[go: up one dir, main page]

WO2015130115A1 - Nouvel anticorps spécifique de la tspan8 et ses utilisations - Google Patents

Nouvel anticorps spécifique de la tspan8 et ses utilisations Download PDF

Info

Publication number
WO2015130115A1
WO2015130115A1 PCT/KR2015/001905 KR2015001905W WO2015130115A1 WO 2015130115 A1 WO2015130115 A1 WO 2015130115A1 KR 2015001905 W KR2015001905 W KR 2015001905W WO 2015130115 A1 WO2015130115 A1 WO 2015130115A1
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
tspan8
antibody
amino acid
lel
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KR2015/001905
Other languages
English (en)
Inventor
Sukmook Lee
Taek-Keun Kim
Chang Sik Park
Mee Hyun JEOUNG
Min Kyoung KI
Woo Ran LEE
Nam Kyung Go
Jong Rip Choi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Scripps Korea Antibody Institute
Original Assignee
Scripps Korea Antibody Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Scripps Korea Antibody Institute filed Critical Scripps Korea Antibody Institute
Priority to KR1020167026871A priority Critical patent/KR101906558B1/ko
Publication of WO2015130115A1 publication Critical patent/WO2015130115A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present invention also provides a pharmaceutical composition for preventing or treating cancer and/or cancer metastasis, comprising said antibody.
  • the present invention also provides said antibody for use in in-vitro diagnosis of cancer and/or cancer metastasis.
  • the present invention also provides a method for diagnosing cancer and/or cancer metastasis, comprising detecting TSPAN8 through antigen-antibody complexes in an isolated biological sample from a subject with suspected cancer and/or cancer metastasis.
  • the present invention also provides a use of said material for inhibiting the expression of TSPAN8 for preparation of a composition for suppressing of cancer and/or cancer metastasis.
  • the present invention also provides said fusion protein of LEL of TSPAN8 and Fc for use in preparation of a pharmaceutical composition for preventing or treating cancer and/or cancer metastasis.
  • Figures 3a to 3d show knockdown of TSPAN8 reduced HCT-8 colorectal cancer cell invasion without affecting proliferation and adhesion to HUVECs.
  • Figure 3a Western blot analysis of cell lysates from TSPAN8 siRNA-transfected HCT-8 cells using an anti-TSPAN8 antibody.
  • Figure 3b Invasion activity was measured in TSPAN8 siRNA-transfected HCT-8 cells using Matrigel-coated Transwell chambers as described in Materials and Methods. Images are representative fields of invasive cells on the membrane. Bar graph shows results as a percentage of the control.
  • Figure 3c Cell Counting Kit-8 (CCK-8) assay of TSPAN8 siRNA-transfected HCT-8 cell proliferation after 48 h incubation.
  • Figure 3d Adhesion assay of TSPAN8 siRNA-transfected HCT-8 cell adhesion to human umbilical vein endothelial cells (HUVEC). Data are shown as means ⁇ SEM. *P ⁇ 0.05.
  • FIG. 4b Binding specificity of TSPAN8 scFv-Fc clones was determined by indirect ELISA using Fc, TSPAN1-LEL-Fc, TSPAN8-SEL-Fc, and TSPAN8-LEL-Fc.
  • Figure 4c Western blot analysis of TSPAN8 scFv-Fc clones bound to TSPAN8-LEL under non-reduced conditions.
  • FIG. 6 shows that TSPAN8 scFv-Fc inhibits the invasion of other metastatic cancer cell lines including U87 and PANC-1.
  • Invasion assays were used to determine the effects of TSPAN8 scFv-Fc (100 ⁇ g/ml) on the metastatic U87 brain glioma and PANC-1 pancreatic cancer cell lines. Images are representative fields of invasive cells on the membrane. Results are expressed as a percentage of the control. Data are shown as means ⁇ SEM. * P ⁇ 0.05.
  • recombinant human antibodies specific to the LEL of TSPAN8 were selected from a human synthetic antibody library and found to more potently inhibit the invasion of metastatic colorectal cancer cells than that of non-metastatic colorectal cancer cells.
  • Inventors of the present application then used immunohistochemical staining to validate the clinical relevance of TSPAN8 for ovarian cancer.
  • Fab' is different from Fab in that it further comprises at least one cysteine residue at the C-terminus of the CH1 domain of the heavy chain.
  • F(ab')2 consists of two molecules of Fab' with a disulfide bond between the cysteine residues of the hinge region.
  • Fv (variable fragment) composed of one variable region of each of the heavy and the light chain, is the smallest antibody fragment containing the original specificity of the parent immunoglobulin.
  • Disulfide-stabilized Fv (dsFv) is formed by linking the variable region of the heavy chain to the variable region of the light chain via a disulfide bond.
  • monoclonal antibody refers to an antibody molecule with a uniform molecular composition, obtained from a substantially identical population of antibodies, which shows binding specificity and affinity for a single epitope.
  • an immunoglobulin has a basic structural unit composed two heavy and two light chains.
  • Each heavy chain comprises one variable region (also known as “region") and three constant domains while each light chain is composed of one variable region and one constant domain.
  • the variable region of each of the light and the heavy chain comprises three complementarity-determining regions (hereinafter referred to as "CDRs") and four framework regions.
  • CDRs function to bind to an epitope of an antibody.
  • CDRs on each chain start from the N terminus and are arranged sequentially as CDR1, CDR2, and CDR3. They are discriminated by the chain on which they are positioned.
  • human antibody is a molecule which consists entirely of the amino acid sequence of all components of human immunoglobulin, including CDRs, framework regions, and the like.
  • human antibodies In the therapy of human diseases, human antibodies have at least three potential advantages.
  • human antibodies more preferably interact with the human immune system to more effectively destroy target cells by, for example, complement-dependent cytotoxicity (CDC) or antibody dependent cell-mediated cytotoxicity (ADCC).
  • CDC complement-dependent cytotoxicity
  • ADCC antibody dependent cell-mediated cytotoxicity
  • Another advantage is that the human immune system does not recognize human antibodies as foreign molecules.
  • the half-lives of human antibodies are similar to those of naturally occurring antibodies in the human circulatory system even when they are administered in smaller doses or with less frequency.
  • TSPAN8 plays a key role in regulating cell motility; however, the molecular mechanisms underlying this function have not been clearly identified.
  • the inventors of the present application suggest that TSPAN8-LEL (amino acids 110-205) may be a unique domain that regulates cancer cell invasion during tumor metastasis.
  • TSPAN8-LEL amino acids 110-205
  • TSPAN8-LEL amino acids 110-205
  • HCT-8 colorectal cancer cell line inhibited HCT-8 cell invasion, while proliferation and adhesion to HUVECs were not affected.
  • HCT-8 cell invasion was inhibited by TSPAN8-LEL but not by TSPAN8-SEL.
  • knockdown of TSPAN8 in the SK-OV3 ovarian cancer cell line also inhibited SK-OV3 cell invasion.
  • Tetraspanins play a key role as molecular facilitators that form molecular webs to bring together large molecular complexes and facilitate their efficient function.
  • tetraspanins have been implicated in many different cellular functions, such as adhesion, motility, and cell fusion.
  • Previous studies have shown that TSPAN8 forms a complex with several binding partners, including EpCAM, claudin-7, and/or CD44v6, and regulates the motility of cancer cells.
  • human antibodies targeting TSPAN8-LEL downregulated cell-surface TSPAN8 via an internalization process, suggesting that this process may be suitable for a therapeutic antibody-drug conjugate.
  • a light-chain variable region comprising light-chain CDR1 defined by an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 to 32;
  • the antibody of the present invention may comprise a heavy-chain variable region comprising heavy-chain CDR1 defined by the amino acid sequence of SEQ ID NO: 11, heavy-chain CDR2 defined by the amino acid sequence of SEQ ID NO: 13, and heavy-chain CDR3 defined by the amino acid sequence of SEQ ID NO: 17, and a light-chain variable region comprising light-chain CDR1 defined by the amino acid sequence of SEQ ID NO: 29 light-chain CDR2 defined by the amino acid sequence of SEQ ID NO: 33, and light-chain CDR3 defined by the amino acid sequence of SEQ ID NO: 36.
  • the antibody of the present invention may comprise a heavy-chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light-chain variable region having the amino acid sequence of SEQ ID NO: 42, but is not limited thereto.
  • a human monoclonal antibody comprised of a heavy-chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light-chain variable region having the amino acid sequence of SEQ ID NO: 42 is designated as clone 3.
  • the nucleic acid encoding the antibody may comprise a heavy-chain variable region having nucleic acid sequence of SEQ ID NO: 27 and a light-chain variable region having nucleic acid sequence of SEQ ID NO: 46, but is not limited thereto.
  • the antibodies composed of heterogeneous sequences were found to inhibit cancer and/or cancer metastasis so long as they specifically recognize TSPAN8-LEL. Therefore, they were identified as effectively applicable to the prophylaxis or therapy of cancer and/or cancer metastasis.
  • a dimer or multimer may be formed from two or more constant domains selected from the group consisting of constant domains of IgG, IgA, IgD, IgE and IgM.
  • hybrid means that sequences encoding two or more heavy-chain constant domains of different origins are present in a single-chain immunoglobulin heavy-chain constant domain.
  • domain hybrids may be composed of one to four domains selected from the group consisting of CH1, CH2, CH3 and CH4 of IgG, IgA, IgD, IgE and IgM.
  • a combination or a hybrid may be made from heavy-chain constant domains of the IgG subtypes IgG1, IgG2, IgG3 and IgG4. The combination and the hybrid are as defined above.
  • the antibody of the present invention further comprises a light-chain constant region, it may be derived from the lamda ( ⁇ ) or kappa ( ⁇ ) light chain.
  • the present invention provides a polynucleotide coding for the epitope or the antibody, an expression vector carrying the polynucleotide, and a transformant anchoring the vector therein.
  • the antibody is as described above.
  • the expression vector carrying the epitope or the antibody according to the present invention may include, but is not limited to, a vector that allows the replication and/or expression of the polynucleotide in eukaryotic or prokaryotic cells such as mammalian cells (e.g., humans, monkeys, rabbits, rats, hamsters, mice, etc.), plant cells, yeasts, insect cells and bacterial cells (e.g., E. coli).
  • the vector has a suitable promoter operably linked to the polynucleotide so as to induce the expression of the gene of interest, and at least one selection marker.
  • the polynucleotide may be introduced into a phage, a plasmid, a cosmid, a mini-chromosome, a virus, or a retroviral vector.
  • the composition comprising the antibody as an active ingredient can be useful for suppressing cancer and/or cancer metastasis and further for preventing or treating cancer and/or cancer metastasis.
  • prevention or “prophylaxis” is intended to refer to any action resulting in the suppression or delay of the onset of diseases or metastasis of interest thanks to the administration of the antibody or composition according to the present invention.
  • treatment or “therapy” is intended to refer to any action resulting in an improvement in the symptoms of a disease of interest or the beneficial alteration of the symptoms thanks to the administration of the antibody or composition according to the present invention.
  • the dose of the pharmaceutical composition of the present invention is not imparted with special limitations, but varies depending on various factors including patient's health state and weight, the severity of disease, the kind of drug, the route of administration, and the time of administration.
  • the composition may be administered in a single dose or in multiple doses per day into mammals including rats, domestic animals, humans, etc. via any typically accepted route, for example, orally, rectally, intravenously, subcutaneously, intrauterinely, or intracerebrovascularly.
  • the pharmaceutical composition is administered at a pharmaceutically effective dose to a subject suspected of cancer.
  • the subject may be a mammal, such as a dog, cow, horse, rabbit, mouse, rat, chicken, and human, but is not limited thereto.
  • the pharmaceutical composition may be administered parenterally, subcutaneously, intraperitoneally, intrapulmonarily, or intranasally, or by a suitable method including, if necessary, intralesional injection for topical treatment.
  • the preferred dose of the pharmaceutical composition of the present invention varies depending on various factors including the subject's health state and weight, the severity of the disease, the kind of drug, the route of administration, and the time of administration, and can be readily determined by those skilled in the art.
  • FITC and RITC may be used, and as for the coloring substrate solution, ABTS (2, 2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)), OPD (o-phenylenediamine), or TMB (tetramethyl benzidine) may be used.
  • ABTS 2, 2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)
  • OPD o-phenylenediamine
  • TMB tetramethyl benzidine
  • biological sample includes samples displaying a difference in expression levels of a cancer marker TSPAN8, preferably TSPAN8-LEL, such as tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine, but is not limited thereto.
  • a cancer marker TSPAN8 preferably TSPAN8-LEL
  • the present invention provides a composition for suppressing cancer and/or cancer metastasis, comprising a material for inhibiting the expression of TSPAN8.
  • the material may be a material for inhibiting the expression of LEL of TSPAN8.
  • the material for inhibiting the expression of TSPAN8, preferably TSPAN8-LEL is antisense oligonucleotides or siRNA oligonucleotides specifically binding to material for inhibiting the expression of TSPAN8, preferably TSPAN8-LEL gene, which is represented by SEQ ID NO:10.
  • the alterations may also include alterations such as capping at 3' and 5'.
  • An alteration can further be performed by adding to an end a polyethyleneglycol, amino acid, peptide, inverted dT, nucleic acid, nucleosides, Myristoyl, Lithocolic-oleyl, Docosanyl, Lauroyl, Stearoyl, Palmitoyl, Oleoyl, Linoleoyl, other lipids, steroids, cholesterol, caffeine, vitamins, pigments, fluorescent substances, anticancer agents, toxins, enzymes, radioactive substances, biotin and the like.
  • the composition for suppressing cancer and/or cancer metastasis may treat cancer and/or cancer metastasis by suppressing cancer and/or cancer metastasis.
  • the present invention provides a use of said fusion protein of LEL of TSPAN8 and Fc for preparation of a pharmaceutical composition for preventing or treating cancer and/or cancer metastasis.
  • the membrane was incubated with TBST (10 mM Tris/HCl, pH 7.5; 150 mM NaCl; and 0.05% Tween 20) containing 5% skim milk at room temperature for 1 h followed by incubation with rat anti-TSPAN8 monoclonal antibody (2 ⁇ g/ml; R&D Systems, Inc., Minneapolis, MN, USA), mouse anti-p120 catenin monoclonal antibody (1:1,000; BD Biosciences, San Jose, CA, USA), mouse anti- ⁇ -actin monoclonal antibody (1:5,000; Applied Biological Materials Inc., Richmond, BC, Canada) in TBST containing 5% skim milk at 4°C overnight.
  • TBST 10 mM Tris/HCl, pH 7.5; 150 mM NaCl; and 0.05% Tween 20
  • TSPAN8-LEL (amino acids 110-205; SEQ ID NO: 9) was amplified using the following primers: 5’-TCGCGGCCCAGGCGGCCAAATCTAAGTCTGATCGCATTG-3’ (SEQ ID NO: 1) and 5’-TCGCGGCCGGCCTGGCCATTTTTTGCCAAGAAGTC-3’ (SEQ ID NO: 2).
  • the TSPAN8-SEL (amino acids 34-57) construct was created using the following primers: 5’-TCGCGGCCCAGGCGGCCCGAGTAAGCAATGACTCTCAAG-3’ (SEQ ID NO: 3) and 5’-TCGCGGCCGGCCTGGCCGTCCACAGCAACGTAGGAG-3’ (SEQ ID NO: 4).
  • PCR fragments were digested with Sfi I (NEB, Ipswich, MA, USA) and cloned into a modified pCEP4 mammalian expression vector (Invitrogen) encoding the hinge and CH 2 -CH 3 domain of human IgG1 at the 3’ end of the cloning site (a kind gift of Dr. Chung, Seoul National University, Seoul, South Korea).
  • Ligated products were transformed into calcium chloride-competent E. coli DH5 ⁇ cells, and plasmid DNA was isolated from these cells.
  • HEK293F cells (6 ⁇ 10 8 cells) were transfected with 0.75 mg of each DNA using 1.5 mg of polyethylenimine (Polysciences, Inc., Warrington, PA, USA).
  • Leukocyte adhesion assays were performed as described previously with minor modifications. Briefly, 6 ⁇ 10 4 HUVECs were pre-plated in 24-well dishes. Simultaneously, mock- or TSPAN8-transfected COS-7 cells or scrambled RNA- or TSPAN8 siRNA-transfected HCT-8 cells were labeled with 5, 6-carboxy-fluorescein succimidyl ester (CFSE; Molecular Probes, Eugene, OR, USA), and 6 ⁇ 10 4 , 1.2 ⁇ 10 5 , 2.4 ⁇ 10 5 , or 4.8 ⁇ 10 5 cells were added to each well.
  • CFSE 6-carboxy-fluorescein succimidyl ester
  • the plate was incubated with 0.1 ⁇ g of irrelevant scFv-Fc or TSAN8-LEL scFv-Fcs in PBST containing 3% BSA for 2 h at 37°C. After two washes with PBST, the plate was incubated with HRP-conjugated anti-human lambda light Chain antibody (1:5,000; Bethyl Laboratories Inc., Montgomery, TX, USA) in PBST containing 3% BSA. After three washes with PBST, 100 ⁇ l of 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solution (BD Biosciences) was added to each well. The reaction was stopped by the addition of an equal volume of 1N H 2 SO 4 to the microplate. Optical density was measured at 450 nm using a microplate reader (VICTORTM X4, Perkin Elmer).
  • TSPAN8-LEL scFv-Fc downregulated the cell-surface TSPAN8 on HCT116 colorectal and SK-OV3 ovarian cancer cell lines, whereas it had little effect on the expression level of TSPAN8 on the surface of either cell line under fixed conditions (Fig. 12), suggesting the internalization-dependent downregulation of cell-surface TSPAN8.
  • TSPAN8-LEL IgG antibody binds to a region (amino acids 31-96) of TSPAN8-LEL
  • TSPAN8-LEL IgG internalizes and down-regulates TSPAN8 on epithelial ovarian cancers
  • TSPAN8-LEL IgG To examine the effect of TSPAN8-LEL IgG on the down-regulation of TSPAN8 on epithelial ovarian cancers, following the treatment of the SK-OV3 cells with TSPAN8-LEL IgG, we performed cell ELISA with HRP-conjugated TSPAN8-LEL IgG to measure TSPAN8 expression on the surface of the cells (Fig. 16A). Here, we confirmed that HRP conjugation little affected the antibody binding to TSPAN8-LEL by comparing the reactivity of TSPAN8-LEL IgG or HRP-comjugated TSPAN8-LEL IgG to TSPAN8-LEL using ELISA (data not shown).
  • TSPAN8-LEL IgG ovarian cancer cell metastasis animal models by injecting luciferase-overexpressing SK-OV3 cells (SK-OV3-luc) into the peritoneal cavity of nude mice. Then, control IgG or TSPAN8-LEL IgG was intravenously injected twice a week from one day prior to the cell injection until 42 days and monitored the metastasis using bioluminescence imaging (Fig. 17A).
  • the present invention Based on the novel finding that the LDL of TSPAN8 plays an important role in cancer and cancer metastasis, the present invention identified the function of a novel antibody against LDL of TSPAN8 in suppressing cancer and/or cancer metastasis. Accordingly, the present invention provides a novel target domain which will be available for suppressing cancer and/or cancer metastasis. Further, the antibody of the present invention can be effective for preventing or treating cancer and/or cancer metastasis.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Cell Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Oncology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Physics & Mathematics (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Hospice & Palliative Care (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Epidemiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Mycology (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)

Abstract

La présente invention concerne un nouvel anticorps se liant spécifiquement à la TSPAN8 (tétraspanine 8) et ses utilisations. Plus particulièrement, la présente invention concerne un anticorps se liant particulièrement à la LEL (grande boucle extracellulaire) de la TSPAN8, un procédé de préparation de l'anticorps, une composition de suppression du cancer et/ou des métastases cancéreuses comprenant l'anticorps, un procédé de suppression du cancer et/ou des métastases cancéreuses par l'administration de l'anticorps ou de la composition, une composition de prévention ou de traitement du cancer et/ou des métastases cancéreuses comprenant l'anticorps, un procédé de prévention ou de traitement du cancer et/ou des métastases cancéreuses par l'administration de l'anticorps ou de la composition, une composition de diagnostic du cancer et/ou des métastases cancéreuses comprenant l'anticorps, une trousse de diagnostic du cancer comprenant la composition, un procédé de diagnostic du cancer utilisant la composition, une composition pour la suppression du cancer et/ou des métastases du cancer comprenant un matériau pour inhiber l'expression de TSPAN8, une trousse pour le cancer et/ou les métastases cancéreuses comprenant la composition, un procédé de suppression du cancer et/ou des métastases cancéreuses ou de traitement du cancer utilisant la composition, et l'utilisation de la LEL de la TSPAN8 comme épitope pour un anticorps de suppression du cancer et/ou des métastases cancéreuses.
PCT/KR2015/001905 2014-02-28 2015-02-27 Nouvel anticorps spécifique de la tspan8 et ses utilisations Ceased WO2015130115A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020167026871A KR101906558B1 (ko) 2014-02-28 2015-02-27 Tspan8에 특이적인 신규 항체 및 이의 용도

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201461946236P 2014-02-28 2014-02-28
US61/946,236 2014-02-28

Publications (1)

Publication Number Publication Date
WO2015130115A1 true WO2015130115A1 (fr) 2015-09-03

Family

ID=54009364

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2015/001905 Ceased WO2015130115A1 (fr) 2014-02-28 2015-02-27 Nouvel anticorps spécifique de la tspan8 et ses utilisations

Country Status (2)

Country Link
KR (1) KR101906558B1 (fr)
WO (1) WO2015130115A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110734494A (zh) * 2019-10-30 2020-01-31 上海市第一人民医院 抗tspan8单克隆抗体及其用途
WO2022102768A1 (fr) 2020-11-16 2022-05-19 アステラス製薬株式会社 Anticorps bispécifique anti-tspan8-anti-cd3 et anticorps anti-tspan8

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12049493B2 (en) 2017-01-06 2024-07-30 Abl Bio Inc. Anti-alpha-synuclein antibodies and uses thereof
AU2018385230B2 (en) * 2017-12-14 2022-10-13 Abl Bio Inc. Bispecific antibody to a-syn/IGF1R and use thereof
KR102775728B1 (ko) * 2021-04-27 2025-03-06 고려대학교 산학협력단 테트라스파닌-12(Tetraspanin-12, TSPAN12)를 포함하는 뇌종양 진단용 바이오마커 조성물

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003092630A2 (fr) * 2002-05-06 2003-11-13 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Identification d'anticorps monoclonaux humains largement neutralisants a reaction croisee au moyen d'un procede d'adherence sequentielle sur plaque d'antigenes de banques de presentation du phage
WO2012010696A1 (fr) * 2010-07-23 2012-01-26 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthodes de gestion du cancer ciblant co-029

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003092630A2 (fr) * 2002-05-06 2003-11-13 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Identification d'anticorps monoclonaux humains largement neutralisants a reaction croisee au moyen d'un procede d'adherence sequentielle sur plaque d'antigenes de banques de presentation du phage
WO2012010696A1 (fr) * 2010-07-23 2012-01-26 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthodes de gestion du cancer ciblant co-029

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AZORSA ET AL.: "A general approach to the generation of monoclonal antibodies against members of the tetraspanin superfamily using recombinant GST fusion proteins", JOURNAL OF IMMUNOLOGICAL METHODS, vol. 229, no. 1, 1999, pages 35 - 48, XP002194216 *
DATABASE genbank [O] 15 July 2006 (2006-07-15), XP055220402, retrieved from ncbi Database accession no. AAH05246.1 *
DATABASE genbank [O] 2 July 2002 (2002-07-02), XP055220405, retrieved from ncbi Database accession no. BAC01631.1 *
DATABASE genbank [O] 7 July 2013 (2013-07-07), XP003034229, retrieved from ncbi Database accession no. AGP01549.1 *
HEMLER: "Tetraspanin proteins promote multiple cancer stages", NATURE REVIEWS CANCER, vol. 14, no. 1, January 2014 (2014-01-01), pages 49 - 60 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110734494A (zh) * 2019-10-30 2020-01-31 上海市第一人民医院 抗tspan8单克隆抗体及其用途
WO2021083248A1 (fr) * 2019-10-30 2021-05-06 上海市第一人民医院 Anticorps monoclonal anti-tspan8 et son utilisation
WO2022102768A1 (fr) 2020-11-16 2022-05-19 アステラス製薬株式会社 Anticorps bispécifique anti-tspan8-anti-cd3 et anticorps anti-tspan8
US11897954B2 (en) 2020-11-16 2024-02-13 Astellas Pharma Inc. Anti-TSPAN8/anti-CD3 bispecific antibody and anti-TSPAN8 antibody
US12304951B2 (en) 2020-11-16 2025-05-20 Astellas Pharma Inc. Anti-TSPAN8/anti-CD3 bispecific antibody and anti-TSPAN8 antibody

Also Published As

Publication number Publication date
KR101906558B1 (ko) 2018-10-11
KR20160127115A (ko) 2016-11-02

Similar Documents

Publication Publication Date Title
WO2013187724A1 (fr) Nouvel anticorps spécifique de clec14a et utilisations de celui-ci
KR100932698B1 (ko) 폐암의 치료용 약제학적 조성물 및 이를 이용한 폐암의성장, 전이 억제 및 치료방법
WO2019124951A9 (fr) Inhibiteurs d'hétéromères gpcr et leurs utilisations
WO2015130115A1 (fr) Nouvel anticorps spécifique de la tspan8 et ses utilisations
WO2016153276A1 (fr) Peptide liant spécifiquement la neuropiline-1, protéine de fusion fusionnée à celui-ci et utilisation correspondante
WO2014189303A1 (fr) Peptide transtumoral spécifique de la neuropiline et protéine de fusion comprenant ce peptide fusionné
WO2016013870A1 (fr) Procédé pour positionner, dans le cytoplasme, un anticorps au format immunoglobuline complète par pénétration de l'anticorps à travers la membrane cellulaire, et son utilisation
CN110139874B (zh) 抗ceacam6抗体及其使用方法
WO2010087594A9 (fr) Utilisation de cd93 ou d'un fragment soluble de celui-ci
WO2017010744A1 (fr) Composition antigénique de détection d'un auto-anticorps avec réponse spécifique à la protéine exosomale eif3a, et méthode de diagnostic du cancer du foie utilisant la composition antigénique
WO2018174544A2 (fr) Anticorps se liant spécifiquement à muc1 et son utilisation
WO2021091319A1 (fr) Aptamère modifié spécifique du glypicane-3 et son utilisation
WO2021225423A1 (fr) Nouveau ligand d'acide nucléique et son procédé d'identification
WO2014021693A2 (fr) Anticorps monoclonal inédit se liant spécifiquement à la protéine tm4sf5 et son utilisation
WO2017171373A2 (fr) Composition pour réduire la résistance à un agent ciblant l'egfr
WO2020250204A1 (fr) Nouveaux anticorps spécifiques de cthrc1 et leur utilisation
WO2024196162A1 (fr) Nouveaux anticorps anti-r-spondine 2 humaine et leur utilisation
WO2024196163A1 (fr) Nouvel anticorps anti-r-spondine-3 humaine et son utilisation
WO2024155098A1 (fr) Antigène de surface de lymphocyte t régulateur et anticorps se liant de manière spécifique à celui-ci
WO2022131889A1 (fr) Utilisation de protéine taci
WO2021015419A1 (fr) Anticorps se liant de manière spécifique à la bêta-caténine phosphorylée et utilisation associée
WO2019022274A1 (fr) Anticorps se liant spécifiquement à une protéine pauf, et utilisation associée
WO2024232668A1 (fr) Anticorps monoclonal anti-ptgfrn et son utilisation
US20250154240A1 (en) Anti-human cxcl1 antibody
WO2020117017A1 (fr) Anticorps agoniste anti-c-met et utilisation associée

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15755513

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 20167026871

Country of ref document: KR

Kind code of ref document: A

122 Ep: pct application non-entry in european phase

Ref document number: 15755513

Country of ref document: EP

Kind code of ref document: A1