti-CD66c)가 반응하고 있음을 증명하는 결과이다. MDSC에 DNP002가 결합하므로 MDSC특정하는 방법으로 DNP002을 사용할 수 있으며 (도 7)， DNP002는 ADCC효과를 유발하여 MDSC를 제거할 수 있다 (도 8) . 하기 시험결과에서 DNP002가결합한 MDSC는 90.9%이다.  FIG. 7 shows the results of demonstrating that DNP002 (anti-CD66c) reacts in a method of specifying MDSC (except lymphocytes, HLA-DR low / (−), CDllb +, 抑 33+) and a specific MDSC. Since DNP002 binds to MDSC, DNP002 can be used as a method for specifying MDSC (FIG. 7), and DNP002 can remove MDSC by inducing ADCC effect (FIG. 8). In the test results below, MDNP002 bound was 90.9%.
도 8은 CD66b 양성인 다핵구성 MDSC가 DNP002 처리에 의하여 살상되고 그 비율이 감소한 현상을 보여준다. 도 8에서 상단 (control)의 점도표는 DNP002을 처리하기 전 시료이며， 하단 (DNP002)은 DNP002 처리 이후 MDSC가 감소한 결과이다. CD66b는 monocytic MDSC와 granulocyticFIG. 8 shows DNP002 treatment of CD66b positive multinucleated MDSC Killed and reduced in proportion. In FIG. 8, the viscosity table at the top of the control is a sample before the treatment of DNP002, and the bottom (DNP002) is the result of the decrease of the MDSC after the treatment of DNP002. CD66b is monocytic MDSC and granulocytic
MDSC를 구분할 수 있는 마커이다. 하기 시험결과에서 대부분의 MDSC는 CD66b 양성인 granulocytic MDSC이며, granulocytic MDSC가 DNP002에 의하여 효과적으로살상되었다. It is a marker that can distinguish MDSC. In the following test results, most MDSCs are CD66b positive granulocytic MDSCs, and granulocytic MDSCs were effectively killed by DNP002.
위암 환자 19명의 혈액을 분석한 결과， 전체 PBMC 내 MDSC 상의 DNP002 의 양성률은 평균 34.3 -76.7%로 나타났고， 평균 55.1%의 양성률을 보였다. 하기 표 17은 19명의 위암 환자 샘플을 가지고 DNP002 항체의 MDSC 반응성을분석한 것이다.  As a result of analyzing the blood of 19 patients with gastric cancer, the average positive rate of DNP002 on MDSC in total PBMC was 34.3-76.7%, and the average positive rate was 55.1%. Table 17 below analyzes the MDSC reactivity of the DNP002 antibody with 19 gastric cancer patient samples.
【표 17】  Table 17
Figure imgf000042_0001
Figure imgf000042_0001
<실시예 5> DNP002에 의한 MDSC용해 효과 Example 5 MDSC Dissolution Effect by DNP002
5.1. 전체 혈액세포 (whole blood) 내 DNP002에 의한 MDSC용해 효과 DNP002에 의한 MDSC살해 효과를 확인하기 위하여 5명의 위암 환자 혈액에 적혈구 용해 버퍼 IX RBC Lysi s Buf fer (ThermoF i sher , Cat#： 00- 4333-57)를 첨가하여 적혈구를 용해 시킨 후, 12 wel l plate 에 wel l 당 1 X 105 씩 분주하여 준비하였다. DNP002 항체를 각 wel l 에 10 ug/mL 이 되도록 첨가하고 37 °C 인큐베이터에서 하루 배양하였다. 배양 후 PBS로 세척하고, 각 형광이 다르게 결합된 MDSC 표지 항원에 대한 항체 (항 HLA-DR, CDllb, CD33 항체)와 20분간 4°C에서 반응시켰다. PBS로 수세한 후 유세포 분석을 시행하였다. 염색강도는 형광강도에 대한 log 로 측정하고 10승 단위로 표시하였다. 5.1. MDSC dissolution effect by DNP002 in whole blood cells Five gastric cancer patients to confirm MDSC killing effect by DNP002 Red blood cell lysis buffer IX RBC Lysi s Buf fer (ThermoF i sher, Cat #: 00-4333-57) was added to lyse red blood cells, and then divided into 12well plates by 1 X 105 per wel l. . DNP002 antibody was added to each wel l to 10 ug / mL and incubated in a 37 ° C incubator for one day. After incubation, the cells were washed with PBS and reacted at 4 ° C. for 20 minutes with antibodies to anti-MDSC labeled antigens (anti-HLA-DR, CDllb, CD33 antibodies) to which each fluorescence was differently bound. After washing with PBS, flow cytometry was performed. Dyeing intensity was measured in logarithm to fluorescence intensity and expressed in 10 power units.
상기 실험결과로서, 도 8은 DNP002 항체가 혈액 내 MDSC 의 세포사멸을 효과적으로 유도하여 DNP002 항체 처리전에 비하여 MDSC 비율이 현격하게 감소한 대표적인 결과를 나타낸다. 도 9는 도 8과 동일한 시험을 5명의 위암환자 혈액시료에서 각각 시험하고 도표화한 결과이다. Open bar는 DNP002 처리 이전 MDSC 비율을 의미하며 , closed bar는 DNP002 처리 이후 MDSC의 상대적 비율을 의미한다. DNP002 처리 이후에 5명의 환자 시료 모두에서 MDSC비율이 확연히 감소하였음을 확인할수있다.  As a result of the experiment, FIG. 8 shows a representative result of the DNP002 antibody effectively inducing apoptosis of MDSC in the blood, resulting in a significantly reduced MDSC ratio compared to the DNP002 antibody treatment. FIG. 9 is the same test as in FIG. 8, and the test results of five gastric cancer patient blood samples, respectively. Open bar means MDSC ratio before DNP002 treatment and closed bar means relative ratio of MDSC after DNP002 treatment. After treatment with DNP002, the MDSC ratio was significantly reduced in all five patient samples.
5.2. PBMC (말초혈액 단핵 세포) 내 DNP002에 의한 MDSC용해 효과5.2. MDSC Dissolution Effect by DNP002 in PBMC (Peripheral Blood Mononuclear Cells)
DNP002 항체의 MDSC타겟 살해 능력을 보다분명히 밝히고자, 성숙한 호중구 (neutrophi l ) 를 제외하고 MDSC만을 얻어서， 호중구에 의한 영향 없이 DNP002항체의 MDSC사멸 효과를 확인하였다. To clarify the MDSC target killing ability of the DNP002 antibody, only MDSC was obtained except for mature neutrophils, and the effect of MDSC killing of the DNP002 antibody was confirmed without the effect of neutrophils.
구체적으로， 2명의 위암 환자 혈액을 Ficol l-Paque PLUS (Ge heal thcare , Cat#： 17-1440-02) 용액을 이용해 MDSC가 포함된 PBMC 층만을 분리하였다. 이러한 Ficol l 용액을 통한 혈액세포의 비중분리는 효과적으로 성숙 호중구를 제외시켜 보다 정확한 MDSC 사멸효과를 확인할 수 있도록 한다. 준비된 PBMC 는 12 wel l plate에 wel l당 1 X 105씩 분주하고 DNP002 항체를 각 wel l 에 10ug/mL 이 되도록 첨가하고 37 °C 인큐베이터에서 48시간 배양하였다. 이때, PD-1 에 대한 항체인 Nivolumab (Bri stol-Myers Squibb)을 대조군으로 이용하여 MDSC 살해능을 비교하였다. 배양 후 세포를 PBS로 세척하고， 유세포분석을.통해 MDSC군의 증감을 확인하였다 (도 10) . 유세포 분석 결과 DNP002 를 처리한 군은 그렇지 않은 군 대비 평균 약 49% 의 세포 사멸 효과를 보였다. 반면 대조군으로 이용한 Nivolumab은 약 24% 의 MDSC 사멸 효과를 보이는데 그쳤다. MDSC 사멸 효과는 두 환자에서 분리한 MDSC모두에서 동일하게 나타났다. 따라서 본 실험을 통해 DNP002항체에 의한 MDSC살상효과가상당함을 확인할수 있다. Specifically, the blood of two gastric cancer patients was separated from the PBMC layer containing MDSC using Ficol l-Paque PLUS (Ge heal thcare, Cat #: 17-1440-02) solution. Specific gravity separation of blood cells through the Ficol 1 solution effectively excludes mature neutrophils and enables more accurate MDSC killing. The prepared PBMC was dispensed in a 12 wel l plate at 1 X 10 5 per wel, and DNP002 antibody was added to each wel l to 10 ug / mL and incubated in a 37 ° C incubator for 48 hours. At this time, MDSC killing ability was compared using Nivolumab (Bri stol-Myers Squibb), an antibody against PD-1. After incubation, the cells were washed with PBS, and flow cytometry confirmed the increase and decrease of the MDSC group (FIG. 10). As a result of flow cytometry, the group treated with DNP002 showed an average of about 49% cell death compared to the group not treated with DNP002. On the other hand, Nivolumab used as a control showed only about 24% MDSC killing effect. MDSC killing effects were the same in both MDSC isolates in both patients. So this experiment The effect of MDSC killing by DNP002 antibody is significant.
DNP002 에 의한 MDSC 의 용해 효과를 전혈 (실시예 5.1)과 PBMC (말초혈액 단핵세포; 실시예 5.2) 각각에 대해 확인하였다. 전혈 및 PBMC 에는 ADCC 를 유발할 수 있는 환자 본인의 ■ 세포가 있어 , DNP002를 매개로 하여 期 ACAM6 양성인 세포를 ADCC 로 용해할 수 있다. 그런데， 전혈에는 CEACAM6 타깃 항원이 양성인 호중구와 MDSC 가 혼합되어 있어， MDSC 만을 선택적으로 용해하였다고 보기 어렵다. DNP002 에 의한 MDSC 의 선택적인 용해를 명확히 하기 위해， 호중구를 원심분리로 층분리시켜 없앤 PBMC를 대상으로 추가 실험하였다 (실시예 5.2) . 이에 DNP002 에 의한 MDSC 용해가분명함을 확인하였다.  The lysis effect of MDSC by DNP002 was confirmed for whole blood (Example 5.1) and PBMC (peripheral blood mononuclear cells; Example 5.2), respectively. Whole blood and PBMCs have their own cells that can induce ADCC, so that DNP002 can mediate ACAM6-positive cells with ADCC. By the way, whole blood contains a mixture of neutrophils positive for CEACAM6 target antigen and MDSC, and it is hard to say that only MDSC was selectively dissolved. In order to clarify the selective dissolution of MDSC by DNP002, further experiments were performed on PBMCs that had been neutrophils separated by centrifugation (Example 5.2). This confirmed that the MDSC dissolution by DNP002 was clear.
<실시예 6>항체 isotype에 따른 MDSC용해 효과 Example 6 MDSC Dissolution Effect According to Antibody Isotype
DNP002 항체의 MDSC 타겟 살해 능력을 확인하기 위해 3가지 유형의 DNP002 항체 제제를 준비하였다. 항체는 i sotype에 따라 NK 세포에 발현한 FcrRI I I (CD16)의 친화도에 차이가 발생하며, 친화도에 비려하여 항체의존성 세포매개 세포독성 (ADCC)이 증가한다. IgG2 i sotype은 FcrRI I I에 대한 친화도가 매우 낮아 ADCC 효능이 없는 반면, IgGl i sotype은 FcrRI I I에 대한 친화도가 높아 ADCC 효능이 뛰어나다. 항체의 ADCC 효능은 i sotype 뿐만 아니라 항체 297번째 아스파라진 아미노산에 연결된 당쇄구조에 따라 달라지는 것으로 보고 되었으며， 특히 당쇄에 푸코오스가 없는 경우 ADCC 효능이 증가한다 (Shi tara K. , et al , J Immunol Mehtods . 2005 Nov 30； 306 (1-2) IgG subclass- independent improvement of ant i body-dependent cel lular cytotoxi ci ty by fucose removal from Asn297-1 inked ol igosacchar ides) .  Three types of DNP002 antibody preparations were prepared to confirm the MDSC target killing ability of the DNP002 antibody. Antibodies differ in the affinity of FcrRI I I (CD16) expressed in NK cells according to the i sotype, and antibody dependent cell-mediated cytotoxicity (ADCC) increases in view of affinity. IgG2 i sotype has very low affinity for FcrRI I I and thus no ADCC efficacy, whereas IgGl i sotype has high ADCC efficacy because of its high affinity for FcrRI I I. The ADCC potency of antibodies has been reported to depend not only on the i sotype but also on the sugar chain structure linked to the 297th asparagine amino acid, particularly in the absence of fucose in the sugar chain (Shi tara K., et al, J Immunol). Mehtods. 2005 Nov 30; 306 (1-2) IgG subclass-independent improvement of ant i body-dependent cel lular cytotoxi ci ty by fucose removal from Asn297-1 inked ol igosacchar ides).
DNP002 항체의 i sotype 및 탈푸코오스 따른 MDSC 타겟 살상 능력을 확인하기 위해 in vi tro 시험을 수행하였다. 5명의 위암 환자 혈액에 적혈구 용해 버퍼 IX RBC Lysi s Buf f er (ThermoF i sher , Cat#: 00-4333-57) 첨가하여 RBC 용해시킨 후， 12 wel l plate 에 wel l 당 1 X 105씩 분주하여 준비하였다. DNP002 IgGl 타입과 IgG2 타입 및 탈푸코오스 (af lucosylated) IgGl 타입의 3가지 종류의 항체를 각각 웰에 10 ug/mL 이 되도록 첨가하고 37 °C 인큐베이터에서 하룻동안 배양하였다. 배양 후 로 세척하고， 각 형광이 다르게 결합된 MDSC 표지 항원에 대한 항체 (항 HLA-DR, CDllb , 抑 33 항체)와 20분간 4°C에서 반응시켰다. PBS로 수세한 후 유세포 분석을 시행하였다. 염색강도는 형광강도에 대한 log 로 측정하고 10승 단위로 표시하였다. In Vitro tests were performed to determine the ability of the DNP002 antibody to kill MDSC targets according to the i sotype and defucose. Red blood cell lysis buffer IX RBC Lysi s Buf f er (ThermoF i sher, Cat #: 00-4333-57) was added to the blood of five gastric cancer patients, and then RBC was dissolved and dispensed 1 X 105 per wel l into a 12 wel l plate. It was prepared by. Three kinds of antibodies, DNP002 IgGl type, IgG2 type, and af lucosylated IgGl type, were added to the wells to 10 ug / mL, respectively, and incubated in a 37 ° C incubator for one day. After incubation, the cells were washed with and reacted with antibodies to MDSC labeled antigens (anti-HLA-DR, CDllb, 抑 33 antibodies) to which each fluorescence was differently bound at 4 ° C. for 20 minutes. After washing with PBS, flow cytometry Was implemented. Dyeing intensity was measured in logarithm to fluorescence intensity and expressed in 10 power units.
시험대상인 위암 환자 5명 모두에서 IgG2， IgGl, 탈푸코오스 (afucosylated) IgGl 타입 순으로 MDSC 사멸효과가 증가하는 것으로 관찰되었다 (도 11a, lib) . Isotype과 탈푸코오스 (afucosylated, 푸코스 함량이 10%이하)에 따라 MDSC 사멸효과에 차이가 발생하는 것은 항체와 FcrRI I I 간의 친화도에 따른 차이로 판단되며 , ■세포에 의한 ADCC 효과로 MDSC가살상된 것으로 이해할수 있다.  MDSC killing effect was observed in all five gastric cancer patients in order of IgG2, IgGl, afucosylated IgGl type (Fig. 11a, lib). The difference in MDSC killing effect according to isotype and afucosylated (fucose content below 10%) is considered to be due to the affinity between the antibody and FcrRI II. It can be understood as broken.
도 11a에 나타낸 바와 같이, MDSC를 효과적으로 제거할 수 있는 DNP002 제제 유형을 확인한 대표적인 시험결과이다. 상단 (control ) 대비 As shown in FIG. 11A, representative test results confirming the type of DNP002 formulation capable of effectively removing MDSC. Control contrast
DNP002 IgG2는 상당부분의 MDSC가 잔존하고 있으나， DNP002 IgGl은 MDSC 비율을 현격하게 감소시켰다. 본 시험에서 DNP002 IgGl에 의한 MDSC 살상효과가 상당하나, IgG2에 의한 살상효과는 미미하다는 것을 확인할 수 있었고， IgG2 i sotype은 ADCC 효능이 IgGl i sotype에 비하여 없거나/매우 낮으므로 DNP002 IgGl에 의한 MDSC 살상효과는 ADCC에 의한 것으로 추론할 수 있다. Although DNP002 IgG2 has a large amount of MDSC remaining, DNP002 IgGl significantly reduced the MDSC ratio. In this study, the MDSC killing effect by DNP002 IgGl was significant, but the killing effect by IgG2 was insignificant, and the IgG2 i sotype had no / very low ADCC potency compared with IgGl i sotype. The effect can be inferred by ADCC.
도 lib에 나타낸 바와 같이, 도 9와 동일한 시험을 5명의 위암환자 혈액시료에서 시험하고 도표화한 결과이며， 그래프의 가로축은 5명의 위암환자 각각을 나타낸다 (P#l， P#2， P#3, P#4, P#5) . Control , IgG2 , IgGl , IgGl-afucosylated 순으로 MDSC 살상효과가 관찰되었다. IgG2 i sotype은 ADCC 기능이 없어 MDSC살상효과가 없거나 미미하나 ADCC 기능이 있는 IgGl과 탈푸코오스화로 ADCC 기능을 강화한 IgGl-afucosylated에서는 MDSC살상효과가뛰어나다. <실시예 7> DNP002 와 자연 살해 세포의 병용에 의한 암세포 사멸 효과  As shown in FIG. Lib, the same test as in FIG. 9 was tested and plotted in blood samples of 5 gastric cancer patients, and the horizontal axis of the graph represents each of 5 gastric cancer patients (P # l, P # 2, P # 3). , P # 4, P # 5). MDSC killing effects were observed in the order of Control, IgG2, IgGl, IgGl-afucosylated. IgG2 i sotype has no ADCC function and no or minimal effect on MDSC killing. However, IgGSC with ADCC function and IgGl-afucosylated enhanced ADCC function by afucose have superior MDSC killing effect. Example 7 Cancer Cell Killing Effect by Combination of DNP002 and Natural Killer Cells
DNP002항체와자연 살해 세포 (Natural ki l ler cel l )의 병용 효과를 확인하기 위해 in vi tro 시험을 수행하였다. 3명의 정상 혈액으로부터 Fi col 1-Paque PLUS (Ge healthcare , Cat# : 17-1440-02) 용액을 이용하여 PBMC 를 분리한 뒤， CD56 mi cro bead (Mi ltenyi Biotec , Cat# : 130-050- 401)를 이용하여 CD56 양성 자연 살해 세포만을 분리하였다. 도 12a 및 도 12b에서 가로축은 각각 상기 자연 살해 세포의 기원인 혈액의 공여자 (donor) 3명을 의미한다. 96 wel l plate 에 DNP002 의 타깃 항원인 CEACAM6 가 양성인 위암 세포주 A549 와 췌장암 세포주 AsPC-1을 wel l 당 lxlO4씩 분주하여 준비한 뒤， 앞서 분리한 자연 살해 세포를 wel l 당 2xl05 씩 분주하고 DNP002 항체를 10ug/mL 이 되도록 처리하여 37 °C 에서 6시간배양하였다. In Vitro tests were performed to determine the combined effect of DNP002 antibody and natural killer cells (Natural ki ler cel l). PBMC was isolated from three normal blood using Fi col 1-Paque PLUS (Ge healthcare, Cat #: 17-1440-02) solution, and then CD56 mi cro bead (Milteni Biotec, Cat #: 130-050- 401) was used to isolate only CD56 positive natural killer cells. In FIG. 12A and FIG. 12B, the horizontal axis means three donors of blood which are the origin of the natural killer cells, respectively. Gastric cancer cell line A549 and pancreatic cancer cell line AsPC-1 positive for DNP002, which is the target antigen of DNP002, were prepared by dispensing lxlO 4 per wel l, and the previously separated natural killer cells were dispensed by 2xl0 5 per wel l. The antibody was treated to 10 ug / mL and incubated at 37 ° C for 6 hours.
EZ-cytox enhanced cel l vi abi l i ty ki t (Daei l Lab) 를 이용하여 세포 생존율을 측정한 결과, 두 종의 암 세포주 모두에서 DNP002 항체와 자연 살해 세포의 병용에 의한 세포 사멸 효과가 단독처리 시 대비 증가하는 것으로 확인되었다 (도 12a 및 도 12b) .  Cell viability was measured using EZ-cytox enhanced cel l vi abi li ty ki t (Daei l Lab). As a result, apoptosis effect by the combination of DNP002 antibody and natural killer cells was treated in both cancer cell lines. It was found to increase with time (FIGS. 12A and 12B).
이는 DNP002 의 암세포 살해능이 자연 살해 세포와의 병용에 의해 상당히 증폭하였음을 나타낸다. 이를 통해, CEACAM6 양성인 암세포뿐만 아니라， 역시 期 ACAM6 양성인 MDSC 의 효과적인 제거를 위해 ■ 세포 또는 NK세포치료제와의 병용 처리가뛰어날수 있음을나타낸다.  This indicates that the cancer cell killing ability of DNP002 was significantly amplified by the combination with natural killing cells. This suggests that combination treatment with cell or NK cell therapy may be superior for the effective removal of not only CEACAM6 positive cancer cells but also ACAM6 positive MDSCs.
실시예 5.2 결과와 같이 DNP002 에 의한 MDSC의 선택적 용해 결과와 실시예 7의 ■ 세포 또는 NK 세포치료제의 병용효과를 통해, CEACAM6 양성인 암세포 및 CEACAM6 양성인 MDSC 를 모두 표적으로 하여 제거할 수 있음을 알 수 있다. 실시예 5.2와 실시예 7이 각각 MDSC 와 암세포로 다른 표적 세포에 대해 ADCC를 보였지만， 실제 두 종류의 세포가 함께 증가되어 있는 암환자의 경우， DNP002 로 두 종류의 표적을 동시에 제거할 수 있으며, 또한 암세포 및 MDSC 표적을 동시 제거하기 효능을 배가하기 위해 · 세포치료제와의 병용이 기능할수 있음을나타낸다.  As shown in Example 5.2, the result of selective lysis of MDSC by DNP002 and the combined effect of ■ cell or NK cell therapy of Example 7 revealed that both CEACAM6-positive cancer cells and CEACAM6-positive MDSC can be targeted and eliminated. have. Although Example 5.2 and Example 7 showed ADCC against different target cells as MDSC and cancer cells, respectively, in the case of cancer patients in which two types of cells are increased together, DNP002 can simultaneously remove both types of targets. It also indicates that combinations with cell therapies may function to double the efficacy of simultaneously eliminating cancer cells and MDSC targets.
<실시예 8> CEACAM6가 음성인 암환자의 미세종양 환경에서 MDSC의 검출 Example 8 Detection of MDSC in the Microtumor Environment of Cancer Patients with CEACAM6 Negative
CEACAM6 항원은 암세포뿐만 아니라 MDSC 에도 발현되기 때문에 DNP002 항체를 이용하여 암세포뿐만 아니라 MDSC 를 검출할 수 있다. 이를 확인하기 위해 CEACAM6 항원이 양성이거나 음성인 암환자 조직에서 암세포가 아닌 MDSC 가 검출되는 지를 면역조직화학염색 ( Immunohi stochemi stry) 법으로 확인하였다 .  Since CEACAM6 antigen is expressed not only in cancer cells but also in MDSC, the DNP002 antibody can be used to detect not only cancer cells but also MDSCs. To confirm this, immunohistochemical staining (Immunohi stochemi stry) method was used to determine whether MDSCs, but not cancer cells, are detected in tissues of cancer patients with positive or negative CEACAM6 antigen.
면역조직화학염색법은 다음과 같은 방법으로 수행하였다. xylene 에 10 분씩 3회， 100% alcoho에 10분씩 2회， 80%, 70% 알코올 (al cohol )에 5분， Immunohistochemical staining was performed in the following manner. 3 times for 10 minutes in xylene, 2 times for 10 minutes in 100% alcoho, 5 minutes for 80% and 70% alcohol (al cohol)
3분간 deparaf f in 하고 3차 증류수에 세척 한다. 이어서， 0.03% ¾02으로 실온에서 10분간 담궈서 Peroxidase Blocking 후 3차 증류수로 세척한다. 바로 IX 시트레이트 완충액 (Ci trate buf fer , pH 6.0)에 슬라이드를 넣고 20분간 끓여서 수돗물에 중탕해서 서서히 식힌 뒤 3 차 증류수로 세척하고 다시 IX PBS로 일회 세척 후 조직이 있는 부위에 DNP002 의 모클론인 8F5 항체를 슬라이드 1 장당 150 ul (10ug/ml ) 씩 실온에서 90 ± 5 분 동안 반응시킨다. 반응 후 IX PBS로 5 분씩 4 번 세척한다. 이차 항체를 슬라이드 1 장당 100 ul씩 실온에서 20 분간 반응시키고 반응 후 IX PBST로 5 분씩 4 번 세척하고 DAB Chromogen 을 슬라이드 1 장당 100 씩 실온에서 3 분간 발색 시키고 수돗물에 10 분 동안 세척 한다. Mayer’ s Hematoxyl in 을 슬라이드 1 장당 100 ul씩 실온에서 3 분간 대조 염색하고 흐르는 수돗물에 10 분 동안 세척한다. 이어서 dehydrat ion한 뒤에 mount ing을 한다. Deparaf f in for 3 minutes and wash in distilled water. Subsequently, it is immersed in 0.03% ¾0 2 at room temperature for 10 minutes and then washed with distilled water after Peroxidase Blocking. Place the slide in IX citrate buffer (Citrate buf fer, pH 6.0) Boil for 20 minutes, cool in tap water, cool slowly, wash with 3 distilled water, wash once again with IX PBS, and then add 150 ul (10ug / ml) per mole of DNP002, a monoclonal 8F5 antibody, at room temperature. React for ± 5 minutes. After reaction, wash 4 times with IX PBS for 5 min. The secondary antibody is reacted at room temperature for 100 minutes at 100 ul per slide. After the reaction, it is washed 4 times for 5 minutes with IX PBST for 5 minutes, and the DAB Chromogen is developed for 100 minutes at room temperature for 3 minutes and washed with tap water for 10 minutes. Mayer's Hematoxyl in is counterstained at room temperature for 3 minutes at 100 ul per slide and washed in running tap water for 10 minutes. Then dehydrat ion and mount ing.
암세포에서 CEACAM6 가 양성인 폐 선암 ( lung adenocarcinoma)과 암세포 자체에서는 CEACAM6가 음성인 편평상피암 ( lung squamous cel l carcinoma) , 비뇨기계 암 (Ur inary bladder cancer) 및 피부 암 (Melanoma mal ignancy) 조직에 CEACAM6 면역 염색한 결과 암 조직의 비 종양 부위에 CEACAM6 양성인 MDSC가 있는 것을 확인하였다 (도 13) . 이는 암 조직 또는 암세포의 자체에서의 CEACAM6 항원 발현과 상관없이 암조직 주변의 침윤된 MDSC를 표적으로 진단 및 치료에 활용할 있음을 나타낸다.  CEACAM6 positive in cancer cells and lung adenocarcinoma in cancer cells and CEACAM6 negative in lung cells itself Staining confirmed that there was a CEACAM6 positive MDSC at the non-tumor site of cancer tissue (FIG. 13). This indicates that infiltrated MDSCs around cancer tissues can be used for diagnosis and treatment as targets regardless of CEACAM6 antigen expression in cancer tissues or cancer cells themselves.
암세포 세포표면의 CEACAM6 양성도와 상관없이， 암환자에서는 MDSC 가 증가되는 경향을 보이며, 이는 실시예 8의 결과와 같이 미세종양환경에 침윤되어 있는 MDSC를 검출하고 확인할 수 있다. 이는 MDSC 를 선택적으로 용해할 수 있음을 보인 실시예 5.2의 결과와 함께 고려하였을 때, 암세포 상의 CEACAM6 양성도와 상관없이 MDSC 를 표적으로 진단 및 치료목적에 사용할 수 있음을 나타낸다. 암 종에 따라 암세포 세포표면의 CEACAM6 발현 유무를 다를 수 있으나, 이와 상관없이 대부분의 암 종에서 MDSC는 증가되기 때문에， DNP002를 이용하여 MDSC를 표적 치료를 통해 대부분의 암 종을 대상으로 한 범용의 진단 및 치료적 목적으로 이용할 수 있음을 나타낸다.  Regardless of the CEACAM6 positivity of cancer cell surface, MDSC tends to be increased in cancer patients, which can detect and confirm MDSC infiltrated into the microtumor environment as shown in Example 8. This indicates that MDSC can be used as a target for diagnosis and treatment regardless of the degree of CEACAM6 positivity on cancer cells when considered in conjunction with the results of Example 5.2 showing that MDSC can be selectively dissolved. Depending on the carcinoma, the expression of CEACAM6 on the surface of cancer cells may be different. However, MDSC is increased in most carcinomas regardless of cancers. It can be used for diagnostic and therapeutic purposes.

Claims

【청구의 범위】 [Range of request]
【청구항 1]  [Claim 1]
골수유래 면역억제 세포 (myeloid-derived suppressor cell, Myeloid-derived suppressor cells,
MDSC)에서 발현되는 CD66c에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편을 포함하는， 면역 활성 증강제. An immune activity enhancing agent comprising an antibody or antigen-binding fragment thereof that specifically binds to CD66c expressed in MDSC).
【청구항 2]  [Claim 2]
제 1항에 있어서 , 상기 면역 활성 증강제는 MDSC의 활성 , 생성 또는 사멸을 조절함으로써 MDSC의 면역 억제능을 제거 또는 감소시키는 것인 면역 활성 증강제.  The immune activity enhancer of claim 1, wherein the immune activity enhancer removes or reduces the immune suppressive ability of MDSC by modulating the activity, production, or death of MDSC.
【청구항 3]  [Claim 3]
제 1항에 있어서， 상기 면역 활성 증강제는 MDSC의 세포 사멸을 유도함으로써 면역 억제능을 제거 또는 감소시키는 것인 면역 활성 증강제. The immune activity enhancer of claim 1, wherein the immune activity enhancer removes or reduces immune suppression by inducing cell death of MDSC.
【청구항 4] [Claim 4]
제 1항에 있어서, 상기 면역 활성 증강제는, T세포, ■세포， 또는 조절 T세포 (regulatory T cell)의 활성에 대한 MDSC의 억제능을 조절하는 것인 면역 활성 증강제.  According to claim 1, wherein the immune activity enhancer, immune activity enhancer that modulates the inhibitory ability of MDSCs on the activity of T cells, ■ cells, or regulatory T cells (regulatory T cells).
【청구항 5】  [Claim 5]
제 1항에 있어서， 상기 항체는 마우스 항체， 키메라 항체 또는 인간화 항체인 면역 활성 증강제.  The immune activity enhancing agent according to claim 1, wherein the antibody is a mouse antibody, chimeric antibody or humanized antibody.
【청구항 6]  [Claim 6]
제 1항에 있어서, 상기 항체는 IgGl, IgG2, IgG3 또는 IgG4 Fc 도메인을 포함하는 것인 면역 활성 증강제 .  The immune activity enhancer of claim 1, wherein the antibody comprises an IgGl, IgG2, IgG3 or IgG4 Fc domain.
【청구항 7]  [Claim 7]
제 1항에 있어서 , 상기 항체는 탈푸코오스화 항체인 면역 활성 증강제 .  The immune activity enhancing agent according to claim 1, wherein the antibody is a defucosylated antibody.
【청구항 8] [Claim 8]
제 1항에 있어서， 상기 항체는 다음의 상보성 결정부위 (complementarity determining region； CDRs)를 포함하는， 항- CD66c 항체 또는 이의 항원 결합 단편인 면역 활성 증강제:  The immune activity enhancing agent of claim 1, wherein the antibody is an anti-CD66c antibody or antigen binding fragment thereof, comprising the following complementarity determining regions (CDRs):
서열번호 1 또는 서열번호 9의 아미노산 서열을 포함하는 CDR-H1, 서열번호 2 또는 서열번호 10의 아미노산 서열을 포함하는 CDR-H2, 서열번호 3의 아미노산 서열을 포함하는 CDR-H3,  CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, or CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3,
서열번호 4, 서열번호 11 또는 서열번호 12의 아미노산 서열을 포함하는 CDR-L1, 2019/221574 1»（：1^1{2019/006007 CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 11 or SEQ ID NO: 12, 2019/221574 1 »（： 1 ^ 1 {2019/006007
서열번호 5의 아미노산서열을포함하는 00^12, 및 00 ^ 12 comprising the amino acid sequence of SEQ ID NO: 5, and
서열번호 6또는서열번호 13의 아미노산서열을포함하는 0에_13임 . 0 to 13 comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 13.
【청구항 9] [Claim 9]
제 1항에 있어서， 상기 항체의 중쇄 가변영역은서열번호 22, 23, 24, 25, 26 또는 27의 아미노산 서열을 포함하는 프레임워크（\나¾1）， 서열번호 The heavy chain variable region of claim 1, wherein the heavy chain variable region of the antibody comprises a framework including amino acid sequence of SEQ ID NO: 22, 23, 24, 25, 26, or 27 (\ NA¾1), SEQ ID NO:
32， 33， 34， 35, 36 또는 37의 아미노산 서열을 포함하는 프레임워크（V- 서열번호 42, 43, 44, 45, 46 또는 47의 아미노산 서열을 포함하는 프레임워크（） ¾3）， 및 서열번호 52， 53， 54， 55, 56 또는 57의 아미노산 서열을 포함하는 프레임워크 ^¾4）로 이루어진 군에서 선택된 적어도 하나의 프레임 워크를 포함하는
Figure imgf000049_0001
항체 또는 이의 항원 결합 단편인 면역 활성 증강제. A framework comprising the amino acid sequence of 32, 33, 34, 35, 36 or 37 (V-SEQ ID NO: 42, 43, 44, 45, 46 or 47) At least one framework selected from the group consisting of a framework ^ ¾4) comprising the amino acid sequence of No. 52, 53, 54, 55, 56 or 57;
Figure imgf000049_0001
An immune activity enhancing agent that is an antibody or antigen-binding fragment thereof.
【청구항 10】  [Claim 10]
제 1항에 있어서， 상기 항체의 경쇄 가변영역은， 서열번호 28, 29, 30 또는 31의 아미노산 서열을 포함하는 프레임워크（丄 ）， 서열번호 38, 39, 40 또는 41의 아미노산 서열을 포함하는 프레임워크（丄-요2）， 서열번호 48, 49, 50, 또는 51의 아미노산 서열을 포함하는 프레임워크此斗1?3）, 및 서열번호 58, 59, 60 또는 61의 아미노산 서열을 포함하는 프레임워크（丄_ ?묘4）로 이루어지는 군에서 선택된 적어도 하나의 프레임 워크를 포함하는 것인 항- 06 항체 또는 이의 항원 결합단편인 면역 활성 증강제 .  The light chain variable region of claim 1, wherein the light chain variable region of the antibody comprises a framework comprising amino acid sequences of SEQ ID NOs: 28, 29, 30, or 31, and comprises amino acid sequences of SEQ ID NOs: 38, 39, 40, or 41; A framework (VII-III), a framework comprising the amino acid sequence of SEQ ID NO: 48, 49, 50, or 51) 1-3, and an amino acid sequence of SEQ ID NO: 58, 59, 60 or 61 An immune activity enhancing agent, which is an anti-06 antibody or antigen-binding fragment thereof, comprising at least one framework selected from the group consisting of a framework (Fig. 4).
【청구항 11】  [Claim 11]
제 1항에 있어서 , 상기 항체는서열번호 7, 14, 15, 16， 17또는 18의 아미노산 서열을 포함하는 중쇄 가변영역; 및 서열번호 8， 19, 20, 또는 21의 아미노산 서열을 포함하는 경쇄 가변영역을 포함하는
Figure imgf000049_0002
항체 또는 이의 항원 결합단편인 면역 활성 증강제 . The method of claim 1, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, 14, 15, 16, 17 or 18; And a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8, 19, 20, or 21
Figure imgf000049_0002
Immune activity enhancers that are antibodies or antigen binding fragments thereof.
【청구항 12】  [Claim 12]
제 1항에 있어서 , 상기 항체는온도 62°（：에서 새 결합에 의한 형광값 변이율이 200% 미만인
Figure imgf000049_0003
항체 또는 이의 항원 결합 단편인 면역 활성 증강제 . 2. The antibody of claim 1, wherein the antibody has a fluorescence variation rate of less than 200% due to new binding at a temperature of 62 °.
Figure imgf000049_0003
An immune activity enhancing agent which is an antibody or antigen binding fragment thereof.
【청구항 13】
Figure imgf000049_0004
[Claim 13]
Figure imgf000049_0004
단편인 면역 활성 증강제. Immune activity enhancers that are fragments.
【청구항 14】 제 1항 내지 제 13항 중 어느 한 항에 따른 면역 활성 증강제를 포함하는， MDSC 관련 질환 또는 증상의 예방， 치료 또는 개선용 약학 조성물. [Claim 14] A pharmaceutical composition for preventing, treating or ameliorating an MDSC related disease or condition, comprising the immune activity enhancing agent according to any one of claims 1 to 13.
【청구항 15】  [Claim 15]
제 14항에 있어서， 상기 MDSC 관련 질환은 림프구를 제외한 HLA-DR The method of claim 14, wherein the MDSC-related disease is HLA-DR excluding lymphocytes
Low/(-)， CDllb+, CD33+ 표현형을 보이며 CD66c 양성인 MDSC의 세포수가 정상세포에 비해 증가된 질환인 약학조성물. A pharmaceutical composition with low / (-), CDllb +, and CD33 + phenotypes and an increased number of CD66c positive MDSC cells compared to normal cells.
【청구항 16】  [Claim 16]
제 14항에 있어서， 상기 T세포, ■세포 (natural killer cell), 또는 조절 T세포 (regulatory T cell)의 활성에 대한 MDSC의 억제능을 조절하는 것인 약학조성물.  15. The pharmaceutical composition according to claim 14, wherein the pharmaceutical composition modulates the inhibitory ability of MDSC on the activity of the T cell, the natural killer cell, or the regulatory T cell.
【청구항 17】  [Claim 17]
제 14항에 있어서， 상기 MDSC 관련 질환은 암 또는 염증성 질환인 약학조성물.  The pharmaceutical composition of claim 14, wherein the MDSC-related disease is cancer or an inflammatory disease.
【청구항 18】  [Claim 18]
제 14항에 있어서 , 상기 MDSC 관련 질환은 Trypanosoma cruzi , Listeria monocytogenes , Leishmania major , helminths , Candida albicans , 또는 Porphyromonas gingivalis의 감염， 톡소플라즈마증, 또는 다균성 패혈증인 약학조성물.  15. The pharmaceutical composition according to claim 14, wherein the MDSC related disease is Trypanosoma cruzi, Listeria monocytogenes, Leishmania major, helminths, Candida albicans, or Porphyromonas gingivalis infection, toxoplasmosis, or multi-septic sepsis.
【청구항 19】  [Claim 19]
제 14항에 있어서, 상기 MDSC 관련 질환은, 위암， 유방암, 폐암, 대장암, 간암， 담낭암， 신장암, 췌장암， 갑상선암， 전립선암， 난소암， 자궁경부암， 방광암인， 급성골수구성백혈병 (acute myeloid leukemia) , 급성림프구성 백혈병 (acute lymphoblastic leukemia) , 급성단구성백혈병 (acute monocytic leukemia) , 또는 호지킨림프종 (Hodgkin’ s lymphoma) , 또는 비호지킨 림프종인 약학조성물 The acute myeloid leukemia according to claim 14, wherein the MDSC-related disease is gastric cancer, breast cancer, lung cancer, colon cancer, liver cancer, gallbladder cancer, kidney cancer, pancreatic cancer, thyroid cancer, prostate cancer, ovarian cancer, cervical cancer, or bladder cancer. Pharmaceutical composition, which is myeloid leukemia, acute lymphoblastic leukemia, acute monocytic leukemia, or Hodgkin's lymphoma, or non-Hodgkin's lymphoma
【청구항 20] [Claim 20]
제 19항에 있어서， 상기 암세포의 성장 또는 암전이 유도를 억제하는 것인 약학조성물.  The pharmaceutical composition of claim 19, wherein the pharmaceutical composition inhibits the growth or cancer metastasis induction of the cancer cells.
【청구항 21]  [Claim 21]
제 14항에 있어서， 상기 항체는 MDSC에서 발현되는 CD66c 및 CD66b를 인지하는 IgGl타입 항체인 약학조성물.  The pharmaceutical composition according to claim 14, wherein the antibody is an IgGl type antibody that recognizes CD66c and CD66b expressed in MDSC.
【청구항 22] 제 14항에 있어서, 상기 약학 조성물은 MDSC에서 발현되는 CD66c에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편에 더하여， ■세포 또는■세포유래 세포치료제를추가로포함하는 것인 약학조성물. [Claim 22] The pharmaceutical composition according to claim 14, wherein the pharmaceutical composition further comprises a cell or cell-derived cell therapeutic agent in addition to an antibody or antigen-binding fragment thereof that specifically binds CD66c expressed in MDSC.
【청구항 23】  [Claim 23]
골수유래 면역억제 세포 (myeloid-derived suppressor cell, Myeloid-derived suppressor cells,
MDSC)에서 발현되는 CD66c에 특이적으로 결합하는 항체 또는 이의 항원 결합 단편을 포함하며, CD66c 양성인 MDSC를 탐지하여 MDSC 관련 질환의 진단용조성물. A composition for the diagnosis of MDSC-related diseases by detecting an MDSC that is CD66c positive, comprising an antibody or antigen-binding fragment thereof that specifically binds CD66c expressed in MDSC).
【청구항 24]  [Claim 24]
제 23항에 있어서 , 상기 진단의 시료는 대상의 생체 시료인 조성물. The composition of claim 23, wherein the sample of diagnosis is a biological sample of a subject.
【청구항 25] [Claim 25]
제 23항에 있어서, 상기 MDSC 관련 질환은 림프구를 제외한 HLA- DRLow/(-), CDllbi, CD33+ 표현형을 보이며 CD66c 양성인 MDSC가 증가된 질환인 조성물.  24. The composition of claim 23, wherein the MDSC related disease is a disease that exhibits an HLA-DRLow / (-), CDllbi, CD33 + phenotype excluding lymphocytes and an increased CD66c positive MDSC.
【청구항 26】  [Claim 26]
제 23항에 있어서 , 상기 MDSC 관련 질환은 Trypanosoma cruzi , Listeria monocytogenes, Leishmania major , helminths , Candida albicans , 또는 Porphyromonas gingivalis의 감염, 톡소플라즈마증， 다균성 패혈증, 위암, 유방암， 폐암， 대장암, 간암 및담낭암， 신장암， 췌장암， 갑상선암， 전립선암， 난소암， 자궁경부암, 방광암인， 급성골수구성백혈병 (acute myeloid leukemia) , 급성림프구성 백혈병 (acute lymphoblastic leukemia) , 급성단구성백혈병 (acute monocytic leukemia) , 호지킨림프종 (Hodgkin’ s lymphoma), 또는 비호지킨 림프종인 조성물.  24. The method of claim 23, wherein the MDSC-associated disease is an infection of Trypanosoma cruzi, Listeria monocytogenes, Leishmania major, helminths, Candida albicans, or Porphyromonas gingivalis, Toxoplasmosis, Polymorphic sepsis, Gastric cancer, Breast cancer, Lung cancer, Colon cancer, Liver cancer and Gallbladder cancer, kidney cancer, pancreatic cancer, thyroid cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, acute myeloid leukemia, acute lymphoblastic leukemia, acute monocytic leukemia , Hodgkin's lymphoma, or non-Hodgkin's lymphoma.
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