WO2024085726A1 - Anti-cancer composition comprising anti-vsig4 antibody - Google Patents

Anti-cancer composition comprising anti-vsig4 antibody Download PDF

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WO2024085726A1
WO2024085726A1 PCT/KR2023/016397 KR2023016397W WO2024085726A1 WO 2024085726 A1 WO2024085726 A1 WO 2024085726A1 KR 2023016397 W KR2023016397 W KR 2023016397W WO 2024085726 A1 WO2024085726 A1 WO 2024085726A1
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amino acid
acid sequence
cancer
represented
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French (fr)
Korean (ko)
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권병세
이중원
김혜정
강경정
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주식회사 유틸렉스
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]

Definitions

  • the present invention provides a composition for preventing or treating cancer comprising a humanized anti-VSIG4 antibody or binding fragment thereof; and a composition that additionally contains an anti-PD-L1 antibody and exhibits a significant cancer prevention or treatment effect.
  • GLOBOCAN 2020 produced by the International Agency for Research on Cancer, estimates that there were approximately 19.3 million new cancer cases (18.1 million excluding non-melanoma skin cancer) and nearly 10 million cancer deaths (excluding non-melanoma skin cancer) worldwide in 2020. It is reported that 9.9 million people (excluding non-melanoma skin cancer) occurred.
  • Cancer treatment technology has continued to develop, but existing treatment methods all have limitations. In order to determine the need for developing new treatments, it is necessary to establish a comprehensive strategy that considers the survival rate for each cancer type.
  • Immunotherapy which is currently undergoing active research and development, is expected to minimize the side effects of existing treatments by strengthening the immune system of cancer patients and allowing the body to destroy cancer cells on its own.
  • more than 70% of patients show resistance to immunotherapy, making it difficult to expect a therapeutic effect.
  • One of the reasons for this low treatment effect is that the tumor microenvironment is not taken into consideration, so recent anticancer drug treatment is approached with a strategy that takes the tumor microenvironment into consideration.
  • the tumor micro-environment is favorable for tumor growth and metastasis.
  • the tumor microenvironment is well known to have suppressed immune function and metabolic stress, which acts as a major obstacle to anticancer treatment.
  • the tumor microenvironment is classified as “cold” or “hot” depending on the degree of T cell infiltration. Hot tumors are infiltrated with T cells and are immune activated, whereas cold tumors are characterized by the absence or exclusion of T cells. In these cold tumors, it is difficult for effector immune cells to infiltrate and resistance to various immune checkpoint inhibitors (ICIs) appears.
  • ICIs immune checkpoint inhibitors
  • One of the reasons why many immune checkpoint inhibitors that have been attempted previously have low actual therapeutic effects is because they do not take into account the tumor microenvironment. Therefore, to increase the response rate to immunotherapy, removal or reprogramming of the abnormal tumor microenvironment that converts noninflamed cold tumors into hot tumors can be an important anticancer treatment approach.
  • Tumor-associated macrophages a specific type of macrophage found in the tumor microenvironment, are known to be important cells that create a chronic inflammatory state in cancer.
  • Tumor-related macrophages migrate to tumor tissues in response to tumor-related inflammation and, unlike other common macrophages, lack cytokine activity.
  • Tumor-related macrophages secrete immune regulatory cytokines such as IL-4, IL-13, and IL-10, and vascular endothelial growth factor (VEGF), which promotes angiogenesis and epidermal growth that helps tumor growth. It has been reported that epidermal growth factor (EGF) is also secreted. For this reason, since the increase in tumor-related macrophages is directly related to the poor prognosis of cancer, there is a need for new anticancer treatments targeting tumor-related macrophages.
  • cytokines such as IL-4, IL-13, and IL-10
  • VEGF vascular endothelial growth factor
  • VSIG4 (V-set immunoglobulin-domain-containing 4) is mainly expressed in tissue-resident macrophages (M2 type macrophages), especially Kupffer cells in liver tissue.
  • M2 type macrophages tissue-resident macrophages
  • VSIG4 is expressed in tumor-associated macrophages (TAM), which inhibits the proliferation of antigen-specific T cells, suppresses inflammatory cytokine production, and induces proteins involved in immune tolerance. This creates a favorable environment for tumors. It is associated with worse survival in carcinomas showing increased VSIG4 expression, such as non-small cell lung cancer (NSCLC), multiple myeloma, ovarian cancer, and glioma. This suggests that VSIG4 plays an important role in tumor immune evasion.
  • NSCLC non-small cell lung cancer
  • the present inventors While researching a new anticancer agent that can improve the tumor-related microenvironment, the present inventors discovered that the anti-VSIG4 antibody of the present invention, which targets VSIG4, which plays an important role in tumor immune evasion, was found to have antibody-dependent cytotoxicity (Antibody-Dependent Cellular). It was confirmed that it can have the effect of improving the tumor microenvironment without showing cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), especially when administered in combination with anti-PD-L1 antibody. The present invention was completed after confirming that it can exhibit excellent anti-cancer effects.
  • ADCC cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the object of the present invention is a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6;
  • a pharmaceutical composition for preventing or treating cancer containing a humanized anti-VSIG4 antibody or an antigen-binding fragment thereof, or an adjuvant anti-cancer treatment for enhancing the efficacy of an anti-PD-L1 antibody is a pharmaceutical composition for preventing or treating cancer containing a humanized anti-VSIG4 antibody or an antigen-binding fragment thereof, or an adjuvant anti-cancer treatment for enhancing the efficacy of an anti-PD-L1 antibody.
  • the object of the present invention is a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6;
  • the present invention provides a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; A humanized anti-VSIG4 antibody or antigen-binding fragment thereof comprising; and b) an anti-PD-L1 antibody.
  • the present invention provides a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; A humanized anti-VSIG4 antibody or antigen-binding fragment thereof comprising; and b) an anti-PD-L1 antibody, and provides a combination administration agent for preventing or treating cancer.
  • the present invention provides a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6;
  • the present invention provides a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6;
  • an adjuvant anti-cancer treatment for enhancing the efficacy of an anti-PD-L1 antibody, comprising a humanized anti-VSIG4 antibody or an antigen-binding fragment thereof.
  • the present invention provides a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; A humanized anti-VSIG4 antibody or antigen-binding fragment thereof comprising; and b) administering an anti-PD-L1 antibody to a subject.
  • the present invention provides a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6;
  • a method for enhancing the efficacy of an anti-PD-L1 antibody comprising administering to a subject a humanized anti-VSIG4 antibody or antigen-binding fragment thereof.
  • the humanized anti-VSIG4 antibody or antigen-binding fragment thereof of the present invention can exhibit an anti-cancer effect by regulating immune cells in the human body and improving the microenvironment of cancer tissue, and when administered together with an anti-PD-L1 antibody, it has a significant anti-cancer effect. It can be expressed. Therefore, the present invention can be used in various cancer prevention or treatment fields, especially in the prevention or treatment of cancer types that have a poor prognosis with respect to immune checkpoint inhibitor therapy.
  • 1 is a schematic diagram of the conversion analysis method performed in the present invention.
  • Figure 2 shows the results of analysis of the binding activity of EU103 to HeLa-hVSIG4 cells, a cell line overexpressing VSIG4.
  • Figure 3 is a diagram showing the results of confirming the expression of VSIG4 in anti-inflammatory macrophages and the expression of VSIG4 in differentiated anti-inflammatory macrophages through FACS analysis.
  • Figure 4 shows the results of confirming the binding activity of EU103 to VSIG4 of anti-inflammatory macrophages.
  • Figure 5 is a diagram showing the results of evaluating the ADCC activity of EU103 in HeLa cells, a cell line overexpressing VSIG4 (EU103-IgG1: introduction of wild-type IgG1).
  • Figure 6 is a diagram showing the results of confirming the binding between EU103 and C1q through enzyme-linked immunosorbent assay (ELISA) (EU103-IgG1: introduction of wild-type IgG1).
  • ELISA enzyme-linked immunosorbent assay
  • Figure 7 is a diagram showing the results of confirming the proliferation of CD4 + / CD8 + T cells according to EU103 treatment concentration.
  • Figure 8 is a diagram showing the results confirming the conversion effect of EU103 in tumor-related macrophages isolated from ovarian cancer patients.
  • Figure 9 is a diagram showing the results confirming the tumor growth inhibition effect upon EU103 administration in the PBMC humanized mouse A549-luci tumor orthotopic model.
  • Figure 10 shows the results of a toxicity test confirming Kupffer cell depletion following treatment with EU103.
  • Figure 11 is a diagram showing the results of confirming changes in cytokine and arginase levels in Kupffer cells (hKC), TAM, and M2 macrophages according to EU103 treatment.
  • Figure 12 is a diagram showing the results confirming the tumor growth inhibition effect upon co-administration of EU103 and Tecentriq in the PBMC humanized mouse A549-luci tumor orthotopic model (A, B: Confirmation of tumor size change in the mouse model, C) : Tumor size measurement on the 43rd day after the end of the experiment).
  • the present invention provides a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; A humanized anti-VSIG4 antibody or antigen-binding fragment thereof comprising; or
  • heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1
  • heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2
  • heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3
  • light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4
  • light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5
  • light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6
  • a humanized anti-VSIG4 antibody or antigen-binding fragment thereof comprising comprising
  • b) comprising an anti-PD-L1 antibody comprising an anti-PD-L1 antibody
  • It relates to a pharmaceutical composition for preventing or treating cancer.
  • the humanized anti-VSIG4 antibody or antigen-binding fragment thereof of the present invention may include a heavy chain variable region represented by SEQ ID NO: 7 and/or a light chain variable region represented by SEQ ID NO: 8.
  • the hydrophobic index of the amino acid may be considered.
  • Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); leucine (+3.8); phenylalanine (+2.8); Cysteine/Cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); histidine (-3.2); glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); and arginine (-4.5).
  • the hydrophobic amino acid index is very important in imparting interactive biological functions to proteins or peptides. It is a known fact that similar biological activity can be maintained only when substituted with an amino acid having a similar hydrophobicity index.
  • substitution is made between amino acids showing a difference in the hydrophobicity index, preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
  • hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Asphaltate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); threonine (-0.4); Proline (-0.5 ⁇ 1); Alanine (-0.5); histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); leucine (-1.8); isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).
  • substitution is made between amino acids showing a difference in hydrophilicity value, preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
  • Amino acid exchanges in peptides that do not overall alter the activity of the molecule are known in the art.
  • the most common exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
  • amino acid sequences represented by SEQ ID NOs: 1 to 9 of the present invention are interpreted to also include sequences showing substantial identity with the sequences described in the sequence listing.
  • the above substantial identity is obtained by aligning the amino acid sequences represented by SEQ ID NOs. 1 to 9 of the present invention and any other sequences to correspond as much as possible, and analyzing the aligned sequences using an algorithm commonly used in the art.
  • sequences showing at least 80% or more homology more preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more. it means.
  • any method known in the art can be used without limitation.
  • the antigen-binding fragment is Fab, Fab', (Fab') 2 , Fv, scFv (single chain sc), bis-scFv, scFv-Fc fragment, Fab 2 , Fab 3 , minibody, dia. It may be selected from the group consisting of sieve, triase, tetrasieve and nanosieve. Said fragment has at least one of the characteristic CDRs of the antibody according to the invention.
  • Fab has a structure comprising the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region of the heavy chain, and has one antigen binding site.
  • Fab' differs from Fab in having a hinge containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain.
  • F(ab)' antibodies are produced because cysteine residues in the hinge region of Fab' form disulfide bonds.
  • Fv is the smallest antibody fragment with only the heavy and light chain variable regions.
  • dsFv double-chain Fv
  • ssFv single-chain Fv
  • antibody fragments can be obtained using proteinases (e.g., Fab can be obtained by restriction digestion of the whole antibody with papain, and F(ab)' fragments can be obtained by restriction digestion with pepsin. can be), preferably it can be produced by genetic engineering techniques.
  • the "antigen-binding fragment” will consist of or comprise a partial sequence of the heavy or light chain variable chain of the antibody from which it is derived, said partial sequence having the same binding specificity with respect to the target as the antibody from which it is derived. and sufficient affinity, preferably at least equal to 1/100, more preferably at least 1/10, of the affinity of the antibody from which it is derived.
  • the whole humanized anti-VSIG4 antibody of the invention may include subtypes or variants of IgA, IgD, IgE, IgM and IgG, and may particularly include IgG1.
  • Heavy chain constant regions are of gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ), and epsilon ( ⁇ ) types and subclasses gamma 1 ( ⁇ 1), gamma 2 ( ⁇ 2), gamma 3 ( ⁇ 3); It has gamma 4 ( ⁇ 4), alpha 1 ( ⁇ 1) and alpha 2 ( ⁇ 2).
  • the constant region of the light chain may include the kappa ( ⁇ ) and lambda ( ⁇ ) types.
  • the humanized anti-VSIG4 antibody of the present invention may include the Fc sequence of a human IgG1 antibody.
  • the humanized anti-VSIG4 antibody of the present invention may be an antibody with an improved Fc region that regulates antibody function, and may exhibit IgG1 Fc antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (ADCC). CDC) can be characterized as being suppressed. Accordingly, the humanized anti-VSIG4 antibody of the present invention is characterized by comprising an engineered IgG1 Fc region represented by SEQ ID NO: 9, and is at least about 80%, 85%, 90%, 95%, 96%, 97% thereof. , 98%, or 99% or more sequence identity may be included. wherein the variants include additions, deletions, and/or substitutions.
  • the humanized anti-VSIG4 antibody of the present invention does not exhibit toxicity to Kupffer cells, and exhibits very low antibody-dependent cytotoxicity and complement-dependent cytotoxicity due to the removal of the binding ability to Fc ⁇ R and C1q, and at the same time, T It can promote cell proliferation and convert tumor-associated macrophages (TAMs) into inflammatory macrophages that suppress tumors. As a result, the tumor microenvironment can be improved and excellent anticancer effects are achieved.
  • the humanized anti-VSIG4 antibody of the present invention can convert a noninflamed cold tumor into a hot tumor and remove the abnormal tumor microenvironment, effectively improving the treatment response to anticancer immunotherapy agents.
  • the present invention provides a composition for preventing or treating cancer further comprising an anti-PD-L1 antibody.
  • a humanized anti-VSIG4 antibody or binding fragment thereof of the invention and a cancer prevention or pharmaceutical composition containing an anti-PD-L1 antibody, or a combined administration agent for cancer prevention or treatment exhibits a significantly increased anticancer effect compared to the administration of an anti-VSIG4 antibody or a binding fragment thereof or an anti-PD-L1 antibody alone. You can.
  • Combination therapy refers to a situation in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents).
  • a humanized anti-VSIG4 antibody or binding fragment thereof; and anti-PD-L1 antibody can be administered simultaneously.
  • the humanized anti-VSIG4 antibody or antigen-binding fragment thereof; and anti-PD-L1 antibody may be administered sequentially.
  • the anti-PD-L1 antibody that can be administered in combination with the humanized anti-VSIG4 antibody or binding fragment thereof simultaneously or sequentially may include, without limitation, antibodies known in the art, and includes antigen-binding fragments thereof. However, it may preferably be atezolizumab, durvalumab, or avelumab, and in a preferred embodiment of the present invention, atezolizumab (Tecentriq) was used to confirm the effect.
  • Cancer of the present invention can be used interchangeably with tumor, neoplasm, and carcinoma, and includes a humanized anti-VSIG4 antibody or binding fragment thereof; Alternatively, it may include, without limitation, cancer species that can exhibit anti-cancer effects by humanized anti-VSIG4 antibody or binding fragment thereof and anti-PD-L1 antibody.
  • the cancer of the present invention may preferably be a VSIG4-overexpressing cancer, for example, multiple myeloma, ovarian cancer, lung cancer, non-small cell lung cancer, glioma, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer.
  • stomach cancer head and neck cancer
  • blood cancer laryngeal cancer
  • liver cancer lymphoma
  • melanoma mesothelioma
  • primary peritoneal cancer salivary gland cancer
  • sarcoma thyroid cancer
  • pancreatic cancer renal cell carcinoma
  • glioblastoma and prostate cancer. It could be more than that.
  • humanized anti-VSIG4 antibody or antigen-binding fragment thereof of the present invention when administered in combination with the anti-PD-L1 antibody, they may be included or administered in the composition at 1:1 to 5 (w/w)%, preferably Can be contained or administered in a ratio of 1:1 to 3 (w/w)%, or 1:1 (w/w)%.
  • the present invention provides a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; A humanized anti-VSIG4 antibody or antigen-binding fragment thereof comprising; and b) administering an anti-PD-L1 antibody to a subject.
  • administration generally refers to administering a composition to an entity or system to deliver the agent contained in or contained within the composition.
  • administration may be ocular, buccal, parenteral, topical, etc.
  • administration may be or include one or more of the following: bronchial (e.g., via bronchial instillation), buccal, dermal (e.g., dermal, intradermal, intradermal, transdermal, etc.
  • administration may involve only a single dose. In some embodiments, administration may involve applying a fixed number of doses.
  • administration may include intermittent (e.g., multiple doses spaced over time) administration and/or periodic (e.g., individual doses separated by a regular period of time) dosing.
  • administration may involve continuous doses (e.g., infusion) for at least a selected period of time.
  • the pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc. for oral administration.
  • buffers, preservatives, and analgesics can be used.
  • Topics, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives, etc. can be used.
  • the dosage form of the pharmaceutical composition according to the present invention can be prepared in various ways by mixing it with a pharmaceutically acceptable carrier as described above.
  • oral administration it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be manufactured in the form of unit dosage ampoules or multiple dosage forms. there is.
  • examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil may be used.
  • fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.
  • the pharmaceutical composition according to the present invention is influenced by various factors, including the activity of the specific active ingredient used, age, body weight, general health, gender, diet, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated.
  • the dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route and period, but may be appropriately selected by a person skilled in the art.
  • the amount that can obtain the maximum effect with the minimum amount without side effects can be administered, more preferably 1 to 10,000 ⁇ g/kg of body weight/day, and even more preferably 10 to 1,000 mg.
  • the effective dose is /kg body weight/day and can be administered repeatedly several times a day. The above dosage does not limit the scope of the present invention in any way.
  • the subject may include an organism, typically a mammal, and preferably a human.
  • the individual may be an individual suffering from or known to have a related disease, symptom, or disorder.
  • the present invention provides a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; One compartment containing a humanized anti-VSIG4 antibody or antigen-binding fragment thereof; and b) a two-compartment containing an anti-PD-L1 antibody.
  • the humanized anti-VSIG4 antibody or antigen-binding fragment thereof and anti-PD-L1 antibody included in the kit of the present invention may be administered simultaneously or sequentially depending on the purpose of treatment.
  • the kit may further include a package insert containing instructions for using the humanized anti-VSIG4 antibody or antigen-binding fragment thereof and the anti-PD-L1 antibody, and the components may be present in the same container or in separate containers.
  • Suitable containers include, for example, bottles, vials, bags, and syringes.
  • the container may be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or metal alloy (e.g., stainless steel or hastelloy).
  • the container holds the formulation and a label on or associated with the container may indicate directions for use.
  • the kit may additionally contain other materials required from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and drug product instructions with instructions for use.
  • the article of manufacture further comprises one or more other agents (e.g., additional chemotherapeutic agents, or anti-neoplastic agents).
  • Suitable containers for one or more agents include, for example, bottles, vials, bags, and syringes.
  • another object of the present invention is a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6;
  • an adjuvant anti-cancer treatment for enhancing anti-PD-L1 antibody efficacy comprising a humanized anti-VSIG4 antibody or an antigen-binding fragment thereof.
  • the humanized anti-VSIG4 antibody or antigen-binding fragment thereof can transform the tumor microenvironment into an environment that can increase compliance with immunotherapy, and as a result, the humanized anti-VSIG4 antibody or antigen-binding fragment thereof It was confirmed that simultaneous administration of anti-PD-L1 antibody and anti-PD-L1 antibody can enhance the anticancer effect compared to administration of anti-PD-L1 antibody alone. Therefore, the humanized anti-VSIG4 antibody or binding fragment thereof of the present invention can be used as an auxiliary method to enhance the efficacy of the anti-PD-L1 antibody.
  • the present invention provides a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; It relates to a method for enhancing the efficacy of an anti-PD-L1 antibody, comprising administering to a subject a humanized anti-VSIG4 antibody or antigen-binding fragment thereof.
  • the method can be performed in vivo, in vitro, or ex vivo .
  • the EU103.2 antibody was prepared by humanization from the mouse anti-VSIG4 antibody mu6H8.
  • CDRs were classified using Kabat numbering, and humanized VHs were projected with the corresponding framework and classified as back-mutants of mu6H8 VH CDRs, VH2, VH27, VH30, VH93, and VH94 (hu6H8.3 VH).
  • CDRs were classified using Kabat numbering, and humanized VLs were projected with the corresponding framework and classified as back-mutants of the mu6H8 VL CDRs, VH2, VH4, VH36, and VH46 (hu6H8.3 VL).
  • VHCDR1 VHCDR2 VHCDR3 VLCDR1 VLCDR2 VLCDR3 GISLTT SEQ ID NO: 1 IFWDDNK (SEQ ID NO: 2) VRVYYKNDGYFDV (SEQ ID NO: 3) KSVTTS (SEQ ID NO: 4) LAS (SEQ ID NO: 5) QQSGELPYT (SEQ ID NO: 6)
  • TAMs were isolated from the ascites of an ovarian cancer patient and treated with EU103, and then it was confirmed whether TAMs could be converted into inflammatory macrophages by EU103 treatment and thereby improve the tumor microenvironment.
  • FIG. 1 A schematic diagram of the conversion analysis method performed in the present invention is shown in Figure 1.
  • VSIG4 was confirmed to be expressed in 26.7% of differentiated anti-inflammatory macrophages.
  • Anti-inflammatory macrophages differentiated as above were treated with EU103 of the present invention at various concentrations, and the binding activity of EU103 was confirmed through FACS analysis, and the results are shown in Figure 4.
  • EU103 showed binding activity to VSIG4 of anti-inflammatory macrophages, and showed saturated binding activity at a concentration of 0.3125 ⁇ g/mL or higher.
  • IgG1 antibodies are known to have the disadvantage of showing strong ADCC (Antibody-Dependent Cellular Cytotoxicity) and CDC (Complement-Dependent Cytotoxicity) effects due to their high binding affinity for Fc ⁇ and complement activity, but they have the advantage of structural stability and high flexibility. Therefore, to preserve the antibody's ability to induce the conversion of anti-inflammatory macrophages to inflammatory macrophages by targeting VSIG4, but also to prevent the reaction mediating the elimination of anti-inflammatory macrophages, mutations were added to the Fc region of IgG1. EU103 was prepared in a form in which binding to Fc ⁇ and C1q, the main proteins mediating ADCC and CDC, was removed.
  • ADCC and CDC activities of IgG1, EU103, and EU103-IgG1 were tested using peripheral blood mononuclear cells (PBMC) used in the classical ADCC assay and HeLa cells overexpressing VSIG4 as target cells.
  • PBMC peripheral blood mononuclear cells
  • EU103 introduced three mutations (human IgG1 L234F/L235E/P331S SEQ ID NO: 9) into the IgG1 Fc region to eliminate ADCC and CDC activities, while EU103-IgG1 has wild-type IgG1 Fc.
  • SEQ ID NO: 9 human IgG1 L234F/L235E/P331S (mutation underlined, bold)
  • ADCC activity of EU103 was not observed when compared with EU103-IgG1 introduced with wild-type IgG1 without Fc region mutation.
  • EU103-IgG1 in which no mutation was introduced, cells were killed in a dose-dependent manner.
  • CDC is initiated by the binding of the antibody Fc domain to the complement protein C1q.
  • ELISA was performed to examine EU103 binding to C1q, and the results are shown in Figure 6.
  • EU103 showed minimal binding to C1q protein, which shows that it has a very low level of CDC activity.
  • CFSE staining can measure the number of cell divisions by number of dilutions.
  • CD4+/CD8+ T cells were isolated from peripheral blood mononuclear cells of healthy donors and activated using anti-CD3 antibody.
  • Tumor-associated macrophages do not exist under normal conditions, but are observed in many tumors and are therefore considered important in anticancer treatment.
  • the specific polarization state of tumor-associated macrophages (TAMs), as anti-inflammatory-like macrophages, are important components of the tumor microenvironment and are a subject of important research in anticancer therapy. Converting anti-inflammatory macrophages to a tumor-suppressing inflammatory macrophage phenotype may be a new therapeutic approach for improving anti-tumor immunity.
  • the role of EU103 in tumor-associated macrophages (TAMs) was analyzed to determine whether EU103 can transform TAMs isolated from the ascites of ovarian cancer patients.
  • CD14+ cells were isolated from the ascites of an ovarian cancer patient, and the isolated tumor-associated macrophages (TAM) expressed CD68 and CD163, and at the same time, the expression of VSIG4 was also observed.
  • the isolated tumor-associated macrophages (TAM) were treated with EU103 for 2 days, and changes in the expression of macrophage surface markers and secretion of cytokines and enzymes in the culture medium were measured.
  • LPS/IFN-g was treated as a positive control, and the experimental results are shown in Figure 8.
  • EU103 can improve the tumor microenvironment (TME) by converting tumor-associated macrophages (TAMs) into inflammatory macrophages that suppress tumors, which means that EU103 can exert anticancer effects by improving the tumor microenvironment. It indicates that there is.
  • TEM tumor-associated macrophages
  • EU103 is an antibody that acts on human VSIG4, and its binding affinity for mouse VSIG4 is low and cannot be observed. Therefore, the anticancer effect of EU103 was evaluated using a humanized mouse model introduced with the human immune system.
  • Humanized mice can be simply defined as research mice in which human cells or tissues are transplanted or genes are introduced into immunodeficient mice.
  • To introduce the human immune system the most common methods are transplantation of human hematopoietic stem cells (hu-HSC, CD34+ humanization) or white blood cells present in human peripheral blood (human peripheral blood leukocytes, hu-PBL, PBMC humanization) is separated and injected.
  • orthotopic models are known to be more clinically relevant and better able to predict drug effects.
  • tumor cell lines transfected with the luciferase gene were transplanted and anticancer effects were evaluated through noninvasive visualization of tumor growth, distribution, and metastatic growth through bioluminescence imaging (BLI).
  • BBI bioluminescence imaging
  • tail vein injection was used to engraft the tumor in the lung.
  • differentiated M2 macrophages were injected together with the lung cancer cell line to induce the formation of tumor-related macrophages.
  • Antibody administration was started when the luminescence intensity of the tumor engrafted in the lung was 1 x 10 7 or higher.
  • the test substance was administered a total of 6 times every 3 days. Tumor cells expressing luciferase were visualized using IVIS once a week after tumor cell transplantation and twice a week after antibody administration. After lung orthotopic induction, luciferase density was measured using the IVIS system to confirm lung-specific tumor cell engraftment, but was not observed in other organs.
  • Human IgG (hIgG) was administered as a control group, and the results of tumor size and body weight changes following EU103 administration are shown in Figure 9.
  • Preliminary monkey toxicity studies were performed using liver Kupffer cells (KC) from cynomolgus monkeys.
  • the experimental group included not only Fc-engineered IgG1 but also various isotypes of EU103 such as IgG1 (Anti-VSIG4-IgG1), IgG2 (Anti-VSIG4-euG2), and IgG4 (Anti-VSIG4-IgG4).
  • IgG1 Anti-VSIG4-IgG1
  • IgG2 Anti-VSIG4-euG2
  • IgG4 Anti-VSIG4-IgG4
  • An experiment was conducted targeting . Wild-type IgG1, IgG4, and Fc-engineered IgG1 EU103 at a concentration of 20 mg/kg were injected into monkeys and the effect on liver Kupffer cells was evaluated. The results are shown in Figure 10.
  • Kupffer cells Preserving Kupffer cells is important for immune homeostasis. Because Kupffer cells express VSIG4, a depletion effect by the ADCC activity of EU103-IgG1 (Anti-VSIG4-IgG1) was expected. Kupffer cells were isolated from the liver of cynomolgus monkeys to analyze depletion of Kupffer cells using FACS. As shown in Figure 10, the test results confirmed that Kupffer cells constitutively express both CD4 and VSIG4. EU103-IgG1 (Anti-VSIG4-IgG1) of IgG1 wild type reduced Kupffer cells by 64%, but EU103 of the present invention had no significant effect on the Kupffer cell population of cynomolgus monkeys. Additionally, no changes in clinical chemistry, including normal ALT and AST, were observed in monkeys administered EU103. In other words, no drug-related adverse reactions were observed in monkeys administered EU103, and clinical signs were normal at 20 mg/kg.
  • Tecentriq (atezolizumab) was used as the anti-PD-L1 antibody.
  • EU103 or Tecentriq alone administration group a dose of 8 mg/kg was administered, and in the combination administration group, EU103 5 mg/kg and Tecentriq 3 mg/kg were administered, and the results are shown in Figure 12 and Table 4.
  • TGI TV (%) [(V i -T i )/V i ]*100
  • T i treated group at endpoint day
  • V i hIgG group at endpoint day
  • the size of the tumor was significantly reduced in the single-administration group and the combined administration group compared to the control group (A, B).
  • the group treated with the combination of EU103 and Tecentriq showed superior antitumor activity than the group treated with EU103 or Tecentriq alone from day 26 onwards.
  • Tumor growth inhibition data was analyzed on day 43, the end date of the test, and the results are shown in Figure 12C.
  • tumor growth inhibition (TGITV (%)) in the EU103 and Tecentriq groups was 80.14% and 72.29%, respectively, and the combination group (Combo) showed an inhibition rate of 94.11%.
  • Monotherapy with EU103 or Tecentriq reduced tumor growth up to 43 days after tumor injection.
  • the combination therapy of EU103 and Tecentriq showed a clear synergistic anti-tumor effect in a humanized orthotopic lung cancer model.
  • the response of the anti-tumor effect of this combination was very immediate upon drug injection.
  • EU103 can efficiently inhibit tumor growth and significantly increase the anti-tumor effect in combination therapy with Tecentriq, an anti-PD-L1 antibody.

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Abstract

The present invention relates to a composition for the prevention or treatment of cancer comprising a humanized anti-VISG4 antibody or an antigen-binding fragment thereof, and a composition further comprising an anti-PD-L1 antibody that exhibits a significant cancer prevention or treatment effect. The humanized anti-VISG4 antibody or binding fragment thereof of the present invention can exhibit an anti-cancer effect by regulating immune cells in the body and improving the micro-environment of cancer tissue, and when administered in combination with an anti-PD-L1 antibody, can exhibit a significant anti-cancer effect. Therefore, the present invention can be utilized in various fields of cancer prevention or treatment, especially in the prevention or treatment of cancer types that have a poor prognosis with respect to immune checkpoint inhibitor therapy.

Description

[규칙 제91조에 의한 정정 01.12.2023]항-VSIG4 항체를 포함하는 항암용 조성물[Correction 01.12.2023 by Rule 91] Anti-cancer composition containing anti-VSIG4 antibody
[규칙 제91조에 의한 정정 01.12.2023]
본 발명은 인간화된 항-VSIG4 항체 또는 이의 결합 단편을 포함하는 암 예방 또는 치료용 조성물; 및 항 PD-L1 항체를 추가적으로 포함하여 현저한 암 예방 또는 치료 효과를 나타내는 조성물;에 관한 것이다.[Correction 01.12.2023 pursuant to Rule 91]
The present invention provides a composition for preventing or treating cancer comprising a humanized anti-VSIG4 antibody or binding fragment thereof; and a composition that additionally contains an anti-PD-L1 antibody and exhibits a significant cancer prevention or treatment effect.
국제 암 연구 기관(International Agency for Research on Cancer)에서 생성한 GLOBOCAN 2020에서는 2020년 전 세계적으로 약 1,930만 건의 새로운 암 사례 (비흑색종 피부암 제외 1,810만 명)와 거의 1,000만 명에 가까운 암 사망 (비흑색종 피부암 제외 990만 명)이 발생했다고 보고하고 있다. GLOBOCAN 2020, produced by the International Agency for Research on Cancer, estimates that there were approximately 19.3 million new cancer cases (18.1 million excluding non-melanoma skin cancer) and nearly 10 million cancer deaths (excluding non-melanoma skin cancer) worldwide in 2020. It is reported that 9.9 million people (excluding non-melanoma skin cancer) occurred.
암에 대한 치료 기술은 지속적으로 발전해 왔으나 기존의 치료방법들은 모두 한계를 가지고 있다. 새로운 치료제 개발의 필요성을 판단하기 위해서는 암 종별 생존율 등을 고려하는 종합적 전략 수립이 필요하다. Cancer treatment technology has continued to develop, but existing treatment methods all have limitations. In order to determine the need for developing new treatments, it is necessary to establish a comprehensive strategy that considers the survival rate for each cancer type.
또한 암 치료와 관련된 기술 개발이 조기 진단 및 초기 암의 치료에는 효과적이지만 진행된 암 환자의 상태는 개선하지는 못하고 있기 때문에 표준 치료에 불응하는 암 환자에게 효력을 나타낼 수 있는 새로운 개념의 항암제에 대한 필요성이 요구된다. In addition, the development of technology related to cancer treatment is effective in early diagnosis and treatment of early cancer, but does not improve the condition of patients with advanced cancer. Therefore, there is a need for a new concept of anticancer drug that can be effective in cancer patients who do not respond to standard treatment. It is required.
최근 연구 및 개발이 활발한 면역치료법은 암 환자의 면역체계를 강화시켜 인체가 스스로 암세포를 파괴하는 방법으로 기존 치료법이 가진 부작용을 최소화할 수 있을 것으로 기대하고 있다. 그러나 현재 면역항암제를 이용한 치료에도 불구하고, 환자의 70% 이상이 면역항암제에 내성을 보이며 여전히 치료 효과를 기대하기 힘들다. 이러한 낮은 치료효과의 원인 중 하나는 종양 미세환경을 고려하지 않았기 때문으로 최근 항암제 치료는 종양 미세환경을 고려한 전략으로 접근하고 있다. Immunotherapy, which is currently undergoing active research and development, is expected to minimize the side effects of existing treatments by strengthening the immune system of cancer patients and allowing the body to destroy cancer cells on its own. However, despite current treatment using immunotherapy, more than 70% of patients show resistance to immunotherapy, making it difficult to expect a therapeutic effect. One of the reasons for this low treatment effect is that the tumor microenvironment is not taken into consideration, so recent anticancer drug treatment is approached with a strategy that takes the tumor microenvironment into consideration.
종양 미세환경 (Tumor micro-environment, TME)은 종양의 성장과 전이에 유리하게 조성된다. 일반적으로 종양 미세환경은 면역능이 억제되고 대사 스트레스가 있는 것으로 잘 알려져 있으며 이는 항암치료에 큰 장애물로 작용한다. 종양 미세환경은 T 세포 침투 정도에 따라 “cold” 또는 “hot”으로 분류된다. Hot tumor는 T 세포가 침투되어 있고 면역 활성화 상태인 반면에 cold tumor는 T 세포가 없거나, 배제되어 있는 특징을 나타낸다. 이러한 cold tumor에서는 효과 면역세포의 침투가 어렵고 다양한 면역관문억제제(immune checkpoint inhibitor, ICI)에 대한 내성이 나타난다. 이전까지 시도된 많은 면역관문억제제가 실제 치료효과가 낮은 이유 중 하나는 이러한 종양 미세환경을 고려하지 않았기 때문이다. 따라서 면역 치료 반응률을 높이기 위해 비침투성(noninflamed) cold tumor를 hot tumor로 전환하는 비정상적인 종양 미세 환경의 제거 또는 재프로그래밍이 중요한 항암 치료의 접근법이 될 수 있다. The tumor micro-environment (TME) is favorable for tumor growth and metastasis. In general, the tumor microenvironment is well known to have suppressed immune function and metabolic stress, which acts as a major obstacle to anticancer treatment. The tumor microenvironment is classified as “cold” or “hot” depending on the degree of T cell infiltration. Hot tumors are infiltrated with T cells and are immune activated, whereas cold tumors are characterized by the absence or exclusion of T cells. In these cold tumors, it is difficult for effector immune cells to infiltrate and resistance to various immune checkpoint inhibitors (ICIs) appears. One of the reasons why many immune checkpoint inhibitors that have been attempted previously have low actual therapeutic effects is because they do not take into account the tumor microenvironment. Therefore, to increase the response rate to immunotherapy, removal or reprogramming of the abnormal tumor microenvironment that converts noninflamed cold tumors into hot tumors can be an important anticancer treatment approach.
종양 미세환경에서 발견되는 특이적인 형태의 대식세포인 종양 관련 대식세포(Tumor-associated macrophage, TAM)는 암의 만성 염증 상태를 만드는 중요한 세포로 알려져 있다. 종양 관련 대식세포는 종양 관련 염증에 반응하여 종양의 조직으로 이동하며, 일반적인 다른 대식세포와는 달리 사이토카인 활성이 부족하다. 종양 관련 대식세포는 면역 조절 사이토카인인 IL-4, IL-13, IL-10 등을 분비하며 혈관 생성을 촉진하는 혈관 내피 성장 인자(vascular endothelial growth factor, VEGF), 종양의 생장을 돕는 표피 성장 인자(epidermal growth factor, EGF) 역시 분비한다고 보고되었다. 이런 이유로 종양 관련 대식세포의 증가는 곧 암의 나쁜 예후와 직접적인 관련이 있기 때문에 종양 관련 대식세포를 대상으로 하는 새로운 항암 치료법에 대한 필요성이 있다. Tumor-associated macrophages (TAMs), a specific type of macrophage found in the tumor microenvironment, are known to be important cells that create a chronic inflammatory state in cancer. Tumor-related macrophages migrate to tumor tissues in response to tumor-related inflammation and, unlike other common macrophages, lack cytokine activity. Tumor-related macrophages secrete immune regulatory cytokines such as IL-4, IL-13, and IL-10, and vascular endothelial growth factor (VEGF), which promotes angiogenesis and epidermal growth that helps tumor growth. It has been reported that epidermal growth factor (EGF) is also secreted. For this reason, since the increase in tumor-related macrophages is directly related to the poor prognosis of cancer, there is a need for new anticancer treatments targeting tumor-related macrophages.
VSIG4(V-set immunoglobulin-domain-containing 4)는 주로 조직 거주 대식세포(M2 형 대식세포)에서 발현하고 있으며, 특히 간조직의 쿠퍼 세포(Kupffer cell)에서 발현된다. 종양 미세 환경에서 VSIG4는 종양 관련 대식세포(Tumor-associated macrophage, TAM)에서 발현하여 항원 특이적인 T 세포의 증식 억제, 염증성 사이토카인 생산 억제 및 면역 관용(Immune Tolerance)에 관여하는 단백질을 유도하는 기전을 통해 종양에 유리한 환경을 조장한다. 비소세포폐암(Non-small cell lung cancer, NSCLC), 다발성 골수종(Multiple myeloma), 난소암(Ovarian cancer) 및 신경교종(Glioma) 과 같이 증가된 VSIG4 발현을 나타내는 암종에서는 더 나쁜 생존율과 관련이 있으며 이는 VSIG4가 종양 면역 회피에 중요한 역할을 함을 시사한다.VSIG4 (V-set immunoglobulin-domain-containing 4) is mainly expressed in tissue-resident macrophages (M2 type macrophages), especially Kupffer cells in liver tissue. In the tumor microenvironment, VSIG4 is expressed in tumor-associated macrophages (TAM), which inhibits the proliferation of antigen-specific T cells, suppresses inflammatory cytokine production, and induces proteins involved in immune tolerance. This creates a favorable environment for tumors. It is associated with worse survival in carcinomas showing increased VSIG4 expression, such as non-small cell lung cancer (NSCLC), multiple myeloma, ovarian cancer, and glioma. This suggests that VSIG4 plays an important role in tumor immune evasion.
[규칙 제91조에 의한 정정 01.12.2023]
본 발명자들은 종양 관련 미세환경을 개선할 수 있는 새로운 항암제에 대하여 연구하던 중, 종양 면역 회피에 중요한 역할을 하는 VSIG4를 타깃으로 하는 본 발명의 항-VSIG4 항체가 항체 의존적 세포독성 (Antibody-Dependent Cellular Cytotoxicity, ADCC) 및 보체 의존적 세포독성 (Complement-Dependent Cytotoxicity, CDC)을 나타내지 않으면서, 종양 미세 환경을 개선하는 효과를 나타낼 수 있음을 확인하였으며, 특히 이를 항-PD-L1 항체와 병용 투여 시 현저히 우수한 항암 효과를 나타낼 수 있음을 확인하고 본 발명을 완성하였다.[Correction 01.12.2023 pursuant to Rule 91]
While researching a new anticancer agent that can improve the tumor-related microenvironment, the present inventors discovered that the anti-VSIG4 antibody of the present invention, which targets VSIG4, which plays an important role in tumor immune evasion, was found to have antibody-dependent cytotoxicity (Antibody-Dependent Cellular). It was confirmed that it can have the effect of improving the tumor microenvironment without showing cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), especially when administered in combination with anti-PD-L1 antibody. The present invention was completed after confirming that it can exhibit excellent anti-cancer effects.
[규칙 제91조에 의한 정정 01.12.2023]
따라서 본 발명의 목적은 서열번호 1의 아미노산 서열로 표시되는 중쇄 CDR1, 서열번호 2의 아미노산 서열로 표시되는 중쇄 CDR2, 서열번호 3의 아미노산 서열로 표시되는 중쇄 CDR3; 및 서열번호 4의 아미노산 서열로 표시되는 경쇄 CDR1, 서열번호 5의 아미노산 서열로 표시되는 경쇄 CDR2, 서열번호 6의 아미노산 서열로 표시되는 경쇄 CDR3; 을 포함하는 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편을 포함하는 암 예방 또는 치료용 약학적 조성물, 또는 항 PD-L1 항체 효능 증진용 항암 보조 치료제를 제공하는 것이다.[Correction 01.12.2023 pursuant to Rule 91]
Therefore, the object of the present invention is a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; To provide a pharmaceutical composition for preventing or treating cancer containing a humanized anti-VSIG4 antibody or an antigen-binding fragment thereof, or an adjuvant anti-cancer treatment for enhancing the efficacy of an anti-PD-L1 antibody.
[규칙 제91조에 의한 정정 01.12.2023]
또한 본 발명의 목적은 a) 서열번호 1의 아미노산 서열로 표시되는 중쇄 CDR1, 서열번호 2의 아미노산 서열로 표시되는 중쇄 CDR2, 서열번호 3의 아미노산 서열로 표시되는 중쇄 CDR3; 및 서열번호 4의 아미노산 서열로 표시되는 경쇄 CDR1, 서열번호 5의 아미노산 서열로 표시되는 경쇄 CDR2, 서열번호 6의 아미노산 서열로 표시되는 경쇄 CDR3; 을 포함하는 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편; 및 b) 항 PD-L1 항체를 포함하는, 암 예방 또는 치료용 약학적 조성물; 또는 암 예방 또는 치료용 키트를 제공하는 것이다.[Correction 01.12.2023 pursuant to Rule 91]
In addition, the object of the present invention is a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; A humanized anti-VSIG4 antibody or antigen-binding fragment thereof comprising; and b) a pharmaceutical composition for preventing or treating cancer, comprising an anti-PD-L1 antibody; Or, it provides a kit for preventing or treating cancer.
[규칙 제91조에 의한 정정 01.12.2023]
상기 목적을 달성하기 위하여, 본 발명은 a) 서열번호 1의 아미노산 서열로 표시되는 중쇄 CDR1, 서열번호 2의 아미노산 서열로 표시되는 중쇄 CDR2, 서열번호 3의 아미노산 서열로 표시되는 중쇄 CDR3; 및 서열번호 4의 아미노산 서열로 표시되는 경쇄 CDR1, 서열번호 5의 아미노산 서열로 표시되는 경쇄 CDR2, 서열번호 6의 아미노산 서열로 표시되는 경쇄 CDR3; 을 포함하는 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편; 및 b) 항 PD-L1 항체를 포함하는, 암 예방 또는 치료용 약학적 조성물을 제공한다.[Correction 01.12.2023 pursuant to Rule 91]
In order to achieve the above object, the present invention provides a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; A humanized anti-VSIG4 antibody or antigen-binding fragment thereof comprising; and b) an anti-PD-L1 antibody.
[규칙 제91조에 의한 정정 01.12.2023]
또한 본 발명은 a) 서열번호 1의 아미노산 서열로 표시되는 중쇄 CDR1, 서열번호 2의 아미노산 서열로 표시되는 중쇄 CDR2, 서열번호 3의 아미노산 서열로 표시되는 중쇄 CDR3; 및 서열번호 4의 아미노산 서열로 표시되는 경쇄 CDR1, 서열번호 5의 아미노산 서열로 표시되는 경쇄 CDR2, 서열번호 6의 아미노산 서열로 표시되는 경쇄 CDR3; 을 포함하는 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편; 및 b) 항 PD-L1 항체를 포함하는, 암 예방 또는 치료용 병용투여 제제를 제공한다.[Correction 01.12.2023 pursuant to Rule 91]
In addition, the present invention provides a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; A humanized anti-VSIG4 antibody or antigen-binding fragment thereof comprising; and b) an anti-PD-L1 antibody, and provides a combination administration agent for preventing or treating cancer.
[규칙 제91조에 의한 정정 01.12.2023]
또한 본 발명은 a) 서열번호 1의 아미노산 서열로 표시되는 중쇄 CDR1, 서열번호 2의 아미노산 서열로 표시되는 중쇄 CDR2, 서열번호 3의 아미노산 서열로 표시되는 중쇄 CDR3; 및 서열번호 4의 아미노산 서열로 표시되는 경쇄 CDR1, 서열번호 5의 아미노산 서열로 표시되는 경쇄 CDR2, 서열번호 6의 아미노산 서열로 표시되는 경쇄 CDR3; 을 포함하는 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편을 포함하는 1구획; 및 b) 항 PD-L1 항체를 포함하는 2구획을 포함하는, 암 예방 또는 치료용 키트를 제공한다.[Correction 01.12.2023 pursuant to Rule 91]
In addition, the present invention provides a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; One compartment containing a humanized anti-VSIG4 antibody or antigen-binding fragment thereof comprising; and b) a two-compartment containing an anti-PD-L1 antibody.
[규칙 제91조에 의한 정정 01.12.2023]
또한 본 발명은 서열번호 1의 아미노산 서열로 표시되는 중쇄 CDR1, 서열번호 2의 아미노산 서열로 표시되는 중쇄 CDR2, 서열번호 3의 아미노산 서열로 표시되는 중쇄 CDR3; 및 서열번호 4의 아미노산 서열로 표시되는 경쇄 CDR1, 서열번호 5의 아미노산 서열로 표시되는 경쇄 CDR2, 서열번호 6의 아미노산 서열로 표시되는 경쇄 CDR3; 을 포함하는 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편을 포함하는, 항 PD-L1 항체 효능 증진용 항암 보조 치료제를 제공한다.[Correction 01.12.2023 pursuant to Rule 91]
In addition, the present invention provides a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; Provided is an adjuvant anti-cancer treatment for enhancing the efficacy of an anti-PD-L1 antibody, comprising a humanized anti-VSIG4 antibody or an antigen-binding fragment thereof.
[규칙 제91조에 의한 정정 01.12.2023]
또한 본 발명은 a) 서열번호 1의 아미노산 서열로 표시되는 중쇄 CDR1, 서열번호 2의 아미노산 서열로 표시되는 중쇄 CDR2, 서열번호 3의 아미노산 서열로 표시되는 중쇄 CDR3; 및 서열번호 4의 아미노산 서열로 표시되는 경쇄 CDR1, 서열번호 5의 아미노산 서열로 표시되는 경쇄 CDR2, 서열번호 6의 아미노산 서열로 표시되는 경쇄 CDR3; 을 포함하는 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편; 및 b) 항 PD-L1 항체를 개체에 투여하는 단계를 포함하는 암 예방 또는 치료방법을 제공한다.[Correction 01.12.2023 pursuant to Rule 91]
In addition, the present invention provides a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; A humanized anti-VSIG4 antibody or antigen-binding fragment thereof comprising; and b) administering an anti-PD-L1 antibody to a subject.
[규칙 제91조에 의한 정정 01.12.2023]
또한 본 발명은 a) 서열번호 1의 아미노산 서열로 표시되는 중쇄 CDR1, 서열번호 2의 아미노산 서열로 표시되는 중쇄 CDR2, 서열번호 3의 아미노산 서열로 표시되는 중쇄 CDR3; 및 서열번호 4의 아미노산 서열로 표시되는 경쇄 CDR1, 서열번호 5의 아미노산 서열로 표시되는 경쇄 CDR2, 서열번호 6의 아미노산 서열로 표시되는 경쇄 CDR3; 을 포함하는 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편을 개체에 투여하는 단계를 포함하는, 항 PD-L1 항체 효능 증진 방법을 제공한다.[Correction 01.12.2023 pursuant to Rule 91]
In addition, the present invention provides a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; Provided is a method for enhancing the efficacy of an anti-PD-L1 antibody, comprising administering to a subject a humanized anti-VSIG4 antibody or antigen-binding fragment thereof.
[규칙 제91조에 의한 정정 01.12.2023]
본 발명의 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편은 인체 내의 면역 세포를 조절하고 암조직의 미세 환경을 개선하여 항암 효과를 나타낼 수 있으며, 항 PD-L1 항체와 함께 투여하면 현저한 항암 효과를 나타낼 수 있다. 따라서 본 발명은 다양한 암 예방 또는 치료 분야, 특히 면역관문억제제 치료법에 대하여 나쁜 예후를 나타내는 암 종의 예방 또는 치료에 활용 가능하다.[Correction 01.12.2023 pursuant to Rule 91]
The humanized anti-VSIG4 antibody or antigen-binding fragment thereof of the present invention can exhibit an anti-cancer effect by regulating immune cells in the human body and improving the microenvironment of cancer tissue, and when administered together with an anti-PD-L1 antibody, it has a significant anti-cancer effect. It can be expressed. Therefore, the present invention can be used in various cancer prevention or treatment fields, especially in the prevention or treatment of cancer types that have a poor prognosis with respect to immune checkpoint inhibitor therapy.
도 1은 본 발명에서 수행된 전환 분석 방법의 개략도이다. 1 is a schematic diagram of the conversion analysis method performed in the present invention.
도 2는 VSIG4 과발현 세포주인 HeLa-hVSIG4 세포에 대한 EU103의 결합 활성 분석 결과를 나타낸 도이다. Figure 2 shows the results of analysis of the binding activity of EU103 to HeLa-hVSIG4 cells, a cell line overexpressing VSIG4.
[규칙 제91조에 의한 정정 01.12.2023]
도 3은 항-염증성 대식세포의 VSIG4 발현 및 분화된 항-염증성 대식세포에서의 VSIG4의 발현을 FACS 분석을 통해 확인한 결과를 나타낸 도이다.[Correction 01.12.2023 pursuant to Rule 91]
Figure 3 is a diagram showing the results of confirming the expression of VSIG4 in anti-inflammatory macrophages and the expression of VSIG4 in differentiated anti-inflammatory macrophages through FACS analysis.
[규칙 제91조에 의한 정정 01.12.2023]
도 4는 항-염증성 대식세포의 VSIG4에 대한 EU103의 결합활성을 확인한 결과를 나타낸 도이다.[Correction 01.12.2023 pursuant to Rule 91]
Figure 4 shows the results of confirming the binding activity of EU103 to VSIG4 of anti-inflammatory macrophages.
도 5는 VSIG4 과발현 세포주 HeLa cell에서 EU103의 ADCC 활성을 평가한 결과를 나타낸 도이다 (EU103-IgG1: 야생형 IgG1 도입). Figure 5 is a diagram showing the results of evaluating the ADCC activity of EU103 in HeLa cells, a cell line overexpressing VSIG4 (EU103-IgG1: introduction of wild-type IgG1).
도 6은 EU103과 C1q의 결합을 효소결합면역흡착검사 (ELISA)을 통해 확인한 결과를 나타낸 도이다(EU103-IgG1: 야생형 IgG1 도입). Figure 6 is a diagram showing the results of confirming the binding between EU103 and C1q through enzyme-linked immunosorbent assay (ELISA) (EU103-IgG1: introduction of wild-type IgG1).
도 7은 EU103 처리 농도별 CD4+/CD8+ T 세포의 증식을 확인한 결과를 나타낸 도이다. Figure 7 is a diagram showing the results of confirming the proliferation of CD4 + / CD8 + T cells according to EU103 treatment concentration.
도 8은 난소암 환자로부터 분리된 종양 관련 대식세포에서 EU103의 전환(conversion) 효과를 확인한 결과를 나타낸 도이다. Figure 8 is a diagram showing the results confirming the conversion effect of EU103 in tumor-related macrophages isolated from ovarian cancer patients.
도 9는 PBMC 인간화 생쥐 A549-luci 종양 동소위(orthotopic) 모델에서 EU103 투여 시 종양 성장 억제 효과를 확인한 결과를 나타낸 도이다. Figure 9 is a diagram showing the results confirming the tumor growth inhibition effect upon EU103 administration in the PBMC humanized mouse A549-luci tumor orthotopic model.
도 10은 EU103의 처리에 따른 쿠퍼 세포(Kupffer cell) 고갈을 확인한 독성 실험 결과이다. Figure 10 shows the results of a toxicity test confirming Kupffer cell depletion following treatment with EU103.
도 11은 EU103 처리에 따른 쿠퍼세포 (Kupffer cell, hKC), TAM 및 M2 대식세포에서 사이토카인 및 아르기나아제 수준의 변화를 확인한 결과를 나타낸 도이다. Figure 11 is a diagram showing the results of confirming changes in cytokine and arginase levels in Kupffer cells (hKC), TAM, and M2 macrophages according to EU103 treatment.
도 12는 PBMC 인간화 생쥐 A549-luci 종양 동소위(orthotopic) 모델에서 EU103와 티센트릭 병용투여 시 종양 성장 억제 효과를 확인한 결과를 나타낸 도이다 (A, B: 마우스 모델에서의 종양 크기 변화 확인, C: 실험 종료 43일차의 종양 크기 측정). Figure 12 is a diagram showing the results confirming the tumor growth inhibition effect upon co-administration of EU103 and Tecentriq in the PBMC humanized mouse A549-luci tumor orthotopic model (A, B: Confirmation of tumor size change in the mouse model, C) : Tumor size measurement on the 43rd day after the end of the experiment).
이하, 본 발명에 대해 상세히 설명한다.Hereinafter, the present invention will be described in detail.
[규칙 제91조에 의한 정정 01.12.2023]
본 발명은 a) 서열번호 1의 아미노산 서열로 표시되는 중쇄 CDR1, 서열번호 2의 아미노산 서열로 표시되는 중쇄 CDR2, 서열번호 3의 아미노산 서열로 표시되는 중쇄 CDR3; 및 서열번호 4의 아미노산 서열로 표시되는 경쇄 CDR1, 서열번호 5의 아미노산 서열로 표시되는 경쇄 CDR2, 서열번호 6의 아미노산 서열로 표시되는 경쇄 CDR3; 을 포함하는 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편; 또는[Correction 01.12.2023 pursuant to Rule 91]
The present invention provides a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; A humanized anti-VSIG4 antibody or antigen-binding fragment thereof comprising; or
[규칙 제91조에 의한 정정 01.12.2023]
a) 서열번호 1의 아미노산 서열로 표시되는 중쇄 CDR1, 서열번호 2의 아미노산 서열로 표시되는 중쇄 CDR2, 서열 번호 3의 아미노산 서열로 표시되는 중쇄 CDR3; 및 서열번호 4의 아미노산 서열로 표시되는 경쇄 CDR1, 서열번호 5의 아미노산 서열로 표시되는 경쇄 CDR2, 서열번호 6의 아미노산 서열로 표시되는 경쇄 CDR3; 을 포함하는 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편; 및 b) 항 PD-L1 항체를 포함하는,[Correction 01.12.2023 pursuant to Rule 91]
a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; A humanized anti-VSIG4 antibody or antigen-binding fragment thereof comprising; and b) comprising an anti-PD-L1 antibody,
암 예방 또는 치료용 약학적 조성물에 관한 것이다. It relates to a pharmaceutical composition for preventing or treating cancer.
[규칙 제91조에 의한 정정 01.12.2023]
본 발명의 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편은 서열번호 7 로 표시되는 중쇄 가변영역 및/또는 서열번호 8 로 표시되는 경쇄 가변영역을 포함할 수 있다.[Correction 01.12.2023 pursuant to Rule 91]
The humanized anti-VSIG4 antibody or antigen-binding fragment thereof of the present invention may include a heavy chain variable region represented by SEQ ID NO: 7 and/or a light chain variable region represented by SEQ ID NO: 8.
[규칙 제91조에 의한 정정 01.12.2023]
본 발명의 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편의 목적하는 효과를 달성하는 한, 상기 서열번호로 표시되는 서열의 변이 서열이 본 발명에 포함될 수 있다. 여기서 변이는 부가, 결실, 및/또는 치환을 포함한다.[Correction 01.12.2023 pursuant to Rule 91]
As long as the desired effect of the humanized anti-VSIG4 antibody or antigen-binding fragment thereof of the present invention is achieved, a variant sequence of the sequence represented by the above sequence number may be included in the present invention. Variations herein include additions, deletions, and/or substitutions.
변이를 도입하는 데 있어서, 아미노산의 소수성 인덱스(hydropathic index)가 고려될 수 있다. 각각의 아미노산은 소수성과 전하에 따라 소수성 인덱스가 부여되어 있다: 아이소루이신 (+4.5); 발린 (+4.2); 루이신(+3.8); 페닐알라닌(+2.8); 시스테인/시스타인 (+2.5); 메티오닌 (+1.9); 알라닌 (+1.8); 글라이신 (-0.4); 트레오닌 (-0.7); 세린 (-0.8); 트립토판 (-0.9); 타이로신 (-1.3); 프롤린 (-1.6); 히스티딘 (-3.2); 글루타메이트 (-3.5); 글루타민 (-3.5); 아스파르테이트 (-3.5); 아스파라긴 (-3.5); 라이신 (-3.9); 및 아르기닌 (-4.5).In introducing mutations, the hydrophobic index of the amino acid may be considered. Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); leucine (+3.8); phenylalanine (+2.8); Cysteine/Cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); histidine (-3.2); glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); and arginine (-4.5).
단백질 또는 펩타이드의 상호적인 생물학적 기능(interactive biological function)을 부여하는 데 있어서 소수성 아미노산 인덱스는 매우 중요하다. 유사한 소수성 인덱스를 가지는 아미노산으로 치환하여야 유사한 생물학적 활성을 보유할 수 있다는 것은 공지된 사실이다. 소수성 인덱스를 참조하여 변이를 도입시키는 경우, 바람직하게는 ±2 이내, 보다 바람직하게는 ±1 이내, 보다 더 바람직하게는 ±0.5 이내의 소수성 인덱스 차이를 나타내는 아미노산 사이에 치환을 한다.The hydrophobic amino acid index is very important in imparting interactive biological functions to proteins or peptides. It is a known fact that similar biological activity can be maintained only when substituted with an amino acid having a similar hydrophobicity index. When introducing a mutation with reference to the hydrophobicity index, substitution is made between amino acids showing a difference in the hydrophobicity index, preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.
한편, 유사한 친수성 값(hydrophilicity value)을 가지는 아미노산 사이의 치환이 균등한 생물학적 활성을 갖는 단백질 또는 펩타이드를 초래한다는 것도 잘 알려져 있다. 다음의 친수성 값이 각각의 아미노산 잔기에 부여되어 있다: 아르기닌 (+3.0); 라이신 (+3.0); 아스팔테이트(+3.0±1); 글루타메이트 (+3.0±1); 세린 (+0.3); 아스파라긴 (+0.2); 글루타민 (+0.2); 글라이신 (0); 쓰레오닌 (-0.4); 프롤린 (-0.5±1); 알라닌 (-0.5); 히스티딘 (-0.5); 시스테인 (-1.0); 메티오닌 (-1.3); 발린 (-1.5); 루이신 (-1.8); 아이소루이신 (-1.8); 타이로신 (-2.3); 페닐알라닌 (-2.5); 트립토판 (-3.4). Meanwhile, it is also well known that substitution between amino acids with similar hydrophilicity values results in proteins or peptides with equivalent biological activity. The following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Asphaltate (+3.0±1); glutamate (+3.0±1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); threonine (-0.4); Proline (-0.5±1); Alanine (-0.5); histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); leucine (-1.8); isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).
친수성 값을 참조하여 변이를 도입시키는 경우, 바람직하게는 ±2 이내, 보다 바람직하게는 ±1 이내, 보다 더 바람직하게는 ±0.5 이내의 친수성 값 차이를 나타내는 아미노산 사이에 치환을 한다.When introducing a mutation with reference to the hydrophilicity value, substitution is made between amino acids showing a difference in hydrophilicity value, preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.
분자의 활성을 전체적으로 변경시키지 않는 펩타이드에서의 아미노산 교환은 당해 분야에 공지되어 있다. 가장 통상적으로 일어나는 교환은 아미노산 잔기 Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly 간의 교환이다.Amino acid exchanges in peptides that do not overall alter the activity of the molecule are known in the art. The most common exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
상술한 생물학적 균등 활성을 갖는 변이를 고려한다면, 본 발명의 서열번호 1 내지 9로 표시되는 아미노산 서열은 서열목록에 기재된 서열과 실질적인 동일성(substantial identity)을 나타내는 서열도 포함하는 것으로 해석된다. 상기의 실질적인 동일성은, 본 발명의 서열번호 1 내지 9로 표시되는 아미노산 서열과 임의의 다른 서열을 최대한 대응되도록 얼라인하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인된 서열을 분석한 경우에, 최소 80% 이상의 상동성, 보다 바람직하게는 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 또는 99% 이상의 상동성을 나타내는 서열을 의미한다. 서열 비교를 위한 얼라인먼트 방법은 당업계에 공지되어 있는 어떤 방법이라도 제한없이 사용할 수 있다. Considering the mutations having the above-mentioned biological equivalent activity, the amino acid sequences represented by SEQ ID NOs: 1 to 9 of the present invention are interpreted to also include sequences showing substantial identity with the sequences described in the sequence listing. The above substantial identity is obtained by aligning the amino acid sequences represented by SEQ ID NOs. 1 to 9 of the present invention and any other sequences to correspond as much as possible, and analyzing the aligned sequences using an algorithm commonly used in the art. In this case, sequences showing at least 80% or more homology, more preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more. it means. As an alignment method for sequence comparison, any method known in the art can be used without limitation.
본 발명에 있어, 상기 항원 결합 단편은 Fab, Fab', (Fab')2, Fv, scFv (단일 사슬의 sc), 비스-scFv, scFv-Fc 단편, Fab2, Fab3, 미니체, 다이아체, 트리아제, 테트라체 및 나노체로 이루어진 군으로부터 선택되는 것일 수 있다. 상기 단편은 본 발명에 따른 항체의 특징적인 CDR 중 적어도 하나를 갖는다. 항체 단편 중에서, Fab는 경쇄 및 중쇄의 가변 영역, 경쇄의 불변영역 및 중쇄의 첫 번째 불변 영역을 포함하는 구조를 갖고, 하나의 항원 결합 부위를 갖는다. Fab'는 중쇄 CH1 도메인의 C-말단에 하나 이상의 시스테인 잔기를 포함하는 힌지를 갖는 점에서 Fab와 다르다. F(ab)' 항체는 Fab'의 힌지 영역의 시스테인 잔기가 디설파이드 결합을 형성하기 때문에 생성된다. Fv는 중쇄 가변 영역 및 경쇄 가변 영역만을 갖는 최소의 항체 단편이다. 이중 사슬 Fv (dsFv)에서 중쇄 가변 영역 및 경쇄 가변 영역은 디설파이드 결합을 통해 서로 결합되고, 단일 사슬 Fv (ssFv)에서 중쇄 가변 영역 및 경쇄 가변 영역은 일반적으로 펩티드 링커를 통해 서로 공유 결합된다. 이들 항체 단편은 프로테이나제를 사용하여 획득될 수 있고 (예로, Fab는 파파인을 사용한 전체 항체의 제한 소화에 의해 획득될 수 있고, F(ab)' 단편은 펩신을 사용한 제한 소화에 의해 획득될 수 있음), 바람직하게 이것은 유전공학기법에 의해 생산될 수 있다. 바람직하게, 상기 "항원-결합 단편"은 이들이 유래한 항체의 중쇄 또는 경쇄 가변 사슬의 부분적 서열로 구성될 것이거나 이를 포함할 것이고, 상기 부분적 서열은 표적과 관련하여 이것이 유래한 항체와 동일한 결합 특이성 및 충분한 친화도, 바람직하게 이것이 유래한 항체의 친화도의 적어도 동등 내지 1/100, 더욱 바람직한 방식으로 적어도 1/10까지의 친화도를 보유하는 것으로 충분하다. In the present invention, the antigen-binding fragment is Fab, Fab', (Fab') 2 , Fv, scFv (single chain sc), bis-scFv, scFv-Fc fragment, Fab 2 , Fab 3 , minibody, dia. It may be selected from the group consisting of sieve, triase, tetrasieve and nanosieve. Said fragment has at least one of the characteristic CDRs of the antibody according to the invention. Among antibody fragments, Fab has a structure comprising the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region of the heavy chain, and has one antigen binding site. Fab' differs from Fab in having a hinge containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. F(ab)' antibodies are produced because cysteine residues in the hinge region of Fab' form disulfide bonds. Fv is the smallest antibody fragment with only the heavy and light chain variable regions. In a double-chain Fv (dsFv), the heavy chain variable region and light chain variable region are linked to each other through disulfide bonds, while in single-chain Fv (ssFv) the heavy chain variable region and light chain variable region are usually covalently linked to each other through a peptide linker. These antibody fragments can be obtained using proteinases (e.g., Fab can be obtained by restriction digestion of the whole antibody with papain, and F(ab)' fragments can be obtained by restriction digestion with pepsin. can be), preferably it can be produced by genetic engineering techniques. Preferably, the "antigen-binding fragment" will consist of or comprise a partial sequence of the heavy or light chain variable chain of the antibody from which it is derived, said partial sequence having the same binding specificity with respect to the target as the antibody from which it is derived. and sufficient affinity, preferably at least equal to 1/100, more preferably at least 1/10, of the affinity of the antibody from which it is derived.
[규칙 제91조에 의한 정정 01.12.2023]
본 발명의 전체(whole) 인간화된 항-VSIG4 항체는 IgA, IgD, IgE, IgM 및　IgG의 하위유형 또는 변이체를 포함할 수 있고, 특히,　IgG1을 포함할 수 있다. 중쇄 불변 영역은 감마(γ), 뮤(μ), 알파(α), 델타(δ) 및 엡실론(ε) 유형 및 하위 부류 감마 1(γ1), 감마 2(γ2), 감마 3(γ3), 감마 4(γ4), 알파1(α1) 및 알파2(α2)를 갖는다. 경쇄의 불변 영역은 카파(κ) 및 람다(λ) 유형을 포함할 수 있다. 본 발명의 인간화된 항-VSIG4 항체는 인간 IgG1 항체의 Fc 서열을 포함하는 것일 수 있다.[Correction 01.12.2023 pursuant to Rule 91]
The whole humanized anti-VSIG4 antibody of the invention may include subtypes or variants of IgA, IgD, IgE, IgM and IgG, and may particularly include IgG1. Heavy chain constant regions are of gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types and subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3); It has gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain may include the kappa (κ) and lambda (λ) types. The humanized anti-VSIG4 antibody of the present invention may include the Fc sequence of a human IgG1 antibody.
[규칙 제91조에 의한 정정 01.12.2023]
본 발명의 인간화된 항-VSIG4 항체는 항체 기능을 조절하는 Fc 영역이 개선된 항체일 수 있으며, IgG1의 Fc 항체 의존성 세포독성 (Antibody dependent cellular cytotoxicity, ADCC) 및 보체 의존성 세포 독성(Complement dependent cytotoxicity, CDC)이 억제된 것을 특징으로 할 수 있다. 따라서 본 발명의 인간화된 항-VSIG4 항체는 서열번호 9로 표시되는 조작된 IgG1 Fc 영역을 포함하는 것을 특징으로 하며, 이와 적어도 약 80%, 85%, 90%, 95%, 96%, 97%, 98%, 또는 99% 이상의 서열 동일성을 갖는 서열을 포함할 수 있다. 여기서 상기 변이체는 부가, 결실, 및/또는 치환을 포함한다.[Correction 01.12.2023 pursuant to Rule 91]
The humanized anti-VSIG4 antibody of the present invention may be an antibody with an improved Fc region that regulates antibody function, and may exhibit IgG1 Fc antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (ADCC). CDC) can be characterized as being suppressed. Accordingly, the humanized anti-VSIG4 antibody of the present invention is characterized by comprising an engineered IgG1 Fc region represented by SEQ ID NO: 9, and is at least about 80%, 85%, 90%, 95%, 96%, 97% thereof. , 98%, or 99% or more sequence identity may be included. wherein the variants include additions, deletions, and/or substitutions.
[규칙 제91조에 의한 정정 01.12.2023]
상기 본 발명의 인간화된 항-VSIG4 항체는 쿠퍼 세포에 독성을 나타내지 않고, FcγR 및 C1q에 대한 결합능이 제거되어 매우 낮은 항체 의존성 세포독성 및 보체 의존성 세포 독성을 나타냄과 동시에, VSIG4에 의해 억제된 T 세포 증식을 촉진시키고, 종양 관련 대식세포(TAM)를 종양을 억제하는 염증성 대식세포로 전환하는 효과를 나타낼 수 있다. 그 결과 종양 미세환경이 개선될 수 있으며, 우수한 항암 효과를 달성한다. 바람직하게는 본 발명의 인간화된 항-VSIG4 항체가 비침투성(noninflamed) cold tumor를 hot tumor로 전환하고, 비정상적인 종양 미세 환경을 제거하여, 면역 항암제의 치료 반응을 효과적으로 향상시킬 수 있다.[Correction 01.12.2023 pursuant to Rule 91]
The humanized anti-VSIG4 antibody of the present invention does not exhibit toxicity to Kupffer cells, and exhibits very low antibody-dependent cytotoxicity and complement-dependent cytotoxicity due to the removal of the binding ability to FcγR and C1q, and at the same time, T It can promote cell proliferation and convert tumor-associated macrophages (TAMs) into inflammatory macrophages that suppress tumors. As a result, the tumor microenvironment can be improved and excellent anticancer effects are achieved. Preferably, the humanized anti-VSIG4 antibody of the present invention can convert a noninflamed cold tumor into a hot tumor and remove the abnormal tumor microenvironment, effectively improving the treatment response to anticancer immunotherapy agents.
따라서 본 발명은 항 PD-L1 항체를 더 포함하는 암 예방 또는 치료용 조성물을 제공한다. Therefore, the present invention provides a composition for preventing or treating cancer further comprising an anti-PD-L1 antibody.
[규칙 제91조에 의한 정정 01.12.2023]
본 발명의 인간화된 항-VSIG4 항체 또는 이의 결합 단편; 및 항 PD-L1 항체를 포함하는 암 예방 또는 약학적 조성물, 또는 암 예방 또는 치료용 병용투여 제제는 항-VSIG4 항체 또는 이의 결합 단편이나 항 PD-L1 항체 단독 투여에 비하여 현저히 증가한 항암 효과를 나타낼 수 있다.[Correction 01.12.2023 pursuant to Rule 91]
A humanized anti-VSIG4 antibody or binding fragment thereof of the invention; And a cancer prevention or pharmaceutical composition containing an anti-PD-L1 antibody, or a combined administration agent for cancer prevention or treatment exhibits a significantly increased anticancer effect compared to the administration of an anti-VSIG4 antibody or a binding fragment thereof or an anti-PD-L1 antibody alone. You can.
[규칙 제91조에 의한 정정 01.12.2023]
병용투여법(combination therapy) 이란 대상체가 두 가지 또는 그 이상의 치료요법적 요법 (이를 테면, 두 가지 또는 그 이상의 치료요법적 제제)에 동시에 노출되는 상황을 지칭한다. 일부 구체예들에서, 인간화된 항-VSIG4 항체 또는 이의 결합 단편; 및 항 PD-L1 항체는 동시에 투여될 수 있다. 일부 구체예들에서, 상기 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편; 및 항 PD-L1 항체는 순차적으로 투여될 수 있다.[Correction 01.12.2023 pursuant to Rule 91]
Combination therapy refers to a situation in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, a humanized anti-VSIG4 antibody or binding fragment thereof; and anti-PD-L1 antibody can be administered simultaneously. In some embodiments, the humanized anti-VSIG4 antibody or antigen-binding fragment thereof; and anti-PD-L1 antibody may be administered sequentially.
[규칙 제91조에 의한 정정 01.12.2023]
본 발명에 있어, 인간화된 항-VSIG4 항체 또는 이의 결합 단편과 동시에 또는 순차적으로 병용 투여할 수 있는 항 PD-L1 항체는 당 분야에 알려진 항체를 제한없이 포함할 수 있고, 이의 항원 결합 단편을 포함할 수 있으나, 바람직하게는 아테졸리주맙, 더발루맙 또는 아벨루맙일 수 있고, 본 발명의 바람직한 일 구현예에서는 아테졸리주맙 (티센트릭)을 이용하여 그 효과를 확인하였다.[Correction 01.12.2023 pursuant to Rule 91]
In the present invention, the anti-PD-L1 antibody that can be administered in combination with the humanized anti-VSIG4 antibody or binding fragment thereof simultaneously or sequentially may include, without limitation, antibodies known in the art, and includes antigen-binding fragments thereof. However, it may preferably be atezolizumab, durvalumab, or avelumab, and in a preferred embodiment of the present invention, atezolizumab (Tecentriq) was used to confirm the effect.
[규칙 제91조에 의한 정정 01.12.2023]
본 발명의 암은 종양, 신생물(neoplasm), 및 암종(carcinoma) 과 상호 교환적으로 사용할 수 있으며, 인간화된 항-VSIG4 항체 또는 이의 결합 단편; 또는 인간화된 항-VSIG4 항체 또는 이의 결합 단편과 항 PD-L1 항체에 의하여 항암 효과를 나타낼 수 있는 암 종을 제한없이 포함할 수 있다. 본 발명의 암은 바람직하게는 VSIG4 과발현 암일 수 있고, 예컨대 상기 암은 다발성 골수종, 난소암, 폐암, 비소세포 폐암, 신경교종, 유방암, 자궁경부암, 결장암, 자궁내막암, 식도암, 나팔관 암, 담낭암, 위암, 두경부암, 혈액 암, 후두암, 간암, 림프종, 흑색종, 중피종, 원발성 복막암, 침샘암, 육종, 갑상선암, 췌장암, 신장 세포 암종, 교모세포종, 및 전립선암으로 이루어진 군에서 선택된 1종 이상일 수 있다.[Correction 01.12.2023 pursuant to Rule 91]
Cancer of the present invention can be used interchangeably with tumor, neoplasm, and carcinoma, and includes a humanized anti-VSIG4 antibody or binding fragment thereof; Alternatively, it may include, without limitation, cancer species that can exhibit anti-cancer effects by humanized anti-VSIG4 antibody or binding fragment thereof and anti-PD-L1 antibody. The cancer of the present invention may preferably be a VSIG4-overexpressing cancer, for example, multiple myeloma, ovarian cancer, lung cancer, non-small cell lung cancer, glioma, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer. , stomach cancer, head and neck cancer, blood cancer, laryngeal cancer, liver cancer, lymphoma, melanoma, mesothelioma, primary peritoneal cancer, salivary gland cancer, sarcoma, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer. It could be more than that.
[규칙 제91조에 의한 정정 01.12.2023]
본 발명의 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편과 항 PD-L1 항체를 병용 투여하는 경우, 이들은 1: 1 내지 5 (w/w)% 로 조성물에 포함되거나 투여될 수 있고, 바람직하게는 1: 1 내지 3 (w/w)%, 또는 1:1(w/w)% 비율로 포함하거나 투여될 수 있다.[Correction 01.12.2023 pursuant to Rule 91]
When the humanized anti-VSIG4 antibody or antigen-binding fragment thereof of the present invention is administered in combination with the anti-PD-L1 antibody, they may be included or administered in the composition at 1:1 to 5 (w/w)%, preferably Can be contained or administered in a ratio of 1:1 to 3 (w/w)%, or 1:1 (w/w)%.
[규칙 제91조에 의한 정정 01.12.2023]
또한 본 발명은 a) 서열번호 1의 아미노산 서열로 표시되는 중쇄 CDR1, 서열번호 2의 아미노산 서열로 표시되는 중쇄 CDR2, 서열번호 3의 아미노산 서열로 표시되는 중쇄 CDR3; 및 서열번호 4의 아미노산 서열로 표시되는 경쇄 CDR1, 서열번호 5의 아미노산 서열로 표시되는 경쇄 CDR2, 서열번호 6의 아미노산 서열로 표시되는 경쇄 CDR3; 을 포함하는 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편; 및 b) 항 PD-L1 항체를 개체에 투여하는 단계를 포함하는, 암 예방 또는 치료 방법을 제공한다.[Correction 01.12.2023 pursuant to Rule 91]
In addition, the present invention provides a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; A humanized anti-VSIG4 antibody or antigen-binding fragment thereof comprising; and b) administering an anti-PD-L1 antibody to a subject.
본 발명에 있어, 투여란 조성물 안에 있는, 또는 내포된 제제를 전달하기 위해 개체 또는 시스템으로 조성물을 투여하는 것을 일반적으로 지칭한다. 당업자는 적절한 환경에서 개체, 예를 들어, 인간에게 투여하기 위해 사용될 수 있는 다양한 경로를 알고 있을 것이다. 예를 들면, 일부 구체예들에서, 투여는 안구, 구강, 장관외, 국소 투여 등이 될 수 있다. 일부 특정 구체예들에서, 투여는 기관지 (예를 들어, 기관지 점적을 통한), 볼협측, 진피 (예를 들어, 진피, 피내, 피내, 경피 국소등 중 하나 또는 이상일 수 있거나 또는 이를 포함할 수 있음), 장으로(enteral), 동맥 내, 피내, 위 내, 골수 내, 근육 내, 비강 내, 복강 내, 척수강 내, 정맥 내, 심실내, 특정 기관 (예를 들면, 간 내), 점막, 비강, 구강, 직장, 피하, 설하, 국소, 기관 (예를 들면, 기관 내 점적을 통해), 질, 유리체 등을 통한 투여가 될 수 있다. 일부 구체예들에서, 투여는 오로지 단일 투여량(dose)만으로 관련될 수 있다. 일부 구체예들에서, 투여는 고정된 횟수의 투여량을 적용하는 것과 관련될 수 있다. 일부 구체예들에서, 투여는 간헐적인 (예를 들어, 시간을 두고 복수의 투여량) 투여 및/또는 주기적 (예를 들어, 일반적인 시간에 의해 분리된 개별 투여량) 투약을 포함할 수 있다. 일부 구체예들에서, 투여는 최소한 선택된 기간동안 연속 투여량(가령, 주입)이 관여할 수 있다.In the present invention, administration generally refers to administering a composition to an entity or system to deliver the agent contained in or contained within the composition. Those skilled in the art will be aware of the various routes that can be used for administration to a subject, such as a human, in appropriate circumstances. For example, in some embodiments, administration may be ocular, buccal, parenteral, topical, etc. In some specific embodiments, administration may be or include one or more of the following: bronchial (e.g., via bronchial instillation), buccal, dermal (e.g., dermal, intradermal, intradermal, transdermal, etc. enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, certain organs (e.g., intrahepatic), mucous membranes , nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., via intratracheal instillation), vaginal, vitreous, etc. administration. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve applying a fixed number of doses. In some embodiments, administration may include intermittent (e.g., multiple doses spaced over time) administration and/or periodic (e.g., individual doses separated by a regular period of time) dosing. In some embodiments, administration may involve continuous doses (e.g., infusion) for at least a selected period of time.
본 발명에 따른 약학적 조성물은 약제학적으로 허용가능한 담체를 포함할 수 있다. 약제학적으로 허용되는 담체는 경구투여시에는 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소, 향료 등을 사용할 수 있으며, 주사제의 경우에는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제 등을 혼합하여 사용할 수 있으며, 국소투여용의 경우에는 기제, 부형제, 윤활제, 보존제 등을 사용할 수 있다. 본 발명에 따른 약학적 조성물의 제형은 상술한 바와 같은 약제학적으로 허용되는 담체와 혼합하여 다양하게 제조될 수 있다. 예를 들어, 경구투여 시에는 정제, 트로키, 캡슐, 엘릭서(elixir), 서스펜션, 시럽, 웨이퍼 등의 형태로 제조할 수 있으며, 주사제의 경우에는 단위 투약 앰플 또는 다수회 투약 형태로 제조할 수 있다. The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc. for oral administration. For injections, buffers, preservatives, and analgesics can be used. Topics, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives, etc. can be used. The dosage form of the pharmaceutical composition according to the present invention can be prepared in various ways by mixing it with a pharmaceutically acceptable carrier as described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be manufactured in the form of unit dosage ampoules or multiple dosage forms. there is.
한편, 제제화에 적합한 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말디톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 또는 광물유 등이 사용될 수 있다. 또한, 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등을 추가로 포함할 수 있다.Meanwhile, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil may be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.
본 발명에 따른 약학적 조성물은 사용된 특정 유효성분의 활성, 연령, 체중, 일반적인 건강, 성별, 정식, 투여시간, 투여경로, 배출율, 약물 배합 및 예방 또는 치료될 특정 질환의 중증을 포함한 여러 요인에 따라 다양하게 변할 수 있고, 상기 약학적 조성물의 투여량은 환자의 상태, 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만 당업자에 의해 적절하게 선택될 수 있다. 바람직하게는 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여할 수 있으며, 더욱 바람직하게는 1~10000㎍/체중kg/day, 더욱더 바람직하게는 10~1000㎎/체중kg/day의 유효용량으로 하루에 수회 반복 투여될 수 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The pharmaceutical composition according to the present invention is influenced by various factors, including the activity of the specific active ingredient used, age, body weight, general health, gender, diet, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated. The dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route and period, but may be appropriately selected by a person skilled in the art. Preferably, considering all of the above factors, the amount that can obtain the maximum effect with the minimum amount without side effects can be administered, more preferably 1 to 10,000 ㎍/kg of body weight/day, and even more preferably 10 to 1,000 mg. The effective dose is /kg body weight/day and can be administered repeatedly several times a day. The above dosage does not limit the scope of the present invention in any way.
상기 개체는 유기체, 전형적으로 포유류를 포함할 수 있으며, 바람직하게는 인간일 수 있다. 상기 개체는 관련 질환, 증상 또는 장애를 앓거나 알고 있는 개체일 수 있다. The subject may include an organism, typically a mammal, and preferably a human. The individual may be an individual suffering from or known to have a related disease, symptom, or disorder.
[규칙 제91조에 의한 정정 01.12.2023]
또한 본 발명은 a) 서열번호 1의 아미노산 서열로 표시되는 중쇄 CDR1, 서열번호 2의 아미노산 서열로 표시되는 중쇄 CDR2, 서열번호 3의 아미노산 서열로 표시되는 중쇄 CDR3; 및 서열번호 4의 아미노산 서열로 표시되는 경쇄 CDR1, 서열번호 5의 아미노산 서열로 표시되는 경쇄 CDR2, 서열번호 6의 아미노산 서열로 표시되는 경쇄 CDR3; 을 포함하는 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편을 포함하는 1구획; 및 b) 항 PD-L1 항체를 포함하는 2구획을 포함하는, 암 예방 또는 치료용 키트를 제공한다.[Correction 01.12.2023 pursuant to Rule 91]
In addition, the present invention provides a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; One compartment containing a humanized anti-VSIG4 antibody or antigen-binding fragment thereof; and b) a two-compartment containing an anti-PD-L1 antibody.
[규칙 제91조에 의한 정정 01.12.2023]
본 발명의 키트에 포함된 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편 및 항 PD-L1 항체는 치료 목적에 따라 동시에 또는 순차적으로 투여될 수 있다.[Correction 01.12.2023 pursuant to Rule 91]
The humanized anti-VSIG4 antibody or antigen-binding fragment thereof and anti-PD-L1 antibody included in the kit of the present invention may be administered simultaneously or sequentially depending on the purpose of treatment.
[규칙 제91조에 의한 정정 01.12.2023]
상기 키트는 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편 및 항 PD-L1 항체를 사용하기 위한 지침서를 포함하는 패키지 삽입물을 추가로 포함할 수 있으며, 상기 성분들은 동일한 용기 또는 별도의 용기 내에 존재할 수 있다. 적합한 용기는, 예를 들어, 병, 바이알, 백 및 주사기를 포함한다. 용기는 다양한 재료, 가령, 유리, 플라스틱 (예를 들어, 폴리비닐 클로라이드 또는 폴리올레핀) 또는 금속 합금 (예를 들어, 스테인리스 강 또는 하스텔로이)으로 형성될 수 있다. 일부 예들에서, 용기는 제제를 보유하며 용기 위의 라벨 또는 용기와 관련된 라벨은 사용지침을 표시할 수 있다. 상기　키트는 다른 완충제, 희석제, 필터, 바늘, 주사기 및 사용 지침서가 있는 약품 지침서를 비롯하여, 상업적 및 사용자 관점에서 필요한 다른 물질을 추가로 포함할 수 있다. 일부 예들에서, 제조 물품은 하나 이상의 또 다른 제제(예를 들어, 추가 화학요법제, 또는 항-종양제)를 추가로 포함한다. 한 가지 또는 그 이상의 작용제에 대한 적합한 용기는, 예를 들어, 병, 바이알, 백 및 주사기를 포함한다.[Correction 01.12.2023 pursuant to Rule 91]
The kit may further include a package insert containing instructions for using the humanized anti-VSIG4 antibody or antigen-binding fragment thereof and the anti-PD-L1 antibody, and the components may be present in the same container or in separate containers. there is. Suitable containers include, for example, bottles, vials, bags, and syringes. The container may be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or metal alloy (e.g., stainless steel or hastelloy). In some examples, the container holds the formulation and a label on or associated with the container may indicate directions for use. The kit may additionally contain other materials required from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and drug product instructions with instructions for use. In some examples, the article of manufacture further comprises one or more other agents (e.g., additional chemotherapeutic agents, or anti-neoplastic agents). Suitable containers for one or more agents include, for example, bottles, vials, bags, and syringes.
[규칙 제91조에 의한 정정 01.12.2023]
한편 본 발명의 또다른 목적은 서열번호 1의 아미노산 서열로 표시되는 중쇄 CDR1, 서열번호 2의 아미노산 서열로 표시되는 중쇄 CDR2, 서열 번호 3의 아미노산 서열로 표시되는 중쇄 CDR3; 및 서열번호 4의 아미노산 서열로 표시되는 경쇄 CDR1, 서열번호 5의 아미노산 서열로 표시되는 경쇄 CDR2, 서열번호 6의 아미노산 서열로 표시되는 경쇄 CDR3; 을 포함하는 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편을 포함하는, 항 PD-L1 항체 효능 증진용 항암 보조 치료제를 제공하는 것이다.[Correction 01.12.2023 pursuant to Rule 91]
Meanwhile, another object of the present invention is a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; To provide an adjuvant anti-cancer treatment for enhancing anti-PD-L1 antibody efficacy, comprising a humanized anti-VSIG4 antibody or an antigen-binding fragment thereof.
[규칙 제91조에 의한 정정 01.12.2023]
본 발명에서는 인간화된 항-VSIG4 항체 또는 이의 결합 단편이 종양 미세 환경을 면역 치료법에 대한 순응도를 높일 수 있는 환경으로 전환할 수 있음을 확인하였고, 그 결과 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편과 항 PD-L1 항체를 동시에 투여하는 경우, 항 PD-L1 항체 단독 투여 대비 항암 효과를 증진시킬 수 있음을 확인하였다. 따라서, 본 발명의 인간화된 항-VSIG4 항체 또는 이의 결합 단편은 항 PD-L1 항체 효능을 증진시키는 보조용법으로 사용될 수 있다.[Correction 01.12.2023 pursuant to Rule 91]
In the present invention, it was confirmed that the humanized anti-VSIG4 antibody or antigen-binding fragment thereof can transform the tumor microenvironment into an environment that can increase compliance with immunotherapy, and as a result, the humanized anti-VSIG4 antibody or antigen-binding fragment thereof It was confirmed that simultaneous administration of anti-PD-L1 antibody and anti-PD-L1 antibody can enhance the anticancer effect compared to administration of anti-PD-L1 antibody alone. Therefore, the humanized anti-VSIG4 antibody or binding fragment thereof of the present invention can be used as an auxiliary method to enhance the efficacy of the anti-PD-L1 antibody.
[규칙 제91조에 의한 정정 01.12.2023]
또한 본 발명은 서열번호 1의 아미노산 서열로 표시되는 중쇄 CDR1, 서열번호 2의 아미노산 서열로 표시되는 중쇄 CDR2, 서열번호 3의 아미노산 서열로 표시되는 중쇄 CDR3; 및 서열번호 4의 아미노산 서열로 표시되는 경쇄 CDR1, 서열번호 5의 아미노산 서열로 표시되는 경쇄 CDR2, 서열번호 6의 아미노산 서열로 표시되는 경쇄 CDR3; 을 포함하는 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편을 개체에 투여하는 단계를 포함하는, 항 PD-L1 항체 효능 증진 방법에 관한 것이다.[Correction 01.12.2023 pursuant to Rule 91]
In addition, the present invention provides a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; It relates to a method for enhancing the efficacy of an anti-PD-L1 antibody, comprising administering to a subject a humanized anti-VSIG4 antibody or antigen-binding fragment thereof.
상기 방법은 in vivo, in vitro, ex vivo에서 수행될 수 있다. The method can be performed in vivo, in vitro, or ex vivo .
[규칙 제91조에 의한 정정 01.12.2023]
본 발명의 항암 보조치료제 및 항 PD-L1 항체 효능 증진 방법에 있어서, 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편 및 항 PD-L1 항체에 관한 설명은 앞서 설명한 바와 같다.[Correction 01.12.2023 pursuant to Rule 91]
In the anti-cancer adjuvant treatment and method for enhancing anti-PD-L1 antibody efficacy of the present invention, the description of the humanized anti-VSIG4 antibody or antigen-binding fragment thereof and anti-PD-L1 antibody is the same as previously described.
상술한 본 발명의 내용은 상호 모순되지 않는 한, 서로 동일하게 적용되며, 당해 기술분야의 통상의 기술자가 적절한 변경을 가해 실시하는 것 또한 본 발명의 범주에 포함된다.The contents of the present invention described above are applied equally to each other unless they contradict each other, and implementation by a person skilled in the art with appropriate changes is also included in the scope of the present invention.
이하 본 발명을 실시예를 통해 상세하게 설명하나 본 발명의 범위가 하기 실시예로만 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail through examples, but the scope of the present invention is not limited to the following examples.
실험예. Experiment example. In vitroIn vitro 또는 or in vivoin vivo conversion 실험 디자인 conversion experiment design
마우스 항-VSIG4 항체 mu6H8로부터 인간화하여 EU103.2 항체를 제조하였다. VH의 인간화된 변이체에 대한 프레임워크는 마우스 6H8 항체 및 Blast (https:/blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) (germline gene: VH2-5/D3-3/JH6c)를 이용하여 만들어졌다. Kabat 번호매김을 이용하여 CDRs을 분류하고, 인간화된 VH는 해당 프레임워크로 기획되었으며, mu6H8 VH CDRs, VH2, VH27, VH30, VH93, 그리고 VH94의 역-돌연변이로 분류되었다(hu6H8.3 VH). The EU103.2 antibody was prepared by humanization from the mouse anti-VSIG4 antibody mu6H8. The framework for humanized variants of VH is the mouse 6H8 antibody and Blast (https:/blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) (germline gene: VH2-5/D3-3/JH6c). ) was created using. CDRs were classified using Kabat numbering, and humanized VHs were projected with the corresponding framework and classified as back-mutants of mu6H8 VH CDRs, VH2, VH27, VH30, VH93, and VH94 (hu6H8.3 VH).
VL의 인간화된 변이체에 대한 프레임워크는 마우스 6H8 항체 및 Blast (https:/blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) (germline gene: A17/JK2)를 이용하여 만들어졌다. Kabat 번호매김을 이용하여 CDRs을 분류하고, 인간화된 VL는 해당 프레임워크로 기획되었으며, mu6H8 VL CDRs, VH2, VH4, VH36, 그리고 VH46의 역-돌연변이로 분류되었다(hu6H8.3 VL).The framework for the humanized variant of VL was created using mouse 6H8 antibody and Blast (https:/blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) (germline gene: A17/JK2) . CDRs were classified using Kabat numbering, and humanized VLs were projected with the corresponding framework and classified as back-mutants of the mu6H8 VL CDRs, VH2, VH4, VH36, and VH46 (hu6H8.3 VL).
이후 EU103.3 항체의 친화력 성숙 실험을 통해 인간화된 항-VSIG4 항체의 클론을 제조하였으며, CDR 서열, 중쇄 및 경쇄 서열을 하기 표 1 및 2 에 나타내었다. 이하, 표 1 및 2의 서열을 포함하는 본 발명의 항체를 “EU103”으로 지칭하였다. Afterwards, a clone of the humanized anti-VSIG4 antibody was prepared through an affinity maturation experiment of the EU103.3 antibody, and the CDR sequence, heavy chain, and light chain sequences are shown in Tables 1 and 2 below. Hereinafter, the antibody of the present invention containing the sequences in Tables 1 and 2 was referred to as “EU103”.
VHCDR1 VHCDR1 VHCDR2VHCDR2 VHCDR3VHCDR3 VLCDR1VLCDR1 VLCDR2VLCDR2 VLCDR3VLCDR3
GISLTT(서열번호 1)GISLTT (SEQ ID NO: 1) IFWDDNK
(서열번호 2)IFWDDNK
(SEQ ID NO: 2) 		VRVYYKNDGYFDV
(서열번호 3)VRVYYKNDGYFDV
(SEQ ID NO: 3) 		KSVTTS
(서열번호 4)KSVTTS
(SEQ ID NO: 4) 		LAS
(서열번호 5)LAS
(SEQ ID NO: 5) 		QQSGELPYT
(서열번호 6)QQSGELPYT
(SEQ ID NO: 6)
중쇄heavy chain QVTLKESGPTLVKPTQTLTLTCTFSGISLTTSGMGVGWIRQPPGKALEWLADIFWDDNKYYNPSLKSRLTITKDTSKNQVVLTM TNMDPVDTATYYCVRVYYKNDGYFDVWGKGTTVTVSS (서열번호 7) QVTLKESGPTLVKPTQTLTLTCTFSGISLTSGMGVGWIRQPPGKALEWLADIFWDDNKYYNPSLKSRLTITKDTSKNQVVLTM TNMDPVDTATYYCVRVYYKNDGYFDVWGKGTTVTVSS (SEQ ID NO: 7)
경쇄light chain DIVLTQSPLSLPVTLGQPASISCRASKSVTTSGYSFMHWYQQRPGQSPRLLIYLASNLEPGVPDRFSGSGSGTDFTLKIFRVEA EDVGVYYCQQSGELPYTFGQGTKLEIK (서열번호 8) DIVLTQSPLSLPVTLGQPASISCRASKSVTTSGYSFMHWYQQRPGQSPRLLIYLASNLEPGVPDRFSGSGSGTDFTLKIFRVEA EDVGVYYCQQSGELPYTFGQGTKLEIK (SEQ ID NO: 8)
EU103이 다른 자극제 없이 M2 또는 TAM (Tumor-associated macrophage)을 M1으로 전환시킬 수 있는지 확인하기 위하여, M2에서 M1로의 전환에 대한 in vitro 또는 in vivo 분석을 수행하였다. To determine whether EU103 can convert M2 or TAM (Tumor-associated macrophage) into M1 without other stimulants, in vitro or in vivo analysis of the conversion from M2 to M1 was performed.
EU103에 의한 대식세포 전환 효과를 평가하기 위해 세 가지 실험 방법을 사용하였다. 먼저, 건강한 공여자 PBMC로부터 분리된 단핵구를 M-CSF로 처리하여 분화시킨 다음, IL-4/IL-13으로 처리하여 M2 대식세포로 분화시켰다. EU103의 전환 효과를 평가하기 위해 배양 배지 변경 후 EU103을 처리하고, 전환 효과를 확인하였다. Three experimental methods were used to evaluate the effect of macrophage transformation by EU103. First, monocytes isolated from healthy donor PBMC were treated with M-CSF to differentiate, and then treated with IL-4/IL-13 to differentiate into M2 macrophages. To evaluate the conversion effect of EU103, EU103 was treated after changing the culture medium, and the conversion effect was confirmed.
둘째, 난소암 환자의 복수에서 TAM을 분리하여 EU103을 처리한 후, EU103 처리에 의하여 TAM 이 염증성 대식세포로 전환되고 이를 통해 종양 미세환경을 개선할 수 있는지 여부를 확인하였다. Second, TAMs were isolated from the ascites of an ovarian cancer patient and treated with EU103, and then it was confirmed whether TAMs could be converted into inflammatory macrophages by EU103 treatment and thereby improve the tumor microenvironment.
셋째, 생체 내에서 EU103의 전환 효과를 평가하기 위해 첫번째 방법의 M2 대식세포에 종양세포를 주입하고 EU103을 처리한 후 그 효과를 확인하였다. Third, to evaluate the conversion effect of EU103 in vivo, tumor cells were injected into M2 macrophages in the first method, treated with EU103, and the effect was confirmed.
본 발명에서 수행된 전환 분석 방법의 개략도를 도 1에 나타내었다. A schematic diagram of the conversion analysis method performed in the present invention is shown in Figure 1.
실시예 1. VSIG4에 대한 EU103의 결합 활성 확인 Example 1. Confirmation of EU103 binding activity to VSIG4
FACS 분석을 이용하여 VSIG4를 과발현하는 HeLa-hVSIG4 세포주와 HeLa 세포주를 이용하여 본 발명 EU103의 결합 활성을 확인하였다. Using FACS analysis, the binding activity of EU103 of the present invention was confirmed using the HeLa-hVSIG4 cell line and the HeLa cell line overexpressing VSIG4.
도 2에 나타낸 바와 같이 HeLa 세포에서는 VSIG4의 발현과 EU103의 상호작용이 관찰되지 않았다. 반면, VSIG4를 과발현하도록 설계된 HeLa-hVSIG4 세포주에서는 VSIG4의 발현과 EU103의 상호작용이 용량 의존적 방식으로 관찰되었다. 이러한 결과를 토대로 EU103이 VSIG4 에 효과적으로 결합할 수 있음을 확인하였다. As shown in Figure 2, no interaction between VSIG4 expression and EU103 was observed in HeLa cells. On the other hand, in the HeLa-hVSIG4 cell line designed to overexpress VSIG4, the interaction of VSIG4 expression and EU103 was observed in a dose-dependent manner. Based on these results, it was confirmed that EU103 can effectively bind to VSIG4.
[규칙 제91조에 의한 정정 01.12.2023]
실시예 2. M2 대식세포의 VSIG4 발현 및 EU103 결합 활성 확인 [Correction 01.12.2023 pursuant to Rule 91]
Example 2. Confirmation of VSIG4 expression and EU103 binding activity of M2 macrophages
[규칙 제91조에 의한 정정 01.12.2023]
FACS 분석을 이용하여 항-염증성 대식세포에서 VSIG4의 발현을 확인하고 이 항-염증성 대식세포에서 EU103의 결합 활성을 확인하였다. 건강한 공여자의 말초 혈액 단핵세포(PBMC)에서 CD14+ 단핵구를 분리하여 7일간 재조합 인간 M-CSF (Recombinant human M-CSF, rhM-CSF)를 처리하고 이 후 2일간 재조합 인간 IL-4 (Recombinant human IL-4, rhIL-4)와 재조합 인간 IL-13 (Recombinant human IL-13, rhIL-13)을 처리하여 항-염증성 대식세포로 분화시켰다. 분화된 항-염증성 대식세포의 VSIG4 발현은 항-VSIG4 항체를 이용하여 염색하였고 FACS 분석을 통해 확인하였으며, 분화된 항-염증성 대식세포에서의 VSIG4의 발현을 확인한 결과를 도 3에 나타내었다.[Correction 01.12.2023 pursuant to Rule 91]
FACS analysis was used to confirm the expression of VSIG4 in anti-inflammatory macrophages and the binding activity of EU103 in these anti-inflammatory macrophages. CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMC) of healthy donors, treated with recombinant human M-CSF (rhM-CSF) for 7 days, and then treated with recombinant human IL-4 (Recombinant human IL-4) for 2 days. -4, rhIL-4) and recombinant human IL-13 (rhIL-13) were treated to differentiate them into anti-inflammatory macrophages. VSIG4 expression in differentiated anti-inflammatory macrophages was stained using an anti-VSIG4 antibody and confirmed through FACS analysis. The results confirming the expression of VSIG4 in differentiated anti-inflammatory macrophages are shown in Figure 3.
[규칙 제91조에 의한 정정 01.12.2023]
도 3에 나타낸 바와 같이, 분화된 항-염증성 대식세포에서는 VSIG4 가 26.7% 발현되는 것을 확인하였다.[Correction 01.12.2023 pursuant to Rule 91]
As shown in Figure 3, VSIG4 was confirmed to be expressed in 26.7% of differentiated anti-inflammatory macrophages.
본 발명의 EU103를 상기와 같이 분화된 항-염증성 대식세포에 다양한 농도로 처리하였으며, EU103의 결합 활성을 FACS 분석을 통해 확인하였고, 그 결과를 도 4에 나타내었다. Anti-inflammatory macrophages differentiated as above were treated with EU103 of the present invention at various concentrations, and the binding activity of EU103 was confirmed through FACS analysis, and the results are shown in Figure 4.
[규칙 제91조에 의한 정정 01.12.2023]
도 4에 나타낸 바와 같이, EU103는 항-염증성 대식세포의 VSIG4에 대한 결합활성을 보였으며, 0.3125 μg/mL 이상의 농도에서는 포화된 결합 활성을 나타내었다.[Correction 01.12.2023 pursuant to Rule 91]
As shown in Figure 4, EU103 showed binding activity to VSIG4 of anti-inflammatory macrophages, and showed saturated binding activity at a concentration of 0.3125 μg/mL or higher.
실시예 3. EU103의 항체 의존적 세포독성 (Antibody-Dependent Cellular Cytotoxicity, ADCC) 및 보체 의존적 세포독성 (Complement-Dependent Cytotoxicity, CDC) 확인 Example 3. Confirmation of Antibody-Dependent Cellular Cytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC) of EU103
IgG1 항체는 Fcγ에 대한 높은 결합 친화도와 보체 활성으로 인해 강력한 ADCC (Antibody-Dependent Cellular Cytotoxicity) 및 CDC (Complement-Dependent Cytotoxicity) 효과를 보이는 단점이 알려져 있으나, 구조적 안정성과 높은 유연성을 갖는 장점이 있다. 그러므로 VSIG4를 표적으로하여 항-염증성 대식세포의 염증성 대식세포로 전환을 유도하는 항체의 기능을 보전하면서도 항-염증성 대식세포의 제거를 매개하는 반응을 방지하기 위하여 IgG1의 Fc 영역에 변이를 추가하여 ADCC 및 CDC를 매개하는 주요 단백질인 Fcγ및 C1q에 대한 결합을 제거한 형태로 EU103을 제조하였다. 고전적 ADCC 분석법에 사용되는 말초혈액 단핵세포(PBMC)를 이용하고 VSIG4 과다 발현 HeLa cell을 대상 세포로 사용하여 IgG1, EU103 및 EU103-IgG1의 ADCC 및 CDC 활성을 검사하였다. 본 실험군에서 EU103는 ADCC 및 CDC 활성의 제거를 위해 IgG1 Fc 영역에 3개의 돌연변이(human IgG1 L234F/L235E/P331S 서열번호 9)를 도입한 반면, EU103-IgG1은 야생형 IgG1 Fc를 보유하고 있다. IgG1 antibodies are known to have the disadvantage of showing strong ADCC (Antibody-Dependent Cellular Cytotoxicity) and CDC (Complement-Dependent Cytotoxicity) effects due to their high binding affinity for Fcγ and complement activity, but they have the advantage of structural stability and high flexibility. Therefore, to preserve the antibody's ability to induce the conversion of anti-inflammatory macrophages to inflammatory macrophages by targeting VSIG4, but also to prevent the reaction mediating the elimination of anti-inflammatory macrophages, mutations were added to the Fc region of IgG1. EU103 was prepared in a form in which binding to Fcγ and C1q, the main proteins mediating ADCC and CDC, was removed. ADCC and CDC activities of IgG1, EU103, and EU103-IgG1 were tested using peripheral blood mononuclear cells (PBMC) used in the classical ADCC assay and HeLa cells overexpressing VSIG4 as target cells. In this experimental group, EU103 introduced three mutations (human IgG1 L234F/L235E/P331S SEQ ID NO: 9) into the IgG1 Fc region to eliminate ADCC and CDC activities, while EU103-IgG1 has wild-type IgG1 Fc.
서열번호 9: human IgG1 L234F/L235E/P331S (돌연변이 밑줄, 볼드 표시)SEQ ID NO: 9: human IgG1 L234F/L235E/P331S (mutation underlined, bold)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE FE GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA S IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE FEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPA S IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
ADCC 활성을 평가한 결과를 도 5에 나타내었다. The results of evaluating ADCC activity are shown in Figure 5.
도 5에 나타낸 바와 같이, Fc 영역 변이 없는 야생형 IgG1을 도입한 EU103-IgG1와 비교하였을 때 EU103의 ADCC 활성이 관찰되지 않았다. 반면 돌연변이가 도입되지 않은 EU103-IgG1의 경우 용량 의존적 방식으로 세포를 사멸시켰다. As shown in Figure 5, ADCC activity of EU103 was not observed when compared with EU103-IgG1 introduced with wild-type IgG1 without Fc region mutation. On the other hand, in the case of EU103-IgG1, in which no mutation was introduced, cells were killed in a dose-dependent manner.
CDC는 항체 Fc 도메인과 보체 단백질 C1q의 결합에 의해서 시작된다. EU103의 CDC 활성을 평가하기 위해 C1q에 대한 EU103 결합을 조사하는 ELISA 를 수행하였으며 그 결과를 도 6에 나타내었다. CDC is initiated by the binding of the antibody Fc domain to the complement protein C1q. To evaluate the CDC activity of EU103, ELISA was performed to examine EU103 binding to C1q, and the results are shown in Figure 6.
도 6에 나타낸 바와 같이, EU103는 C1q 단백질에 대해 미미한 결합을 나타냄을 보여주었고, 이는 매우 낮은 수준의 CDC 활성을 가지는 것을 보여주는 결과이다. As shown in Figure 6, EU103 showed minimal binding to C1q protein, which shows that it has a very low level of CDC activity.
실시예 4. FcRn에 대한 EU103 결합 친화도 평가 Example 4. Evaluation of EU103 binding affinity to FcRn
SPR 분석을 통해 재조합 인간 FcRn에 대한 EU103와 EU103-IgG1 (야생형 IgG1 Fc 보유)의 결합 친화도를 평가하였으며, 그 결과를 하기 표 3에 나타내었다. The binding affinity of EU103 and EU103-IgG1 (having wild-type IgG1 Fc) to recombinant human FcRn was evaluated through SPR analysis, and the results are shown in Table 3 below.
Antibody Antibody Ka (1/Ms)Ka (1/Ms) Kd(1/s)Kd(1/s) KD(M) KD(M)
EU103-IgG1EU103-IgG1 2.045E+42.045E+4 0.0031150.003115 1.523E-71.523E-7
1.777E+41.777E+4 0.0029620.002962 1.666E-71.666E-7
EU103EU103 1.522E+41.522E+4 0.0016370.001637 1.076E-71.076E-7
1.311E+41.311E+4 0.0014780.001478 1.127E-71.127E-7
표 3에 나타낸 바와 같이, EU103-IgG1, EU103간 FcRn 결합에 대한 KD 분석 결과, IgG1의 Fc 영역에 변이를 도입한 EU103가 정상 인간 IgG1과 유사한 결합을 나타내는 것으로 밝혀졌다.As shown in Table 3, as a result of KD analysis of FcRn binding between EU103-IgG1 and EU103, it was found that EU103, which introduced a mutation in the Fc region of IgG1, showed similar binding to normal human IgG1.
실시예 5. 인간 T세포 분열에 미치는 EU103의 영향Example 5. Effect of EU103 on human T cell division
VSIG4와 T 세포 표면의 알려지지 않은 수용체의 결합이 T 세포 분열을 억제함에 따라, carboxyfluorescein succinimidyl ester(CFSE) 염색을 사용하여 FACS 측정법으로 인간 T 세포 분열에 미치는 EU103의 영향을 분석했다. CFSE 염색은 희석 횟수별로 세포 분열 횟수를 측정할 수 있다. 건강한 공여자의 말초 혈액 단핵세포에서 CD4+/CD8+ T 세포를 분리하여 항-CD3 항체를 이용하여 활성화시켰다. As binding of VSIG4 to an unknown receptor on the T cell surface inhibits T cell division, we analyzed the effect of EU103 on human T cell division by FACS measurements using carboxyfluorescein succinimidyl ester (CFSE) staining. CFSE staining can measure the number of cell divisions by number of dilutions. CD4+/CD8+ T cells were isolated from peripheral blood mononuclear cells of healthy donors and activated using anti-CD3 antibody.
도 7에 나타낸 바와 같이, rhVSIG4-His는 항-CD3 항체의 존재하에서도 CD4+/CD8+ T 세포의 증식을 억제하는 결과를 나타냈다. 이 때 EU103를 처리하면 용량 의존적 방식으로 CD4+/CD8+ T 세포의 증식이 증가됨을 확인하였다. 이 결과는 EU103가 VSIG4와 알려지지 않은 수용체 결합을 차단함으로써 VSIG4에 의해 억제된 T 세포 증식을 다시 촉진함을 시사한다.As shown in Figure 7, rhVSIG4-His inhibited the proliferation of CD4+/CD8+ T cells even in the presence of anti-CD3 antibodies. At this time, it was confirmed that treatment with EU103 increased the proliferation of CD4+/CD8+ T cells in a dose-dependent manner. These results suggest that EU103 re-promotes T cell proliferation suppressed by VSIG4 by blocking the binding of VSIG4 to an unknown receptor.
실시예 6. 난소암 유래 종양 관련 대식세포에 대한 EU103 의 전환 효과 확인Example 6. Confirmation of the conversion effect of EU103 on ovarian cancer-derived tumor-related macrophages
종양 관련 대식세포(TAM)는 정상 상태에서 존재하지 않지만 많은 종양에서 관찰되기 때문에 항암 치료에서 중요하게 고려된다. 종양 관련 대식세포(TAM)의 특정 분극 상태는 항-염증성 유사 대식세포로서 종양 미세환경의 중요한 구성요소이며 항암 치료에서 중요한 연구의 대상이다. 항-염증성 대식세포를 종양을 억제하는 염증성 대식세포 표현형으로 전환하는 것이 항-종양 면역의 개선을 위한 새로운 치료방법이 될 수 있다. EU103가 난소암 환자의 복수에서 분리된 종양 관련 대식세포(TAM)를 전환가능한지 종양 관련 대식세포 (TAM)에서 EU103의 역할을 분석하였다. Tumor-associated macrophages (TAMs) do not exist under normal conditions, but are observed in many tumors and are therefore considered important in anticancer treatment. The specific polarization state of tumor-associated macrophages (TAMs), as anti-inflammatory-like macrophages, are important components of the tumor microenvironment and are a subject of important research in anticancer therapy. Converting anti-inflammatory macrophages to a tumor-suppressing inflammatory macrophage phenotype may be a new therapeutic approach for improving anti-tumor immunity. The role of EU103 in tumor-associated macrophages (TAMs) was analyzed to determine whether EU103 can transform TAMs isolated from the ascites of ovarian cancer patients.
난소암 환자의 복수에서 CD14+ 세포를 분리하였고 분리한 종양 관련 대식세포 (TAM)는 CD68과 CD163를 발현하고 있으며 동시에 VSIG4의 발현 또한 관찰하였다. 이렇게 분리된 종양 관련 대식세포(TAM)에 EU103를 2일간 처리하고 대식세포 표면 마커의 발현 변화 및 배양액에서 사이토카인 및 효소의 분비 변화를 측정하였다. 양성 대조군으로는 LPS/IFN-g를 처리하였고, 실험 결과를 도 8에 나타내었다. CD14+ cells were isolated from the ascites of an ovarian cancer patient, and the isolated tumor-associated macrophages (TAM) expressed CD68 and CD163, and at the same time, the expression of VSIG4 was also observed. The isolated tumor-associated macrophages (TAM) were treated with EU103 for 2 days, and changes in the expression of macrophage surface markers and secretion of cytokines and enzymes in the culture medium were measured. LPS/IFN-g was treated as a positive control, and the experimental results are shown in Figure 8.
도 8에 나타낸 바와 같이, EU103 또는 LPS/IFN-g 를 처리함에 따라, M1 대식세포의 마커인 CD86의 발현이 현저히 증가함과 동시에 M2 대식세포의 마커인 CD163의 발현이 감소됨을 확인하였다. 또한 TNF-α, IL-6, IL-10 및 IL-1β의 생성을 증가시켰지만, 아르기나제(Arginase)의 생성은 감소시켰다. 이러한 결과는 EU103의 자극으로 난소암 환자에서 유래한 종양 관련 대식세포(TAM)가 종양을 억제하는 염증성 대식세포로 전환되었음을 나타낸다. 따라서 EU103이 종양 관련 대식세포(TAM)를 종양을 억제하는 염증성 대식세포로 전환시켜 종양 미세환경(TME)을 개선할 수 있음을 확인하였으며, 이는 EU103가 종양 미세환경을 개선하여 항암 효과를 나타낼 수 있음을 나타낸다. As shown in Figure 8, it was confirmed that upon treatment with EU103 or LPS/IFN-g, the expression of CD86, a marker of M1 macrophages, significantly increased, while the expression of CD163, a marker of M2 macrophages, decreased. It also increased the production of TNF-α, IL-6, IL-10, and IL-1β, but decreased the production of arginase. These results indicate that tumor-associated macrophages (TAMs) derived from ovarian cancer patients were converted into tumor-suppressing inflammatory macrophages by stimulation of EU103. Therefore, it was confirmed that EU103 can improve the tumor microenvironment (TME) by converting tumor-associated macrophages (TAMs) into inflammatory macrophages that suppress tumors, which means that EU103 can exert anticancer effects by improving the tumor microenvironment. It indicates that there is.
실시예 7. PBMC 인간화 생쥐 A549-luci 폐암 동소위(orthotopic) 모델에서 항-종양 활성Example 7. Anti-tumor activity in PBMC humanized mouse A549-luci lung cancer orthotopic model
EU103는 인간 VSIG4에 작용하는 항체이며, 생쥐 VSIG4에 대한 결합 친화도는 낮아서 관찰할 수 없다. 따라서 인간의 면역체계가 도입된 인간화 생쥐모델을 사용하여 EU103의 항암효과를 평가하였다. 인간화 생쥐는 면역성이 결여된 생쥐(Immunodeficient mouse)에 사람의 세포 또는 조직을 이식하거나 유전자를 도입한 연구용 생쥐로 간단히 정의할 수 있다. 인간의 면역체계 도입을 위해서 가장 일반적인 방식으로는 인간의 조혈모세포(Human hematopoietic stem cell, hu-HSC, CD34+ humanization)를 이식하거나 인간의 말초혈액에 존재하는 백혈구(Human peripheral blood leukocytes, hu-PBL, PBMC humanization)을 분리하여 주입하는 방식을 이용한다. 피하 종양 모델과 비교하여 동소위 모델은 임상적으로 더 관련성이 있고 약물 효과를 더 잘 예측할 수 있는 것으로 알려져 있다. 이는 종양 세포가 장기에 직접 이식되기 때문에 종양이 기존의 피하 종양 모델보다 훨씬 더 실제 종양 미세환경을 구성할 수 있을 것이라 기대하기 때문이다. 루시페라제 유전자가 형질 감염된 종양 세포주를 이식하여 생물발광 영상화(BLI)를 통해 종양 성장, 분포 및 전이 성장의 비침습적인 시각화를 통해 항암 효과를 평가하였다. 폐암 동소위 모델을 유도하기 위한 방법 중 꼬리정맥 주입방법을 사용하여 폐에 종양을 생착시켰으며 이 때 분화시킨 M2 대식세포를 폐암 세포주와 함께 주입하여 종양 관련 대식세포가 형성되도록 유도하였다. 폐에 생착된 종양의 발광 강도가 1 x 107 이상일 때 항체 투여를 시작하였다. 시험물질은 3일마다 총 6회 투여하였다. 루시페라제를 발현하는 종양 세포는 종양 세포 이식 후 주 1회, 항체 투여 후 주 2회 IVIS를 사용하여 가시화하였다. 폐 동소위 유도 후 루시페라제 밀도를 IVIS 시스템을 통해 측정하여 폐 특이적 종양 세포 생착을 확인했지만 다른 장기에서는 관찰되지 않았다. 대조군으로 인간 IgG (hIgG) 를 투여하였으며, EU103 투여에 따른 종양 크기 및 체중 변화를 확인한 결과를 도 9에 나타내었다. EU103 is an antibody that acts on human VSIG4, and its binding affinity for mouse VSIG4 is low and cannot be observed. Therefore, the anticancer effect of EU103 was evaluated using a humanized mouse model introduced with the human immune system. Humanized mice can be simply defined as research mice in which human cells or tissues are transplanted or genes are introduced into immunodeficient mice. To introduce the human immune system, the most common methods are transplantation of human hematopoietic stem cells (hu-HSC, CD34+ humanization) or white blood cells present in human peripheral blood (human peripheral blood leukocytes, hu-PBL, PBMC humanization) is separated and injected. Compared with subcutaneous tumor models, orthotopic models are known to be more clinically relevant and better able to predict drug effects. This is because the tumor cells are directly transplanted into the organ, so it is expected that the tumor will be able to constitute a much more realistic tumor microenvironment than the existing subcutaneous tumor model. Tumor cell lines transfected with the luciferase gene were transplanted and anticancer effects were evaluated through noninvasive visualization of tumor growth, distribution, and metastatic growth through bioluminescence imaging (BLI). Among the methods to induce the lung cancer orthotopic model, tail vein injection was used to engraft the tumor in the lung. At this time, differentiated M2 macrophages were injected together with the lung cancer cell line to induce the formation of tumor-related macrophages. Antibody administration was started when the luminescence intensity of the tumor engrafted in the lung was 1 x 10 7 or higher. The test substance was administered a total of 6 times every 3 days. Tumor cells expressing luciferase were visualized using IVIS once a week after tumor cell transplantation and twice a week after antibody administration. After lung orthotopic induction, luciferase density was measured using the IVIS system to confirm lung-specific tumor cell engraftment, but was not observed in other organs. Human IgG (hIgG) was administered as a control group, and the results of tumor size and body weight changes following EU103 administration are shown in Figure 9.
도 9에 나타낸 바와 같이, 대조군인 인간 IgG 투여군에서는 종양의 크기가 증가함에 따라 2마리가 사망하였고, 1마리는 정상적이지 않은 종양 생착을 보였다. 그러나, EU103 투여군은 전체 종양 크기 감소를 보였다. 전반적으로 종양 크기는 EU103 투여군에서 유의하게 감소한 반면 체중에는 유의한 변화가 없었다. 이러한 결과는 EU103의 항종양 효과를 나타내는 결과이다. As shown in Figure 9, in the control group administered human IgG, two animals died as the size of the tumor increased, and one animal showed abnormal tumor engraftment. However, the EU103 administration group showed a decrease in overall tumor size. Overall, tumor size was significantly reduced in the EU103 treatment group, while there was no significant change in body weight. These results demonstrate the antitumor effect of EU103.
실시예 8. 시노몰구스 원숭이에서 EU103의 예비 독성 시험Example 8. Preliminary toxicity testing of EU103 in cynomolgus monkeys
시노몰구스 원숭이의 간 쿠퍼 세포 (Kupffer cell, KC)를 이용하여 예비 원숭이 독성 연구를 수행하였다. 독성 확인 실험에서는 Fc-조작된 IgG1 뿐만 아니라 EU103의 다양한 이소타입(Isotype)으로 IgG1(Anti-VSIG4-IgG1), IgG2(Anti-VSIG4-euG2), IgG4(Anti-VSIG4-IgG4)를 포함하는 실험군을 대상으로 실험을 수행하였다. 20mg/kg 농도의 야생형 IgG1, IgG4 및 Fc-조작된 IgG1의 EU103을 원숭이에 주입하고 간 쿠퍼 세포에 대한 효과를 평가하였으며 그 결과를 도 10에 나타내었다. Preliminary monkey toxicity studies were performed using liver Kupffer cells (KC) from cynomolgus monkeys. In the toxicity confirmation experiment, the experimental group included not only Fc-engineered IgG1 but also various isotypes of EU103 such as IgG1 (Anti-VSIG4-IgG1), IgG2 (Anti-VSIG4-euG2), and IgG4 (Anti-VSIG4-IgG4). An experiment was conducted targeting . Wild-type IgG1, IgG4, and Fc-engineered IgG1 EU103 at a concentration of 20 mg/kg were injected into monkeys and the effect on liver Kupffer cells was evaluated. The results are shown in Figure 10.
쿠퍼 세포를 보존하는 것은 면역 항상성에 중요하다. 쿠퍼 세포는 VSIG4를 발현하기 때문에 EU103-IgG1(Anti-VSIG4-IgG1)의 ADCC 활성에 의한 고갈 효과가 예상되었다. FACS를 사용하여 쿠퍼 세포의 고갈을 분석하기 위해 시노몰구스 원숭이의 간에서 쿠퍼 세포를 분리하였다. 도 10에 나타낸 바와 같이, 시험 결과 쿠퍼 세포는 구성적으로 CD4와 VSIG4를 모두 발현하는 것을 확인하였다. IgG1 야생형의 EU103-IgG1(Anti-VSIG4-IgG1)은 쿠퍼 세포를 64% 감소시켰지만, 본 발명의 EU103은 시노몰구스 원숭이의 쿠퍼 세포 집단에 유의미한 영향을 미치지 않았다. 또한 EU103 투여 원숭이의 정상적인 ALT 및 AST 등 임상 화학 변화가 관찰되지 않았다. 즉, EU103 투여 원숭이에서 약물과 관련된 이상반응은 관찰되지 않았고 임상 징후는 20mg/kg에서 정상이었다. Preserving Kupffer cells is important for immune homeostasis. Because Kupffer cells express VSIG4, a depletion effect by the ADCC activity of EU103-IgG1 (Anti-VSIG4-IgG1) was expected. Kupffer cells were isolated from the liver of cynomolgus monkeys to analyze depletion of Kupffer cells using FACS. As shown in Figure 10, the test results confirmed that Kupffer cells constitutively express both CD4 and VSIG4. EU103-IgG1 (Anti-VSIG4-IgG1) of IgG1 wild type reduced Kupffer cells by 64%, but EU103 of the present invention had no significant effect on the Kupffer cell population of cynomolgus monkeys. Additionally, no changes in clinical chemistry, including normal ALT and AST, were observed in monkeys administered EU103. In other words, no drug-related adverse reactions were observed in monkeys administered EU103, and clinical signs were normal at 20 mg/kg.
추가적으로 쿠퍼 세포, 분화된 M2 대식세포, 또는 난소암 환자에서 분리한 종양 관련 대식세포에 EU103를 처리한 후, TAM 및 M2 대식세포에서 사이토카인 및 아르기나아제 수준의 변화를 측정한 결과를 도 11에 나타내었다. Additionally, after treating Kupffer cells, differentiated M2 macrophages, or tumor-related macrophages isolated from ovarian cancer patients with EU103, the results of measuring changes in cytokine and arginase levels in TAM and M2 macrophages are shown in Figure 11. shown in
도 11에 나타낸 바와 같이, 쿠퍼 세포에서는 유의한 변화가 관찰되지 않았으며, 이는 EU103이 쿠퍼 세포의 기능에는 최소한의 영향을 미친다는 것을 시사한다. As shown in Figure 11, no significant changes were observed in Kupffer cells, suggesting that EU103 has minimal effect on Kupffer cell function.
실시예 9. EU103 및 항 PD-L1 항체 병용투여에 따른 항 종양 활성 확인 Example 9. Confirmation of anti-tumor activity following combined administration of EU103 and anti-PD-L1 antibody
실시예 7에서 제조한 모델과 동일한 A549-luci 폐암 동소위 모델을 사용하여 EU103와 항 PD-L1 항체 병용 투여에 따른 시너지 효과를 평가하였다. 항PD-L1 항체는 티센트릭 (atezolizumab)을 사용하였다. EU103 또는 티센트릭 단독 투여군의 경우 각각 8 mg/kg의 용량을 투여하였고, 병용 투여군에서는 EU103 5 mg/kg 및 티센트릭 3 mg/kg을 투여하였으며, 그 결과를 도 12 및 표 4 에 나타내었다. The synergistic effect of combined administration of EU103 and anti-PD-L1 antibody was evaluated using the same A549-luci lung cancer orthotopic model as the model prepared in Example 7. Tecentriq (atezolizumab) was used as the anti-PD-L1 antibody. In the case of EU103 or Tecentriq alone administration group, a dose of 8 mg/kg was administered, and in the combination administration group, EU103 5 mg/kg and Tecentriq 3 mg/kg were administered, and the results are shown in Figure 12 and Table 4.
GroupGroup 실험군experimental group Dosage (mpk)Dosage (mpk) 종양 부피
(Luminescent intensity)a tumor volume
(Luminescent intensity) a 		TGITV (%)b TGI TV (%) b TF micec TF mice c P valued P value d
G1G1 hIgGhIgG 88 2.119e+008±7.106e+0072.119e+008±7.106e+007 -- 00 --
G2 G2 EU103EU103 88 4.208e+007±1.690e+0074.208e+007±1.690e+007 80.1480.14 00 *P<0.05* P <0.05
G3 G3 TecentriqTecentriq 88 5.872e+007±1.732e+0075.872e+007±1.732e+007 72.2972.29 00 *P<0.05* P <0.05
G4G4 EU103+TecentriqEU103+Tecentriq 5+35+3 1.249e+007±4.586e+0061.249e+007±4.586e+006 94.1194.11 00 **P<0.01** P <0.01
a) Mean ± SEMa) Mean ± SEM
b) Tumor growth inhibition (TGITV (%)) =[(Vi-Ti)/Vi]*100b) Tumor growth inhibition (TGI TV (%)) =[(V i -T i )/V i ]*100
Ti: treated group at endpoint day; Vi: hIgG group at endpoint dayT i : treated group at endpoint day; V i : hIgG group at endpoint day
c) TF mice: Tumor free micec) TF mice: Tumor free mice
d) Statistical analysis via One-way ANOVA on tumor volume of the treated group versus hIgG at the endpoint dayd) Statistical analysis via One-way ANOVA on tumor volume of the treated group versus hIgG at the endpoint day
도 12 A 및 B에 나타낸 바와 같이, 대조군과 비교하여 단독 투여군과 병용 투여군에서 종양의 크기가 유의하게 감소하였다 (A, B). 특히, EU103과 티센트릭의 병용 투여군은 26일차 이후부터 EU103 또는 티센트릭 단독 투여군보다 우수한 항종양 활성을 나타냈다.As shown in Figures 12 A and B, the size of the tumor was significantly reduced in the single-administration group and the combined administration group compared to the control group (A, B). In particular, the group treated with the combination of EU103 and Tecentriq showed superior antitumor activity than the group treated with EU103 or Tecentriq alone from day 26 onwards.
시험 종료일인 43일차에 종양 성장 억제 데이터를 분석하고, 그 결과를 도 12 C 에 나타내었다. 43일째에 EU103 투여군과 티센트릭 투여군의 종양 성장 억제 (TGITV(%))는 각각 80.14%와 72.29%였으며, 병용 투여군(Combo)에서는 94.11%의 억제율을 보였다.Tumor growth inhibition data was analyzed on day 43, the end date of the test, and the results are shown in Figure 12C. On day 43, tumor growth inhibition (TGITV (%)) in the EU103 and Tecentriq groups was 80.14% and 72.29%, respectively, and the combination group (Combo) showed an inhibition rate of 94.11%.
EU103 또는 티센트릭의 단독요법은 종양 주사 후 43일까지 종양 성장을 감소시켰다. 특히, EU103과 티센트릭의 병용 요법은 인간화 동소위 폐암 모델에서 명백한 상승적 항-종양 효과를 보였다. 이러한 병용 조합의 항-종양 효과의 반응은 약물 주입과 함께 매우 즉각적으로 나타났다. Monotherapy with EU103 or Tecentriq reduced tumor growth up to 43 days after tumor injection. In particular, the combination therapy of EU103 and Tecentriq showed a clear synergistic anti-tumor effect in a humanized orthotopic lung cancer model. The response of the anti-tumor effect of this combination was very immediate upon drug injection.
즉, EU103는 항-PD-L1 항체인 티센트릭과의 병용 요법에서 종양의 성장을 효율적으로 억제하고 항-종양 효과를 크게 증가시킬 수 있음을 확인하였다. In other words, it was confirmed that EU103 can efficiently inhibit tumor growth and significantly increase the anti-tumor effect in combination therapy with Tecentriq, an anti-PD-L1 antibody.

Claims (17)

  1. [규칙 제91조에 의한 정정 01.12.2023]
    [Correction 01.12.2023 pursuant to Rule 91]
    a) 서열번호 1의 아미노산 서열로 표시되는 중쇄 CDR1, 서열번호 2의 아미노산 서열로 표시되는 중쇄 CDR2, 서열번호 3의 아미노산 서열로 표시되는 중쇄 CDR3; 및 a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and
    서열번호 4의 아미노산 서열로 표시되는 경쇄 CDR1, 서열번호 5의 아미노산 서열로 표시되는 경쇄 CDR2, 서열번호 6의 아미노산 서열로 표시되는 경쇄 CDR3; 을 포함하는 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편; 및 Light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; A humanized anti-VSIG4 antibody or antigen-binding fragment thereof comprising; and
    b) 항 PD-L1 항체를 포함하는, 암 예방 또는 치료용 약학적 조성물. b) A pharmaceutical composition for preventing or treating cancer, comprising an anti-PD-L1 antibody.
  2. [규칙 제91조에 의한 정정 01.12.2023]
    [Correction 01.12.2023 pursuant to Rule 91]
    제1항에 있어서,According to paragraph 1,
    상기 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편은 서열번호 7 로 표시되는 중쇄 가변영역을 포함하는, 암 예방 또는 치료용 약학적 조성물.The humanized anti-VSIG4 antibody or antigen-binding fragment thereof is a pharmaceutical composition for preventing or treating cancer, comprising a heavy chain variable region represented by SEQ ID NO: 7.
  3. [규칙 제91조에 의한 정정 01.12.2023]
    [Correction 01.12.2023 pursuant to Rule 91]
    제1항에 있어서,According to paragraph 1,
    상기 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편은 서열번호 8 로 표시되는 경쇄 가변영역을 포함하는, 암 예방 또는 치료용 약학적 조성물.The humanized anti-VSIG4 antibody or antigen-binding fragment thereof is a pharmaceutical composition for preventing or treating cancer, comprising a light chain variable region represented by SEQ ID NO: 8.
  4. [규칙 제91조에 의한 정정 01.12.2023]
    [Correction 01.12.2023 pursuant to Rule 91]
    제1항에 있어서,According to paragraph 1,
    상기 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편은 서열번호 7 로 표시되는 중쇄 가변영역 및 서열번호 8 로 표시되는 경쇄 가변영역을 포함하는, 암 예방 또는 치료용 약학적 조성물.The humanized anti-VSIG4 antibody or antigen-binding fragment thereof is a pharmaceutical composition for preventing or treating cancer, comprising a heavy chain variable region represented by SEQ ID NO: 7 and a light chain variable region represented by SEQ ID NO: 8.
  5. [규칙 제91조에 의한 정정 01.12.2023]
    [Correction 01.12.2023 pursuant to Rule 91]
    제1항에 있어서, According to paragraph 1,
    상기 인간화된 항-VSIG4 항체는 서열번호 9로 표시되는 조작된 IgG1 Fc 영역을 포함하는 것을 특징으로 하는, 암 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating cancer, wherein the humanized anti-VSIG4 antibody comprises an engineered IgG1 Fc region represented by SEQ ID NO: 9.
  6. [규칙 제91조에 의한 정정 01.12.2023]
    [Correction 01.12.2023 pursuant to Rule 91]
    제5항에 있어서, According to clause 5,
    상기 인간화된 항-VSIG4 항체는 쿠퍼 세포에 비독성인 것을 특징으로 하는, 암 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating cancer, wherein the humanized anti-VSIG4 antibody is non-toxic to Kupffer cells.
  7. [규칙 제91조에 의한 정정 01.12.2023]
    [Correction 01.12.2023 pursuant to Rule 91]
    제5항에 있어서, According to clause 5,
    상기 인간화된 항-VSIG4 항체는 Fcγ및 C1q에 대한 결합능이 제거된 것인, 암 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating cancer, wherein the humanized anti-VSIG4 antibody has its binding ability to Fcγ and C1q removed.
  8. 제1항에 있어서, 상기 항원 결합 단편은 Fab, Fab', (Fab')2, Fv, scFv, 비스-scFv, scFv-Fc 단편, Fab2, Fab3, 미니체, 다이아체, 트리아제, 테트라체 및 나노체로 이루어진 군으로부터 선택되는 것인, 암 예방 또는 치료용 약학적 조성물.The method of claim 1, wherein the antigen-binding fragment is Fab, Fab', (Fab') 2 , Fv, scFv, bis-scFv, scFv-Fc fragment, Fab 2 , Fab 3 , minibody, diabody, triase, A pharmaceutical composition for preventing or treating cancer, which is selected from the group consisting of tetramers and nanomers.
  9. 제1항에 있어서, 상기 항 PD-L1 항체는 아테졸리주맙, 더발루맙 또는 아벨루맙인, 암 예방 또는 치료용 약학적 조성물. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the anti-PD-L1 antibody is atezolizumab, durvalumab, or avelumab.
  10. [규칙 제91조에 의한 정정 01.12.2023]
    [Correction 01.12.2023 pursuant to Rule 91]
    제1항에 있어서,According to paragraph 1,
    상기 암은 VSIG4 과발현 암인, 암 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating cancer, wherein the cancer is a cancer that overexpresses VSIG4.
  11. 제10항에 있어서, 상기 암은 다발성 골수종, 난소암, 폐암, 비소세포 폐암, 신경교종, 유방암, 자궁경부암, 결장암, 자궁내막암, 식도암, 나팔관 암, 담낭암, 위암, 두경부암, 혈액 암, 후두암, 간암, 림프종, 흑색종, 중피종, 원발성 복막암, 침샘암, 육종, 갑상선암, 췌장암, 신장 세포 암종, 교모세포종, 및 전립선암으로 이루어진 군에서 선택된 1종 이상인, 암 예방 또는 치료용 약학적 조성물.The method of claim 10, wherein the cancer is multiple myeloma, ovarian cancer, lung cancer, non-small cell lung cancer, glioma, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, stomach cancer, head and neck cancer, blood cancer, Pharmaceuticals for the prevention or treatment of cancer, one or more types selected from the group consisting of laryngeal cancer, liver cancer, lymphoma, melanoma, mesothelioma, primary peritoneal cancer, salivary gland cancer, sarcoma, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer. Composition.
  12. [규칙 제91조에 의한 정정 01.12.2023]
    [Correction 01.12.2023 pursuant to Rule 91]
    제1항에 있어서, 상기 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편; 및 항 PD-L1 항체는 1: 1 내지 5 (w/w)% 로 포함되는 것인, 암 예방 또는 치료용 약학적 조성물.The method of claim 1 , wherein said humanized anti-VSIG4 antibody or antigen-binding fragment thereof; and anti-PD-L1 antibody in an amount of 1:1 to 5 (w/w)%. A pharmaceutical composition for preventing or treating cancer.
  13. [규칙 제91조에 의한 정정 01.12.2023]
    [Correction 01.12.2023 pursuant to Rule 91]
    a) 서열번호 1의 아미노산 서열로 표시되는 중쇄 CDR1, 서열번호 2의 아미노산 서열로 표시되는 중쇄 CDR2, 서열번호 3의 아미노산 서열로 표시되는 중쇄 CDR3; 및 a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and
    서열번호 4의 아미노산 서열로 표시되는 경쇄 CDR1, 서열번호 5의 아미노산 서열로 표시되는 경쇄 CDR2, 서열번호 6의 아미노산 서열로 표시되는 경쇄 CDR3; 을 포함하는 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편; 및 Light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; A humanized anti-VSIG4 antibody or antigen-binding fragment thereof comprising; and
    b) 항 PD-L1 항체를 포함하는, 암 예방 또는 치료용 병용투여 제제. b) A combination administration agent for cancer prevention or treatment containing an anti-PD-L1 antibody.
  14. [규칙 제91조에 의한 정정 01.12.2023]
    [Correction 01.12.2023 pursuant to Rule 91]
    a) 서열번호 1의 아미노산 서열로 표시되는 중쇄 CDR1, 서열번호 2의 아미노산 서열로 표시되는 중쇄 CDR2, 서열번호 3의 아미노산 서열로 표시되는 중쇄 CDR3; 및 a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and
    서열번호 4의 아미노산 서열로 표시되는 경쇄 CDR1, 서열번호 5의 아미노산 서열로 표시되는 경쇄 CDR2, 서열번호 6의 아미노산 서열로 표시되는 경쇄 CDR3; 을 포함하는 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편을 포함하는 1구획; 및 Light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; One compartment containing a humanized anti-VSIG4 antibody or antigen-binding fragment thereof comprising; and
    b) 항 PD-L1 항체를 포함하는 2구획을 포함하는, 암 예방 또는 치료용 키트. b) A kit for preventing or treating cancer, comprising two compartments containing an anti-PD-L1 antibody.
  15. [규칙 제91조에 의한 정정 01.12.2023]
    [Correction 01.12.2023 pursuant to Rule 91]
    서열번호 1의 아미노산 서열로 표시되는 중쇄 CDR1, 서열번호 2의 아미노산 서열로 표시되는 중쇄 CDR2, 서열 번호 3의 아미노산 서열로 표시되는 중쇄 CDR3; 및 Heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and
    서열번호 4의 아미노산 서열로 표시되는 경쇄 CDR1, 서열번호 5의 아미노산 서열로 표시되는 경쇄 CDR2, 서열번호 6의 아미노산 서열로 표시되는 경쇄 CDR3; 을 포함하는 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편을 포함하는, 항 PD-L1 항체 효능 증진용 항암 보조 치료제.Light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; An adjuvant anticancer treatment for enhancing anti-PD-L1 antibody efficacy, comprising a humanized anti-VSIG4 antibody or an antigen-binding fragment thereof.
  16. a) 서열번호 1의 아미노산 서열로 표시되는 중쇄 CDR1, 서열번호 2의 아미노산 서열로 표시되는 중쇄 CDR2, 서열번호 3의 아미노산 서열로 표시되는 중쇄 CDR3; 및 a) heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and
    서열번호 4의 아미노산 서열로 표시되는 경쇄 CDR1, 서열번호 5의 아미노산 서열로 표시되는 경쇄 CDR2, 서열번호 6의 아미노산 서열로 표시되는 경쇄 CDR3; 을 포함하는 인간화된 항-VISG4 항체 또는 이의 항원 결합 단편; 및 Light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; A humanized anti-VISG4 antibody or antigen-binding fragment thereof comprising; and
    b) 항 PD-L1 항체를 개체에 투여하는 단계를 포함하는, 암 예방 또는 치료 방법. b) A method of preventing or treating cancer, comprising administering an anti-PD-L1 antibody to a subject.
  17. [규칙 제91조에 의한 정정 01.12.2023]
    [Correction 01.12.2023 pursuant to Rule 91]
    서열번호 1의 아미노산 서열로 표시되는 중쇄 CDR1, 서열번호 2의 아미노산 서열로 표시되는 중쇄 CDR2, 서열번호 3의 아미노산 서열로 표시되는 중쇄 CDR3; 및 Heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and
    서열번호 4의 아미노산 서열로 표시되는 경쇄 CDR1, 서열번호 5의 아미노산 서열로 표시되는 경쇄 CDR2, 서열번호 6의 아미노산 서열로 표시되는 경쇄 CDR3; 을 포함하는 인간화된 항-VSIG4 항체 또는 이의 항원 결합 단편을 개체에 투여하는 단계를 포함하는, 항 PD-L1 항체 효능 증진 방법. Light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; A method for enhancing anti-PD-L1 antibody efficacy, comprising administering to a subject a humanized anti-VSIG4 antibody or antigen-binding fragment thereof.
PCT/KR2023/016397 2022-10-21 2023-10-20 Anti-cancer composition comprising anti-vsig4 antibody WO2024085726A1 (en)

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