WO2008004760A1 - Antibody specifically binding to dr5 and composition for preventing or treating cancers comprising the same - Google Patents

Antibody specifically binding to dr5 and composition for preventing or treating cancers comprising the same Download PDF

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Publication number
WO2008004760A1
WO2008004760A1 PCT/KR2007/002644 KR2007002644W WO2008004760A1 WO 2008004760 A1 WO2008004760 A1 WO 2008004760A1 KR 2007002644 W KR2007002644 W KR 2007002644W WO 2008004760 A1 WO2008004760 A1 WO 2008004760A1
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WIPO (PCT)
Prior art keywords
antibody
cancer
seq
trail
scfv
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PCT/KR2007/002644
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French (fr)
Inventor
Yong Sung Kim
Myung Hee Kwon
Seung Hyun Lee
Kyung Jin Park
Hae Won Lee
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Ajou University Industry-Academic Cooperation Foundation
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Priority to JP2009517957A priority Critical patent/JP2009542202A/en
Priority to EP07746791A priority patent/EP2046836A4/en
Priority to US12/307,293 priority patent/US8097704B2/en
Publication of WO2008004760A1 publication Critical patent/WO2008004760A1/en

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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
    • 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
    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to an antibody specifically binding to death receptor 5 (DR5), and a composition for preventing or treating cancers comprising the same.
  • DR5 death receptor 5
  • TRAIL TNF-related apoptosis inducing ligand
  • caner cell-specific therapeutic agents which target DR4 or DR5
  • a method using a recombinant TRAIL for example, 114 th to 281 st amino acid residues of TRAIL
  • a method of developing an agonistic antibody among mouse- or human-derived complete antibodies e.g.: mAb or IgG
  • mAb or IgG mouse- or human-derived complete antibodies
  • TRAIL is very unstable and tends to form a soluble oligomer, whose apoptotic activity is about 20 to 100 times lower, and it causes side effects such as cytotoxicity and immune reaction on normal cells such as astrocytes, hepatocytes and keratinocytes (Jo et al., Nat. Med., 6:564-567, 2000). Further, TRAIL is not capable of inducing more than 50% cell death of malignant tumor cells (Zhang et al., Cancer Gene Ther., 12:228-237, 2005). Hence, a cancer cell that can be killed by TRAIL is designated a TRAIL- sensitive cancer cell, and a cancer cell not killed by TRAIL, a TRAIL-resistant cancer cell.
  • humanized antibodies developed from mouse-derived monoclonal antibodies such as TRA-8 (mouse IgG) (Walczak et al, Nat. Med., 5: 157-161) and AD5-10 (mouse IgG) (Guo et al., J. Biol. Chem., 280:41940-41952, 2005), as well as human-derived monoclonal antibodies such as HGS-ETR2 (human IgGl) (Georgakis et al., Br. J. Haematol, 130:501-510, 2005) and KMTR2 (human IgG4) (Motoki et al., CHn. Cancer Res., 11 :3126- 3135, 2005).
  • TRA-8 mouse IgG
  • AD5-10 mimouse IgG
  • human-derived monoclonal antibodies such as HGS-ETR2 (human IgGl) (Georgakis et al., Br. J. Haematol, 130
  • the above antibodies induce apoptotic cell death of TRAIL- sensitive cancer cells but not TRAIL-resistant cancer cells.
  • the antibodies in the form of an antigen-binding fragment (Fab) or a single chain variable fragment (scFv) having a monovalent antigen binding site does not induce cell death of cancer cells (e.g.: KMTR2)
  • antibodies in the form of IgG having divalent antigen binding site e.g.: HGS-ETR2 and AD5-10) show cytotoxicity or induced cell death when an IgG is used as a cross-linker (Chuntharapai et al., J. Immunol, 166:4891-4898, 2001; Motoki et al., Clin.
  • an antibody specifically binding to death receptor 5 which is selected from the group consisting of: an antibody comprising a heavy chain variable region (V H ) having the amino acid sequences of SEQ ID NOs: 1 to 3 at complementary determining regions (CDRs) and a light chain variable region
  • V L having the amino acid sequences of SEQ ID NOs: 4 to 6 at CDRs; and an antibody comprising a V H having the amino acid sequences of SEQ ID NOs: 7 to 9 at CDRs and a V L having the amino acid sequences of SEQ ID NOs: 10 to
  • a cell transformed with the DNA or an expression vector comprising the DNA in accordance with a further aspect of the present invention, there is provided a cell transformed with the DNA or an expression vector comprising the DNA.
  • composition for preventing or treating a cancer comprising the antibody as an active ingredient.
  • a method of preventing or treating a cancer comprising administering the antibody to a subject.
  • Figs. IA and IB amino acid sequences of HWl and HW6, and DNAs encoding thereof as anti-DR5 scFv antibodies specifically binding to DR5, respectively (wherein, CDRl to CDR3 of a heavy chain variable region, a linker oligopeptide and CDRl to CDR3 of a light chain variable region of each antibody are underlined in order);
  • Fig. 2 the result of ELISA quantifying the antibody titer against DR5, DcRl and BSA in 8 serums showing high antibody titers against DR5;
  • Fig. 3 the result of FACS (fluorescence activated cell sorting) isolating an anti-DR5 scFv antibody library expressed on cell surface by using biotin- labeled DR5 as an antigen;
  • FACS fluorescence activated cell sorting
  • Fig. 4 a diagram of an E. coli expression vector prepared by cloning HWl and HW6 to pKJl vector, respectively;
  • Figs. 5 A and 5B the results of SDS-PAGE and western blotting, respectively, obtained for HWl and HW6 expressed in E. coli and purified;
  • Fig. 6 the result of size-exclusion chromatography of purified HWl and HW6;
  • Figs. 7 A and 7B the results of reducing SDS-PAGE and non-reducing SDS-PAGE of purified HWl and HW6;
  • Figs. 8A to 8D the results of SPR (surface plasmon resonance) showing the cross reactivity of HWl (Figs. 8A and 8B) and HW6 (Figs. 8C and 8D) to antigens, DR5, and DcRl, DcR2, DR4, TNFRl, TNFR2 and CD95, respectively;
  • Figs. 9A and 9B images showing the binding of Alexa633-labeled HWl
  • Fig. 9A or TRAIL (Fig. 9B) to YFP (yellow fluorescence protein)-labeled DR5 (DR5 ⁇ CDYFP) and DcR2 (DcR2 ⁇ CDYFP) in HCTl 16 cells (human colon cancer cell);
  • Figs. 1OA and 1OB the results of competitive ELISA examining whether or not the binding sites of HWl and HW6 to DR5 are identical to those of TRAIL;
  • Figs. 1 IA and 1 IB the results of MTT assay showing the degree of cell death induced in TRAIL-sensitive HCTl 16, HL60 and DU 145 cells as function of the concentration of TRAIL (Fig. 1 IA) and HWl and HW6 (Fig. 1 IB);
  • Figs. 12A to 12D the results of MTT assay showing the degree of cell death induced in TRAIL-resistant HepG2, U87MG and Molt-4 cells as function of the concentration of TRAIL (Figs. 12A and 12C) and HWl and HW6 (Figs. 12B and 12D);
  • Figs. 13A and 13B microscopic images showing the cell death induced in TRAIL-resistant HepG2 cells (Fig. 13A) and Huh7 cell (Fig. 13B) by TRAIL, HWl and HW ⁇ ;
  • Figs. 14A and 14B the results of cytotoxicity test showing that no cytotoxicity to normal human cells, hepatocytes (Fig. 14A) and mammary epithelial cells (Fig. 14B) was caused by increased concentration of TRAIL and HWl;
  • Fig. 15 microscopic images showing that no cytotoxicity to normal human brain astrocytes was caused by HWl or HW6;
  • Fig. 16 the result of MTT assay to quantify the cytotoxicity of varying concentration of HWl and TRAIL in HCTl 16 cells, after treating cell membrane-expressed DR5 and soluble competitor DR5-Fc;
  • Fig. 17 the result of MTT assay to quantify cell death by TRAIL and HWl of TRAIL-sensitive HCTl 16 cells in the presence or absence of sulforaphane;
  • Fig. 18 the result of MTT assay to quantify the cytotoxicity of HWl for TRAIL-sensitive HCTl 16 cells and TRAIL-resistant HepG2 cells in the presence or absence of a cross-linker (CL);
  • Figs. 19A to 19D microscopic images showing that cell death of TRAIL-sensitive cells (HCTl 16 and Dul45) and TRAIL-resistant cells (HepG2 and U87MG) induced by HWl is autophagic cell death (wherein, Fig. 19A is
  • Figs. 19B to 19D are high-magnification images showing multiple-membrane-bound autophagic vacuoles, double-membrane-bound autophagic vacuoles, and autophagic vacuole induction step by fusing empty vacuoles with vacuoles containing cellular organelles, respectively);
  • Figs. 2OA and 2OB fluorescent microscopic images obtained using lysotracker-red DND-99 which specifically stains cellular autophagic vacuoles, showing autophagic cell death induced by HWl on TRAIL-sensitive HCTl 16 cells (Fig. 20A) and TRAIL-resistant U87MG cells (Fig. 20B).
  • DR5 protein means a receptor as a member of tumor necrosis factor (TNF) receptor family, which binds to TRAIL and has a C-terminal cytoplasmic death domain (Pan et al., Science, 277:815-818, 1997).
  • TNF tumor necrosis factor
  • DR5 includes any protein having the above characteristics, e.g., a protein having the amino acid sequences described in United States Patent No. 6,872,568.
  • the term "antibody” may be a whole antibody or a fragment thereof.
  • the whole antibody can be a monomer selected from the group consisting of IgG, IgM, IgA, IgD and IgE, or a multimer formed by combining monomers.
  • a functional fragment of an antibody means an antibody having heavy chain and light chain variable regions of a whole antibody actually recognizing the same epitope recognized by the whole antibody.
  • the functional fragment of an antibody may be a single chain variable fragment (scFv), (ScFv) 2 , Fab, Fab', F(ab') 2 or scFv-Fc, and it is preferably scFv.
  • single chain variable fragment means an antibody fragment of a single chain polypeptide form having a heavy chain variable region and a light chain variable region linked through a linker peptide.
  • the antibody may be produced by any of the conventional methods known to those in the art, such as the phage display method or yeast cell surface expression system.
  • scFv may be prepared by any of the conventional methods known to those in the art, e.g., the method described in United States Patent No.
  • 4,946,778 or 5,258,498, and Fab, Fab 1 and F(ab') 2 fragments may be prepared by recombinant antibody technology, e.g., the method described in International
  • Patent Publication No. WO 92/22324 Patent Publication No. WO 92/22324.
  • the inventive antibody may be derived from any animal which may be mammals excluding human, birds and so on.
  • the antibody may be derived from a human, mouse, donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken.
  • the antibody derived from a human is an antibody having amino acid sequences of human immunoglobulin, and may include an antibody isolated from human immunoglobulin libraries, or an antibody isolated from animals, which are transformed to produce one or more human immunoglobulins and incapable of expressing endogenous immunoglobulin (see United States Patent No. 5,939,598)
  • the inventive antibody may be conjugated with a marker including, but not limited to, an enzyme, fluorescent material, radioactive material, protein and so on.
  • a marker including, but not limited to, an enzyme, fluorescent material, radioactive material, protein and so on.
  • the methods to conjugate the materials are well known in the art.
  • the inventive antibody specifically binds to DR5 protein.
  • the term "specifically bind” means that the inventive antibody does not bind to DR5-like TNFR family receptors such as DcRl (death decoy receptor 1), DcR2, DR4, TNFRl, TNFR2 and CD95.
  • the amino acid sequences of SEQ ID NOs: 1 to 3 or 7 to 9 are CDRl, CDR2 and CDR3, respectively, of the heavy chain variable region of an antibody specifically binding to DR5, while the amino acid sequences of SEQ ID NOs: 4 to 6 or 10 to 12 are CDRl, CDR2 and CDR3, respectively, of the light chain variable region of an antibody specifically binding to DR5.
  • scFv antibodies HWl and HW6, which have the amino acid sequences of SEQ ID NOs: 13 and 14, respectively, and the antibodies comprise CDRl to CDR3 of a heavy chain variable region, linker-oligopeptide and CDRl to CDR3 of a light chain variable region in order ⁇ see Figs. IA and IB).
  • HWl and HW6 specifically bind to DR5, and have binding affinities
  • the inventive antibody is believed to induce autophagic cell death of various cancer cells including TRAIL-resistant cancer cells through specific binding to epitopes (binding sites) of DR5 which do not overlap with the TRAIL-binding epitopes.
  • the present invention further provides a DNA encoding the inventive antibody.
  • the DNA is preferably a DNA encoding the scFv having the amino acid sequence of SEQ ID NO: 13 or 14, and more preferably, a DNA having the nucleotide sequence of SEQ ID NO: 15 or 16.
  • the DNA sequence encoding the inventive antibody can be obtained by any of the conventional methods known in the art. For example, based on the DNA sequence encoding the antibody heavy or light chain, a part thereof, or the corresponding amino acid sequence, an appropriate DNA sequence can be synthesized by the well known oligonucleotide synthesis method, e.g., site- directed mutagenesis and polymerase chain reaction (PCR).
  • oligonucleotide synthesis method e.g., site- directed mutagenesis and polymerase chain reaction (PCR).
  • the present invention provides a cell transformed with the inventive DNA or an expression vector comprising the DNA.
  • the inventive DNA or the expression vector comprising the DNA may be transferred to a host cell by any of the conventional methods, e.g., viral transfection or non-viral method.
  • introduction of the DNA or the expression vector may be conducted in accordance with any of the methods known to those in the art including adenoviral transformation, gene gun, liposome-mediated transformation, retrovirus or lentivirus-mediated transformation, plasmid or adeno-associated virus.
  • the transformed cell may be transplanted together with carriers having gene delivery system, which can release or deliver the designed gene to the cells of a subject for a long period of time.
  • the present invention further provides a method of producing the inventive antibody molecule comprising the steps of: 1) expressing the antibody by culturing a host cell comprising the inventive DNA sequence or an expression vector comprising the DNA under a suitable condition; and 2) isolating the expressed antibody.
  • the antibody molecule may be accumulated in the cell cytoplasm, or in the periplasm or extracellular medium (supernatant) using a proper signal sequence, and the accumulation thereof in the periplasm or extracellular medium is preferred. Further, the produced antibody molecule may be refolded to confer a functional conformation thereto using any of the conventional methods known in the art. In order to produce only a heavy chain or a light chain polypeptide of the antibody molecule, a single vector comprising a heavy chain or light chain polypeptide may be transformed into a host cell.
  • both the first vector encoding a light chain polypeptide and the second vector encoding a heavy chain polypeptide, or a single vector comprising both heavy chain and light chain polypeptides may be transformed into a host cell.
  • the inventive antibody which induces autophagic cell death of DR5 -expressed TRAIL-sensitive and TRAIL-resistant cancer cells by specific binding to DR5
  • Such cancers may be any cancer expressing DR5 and include TRAIL-sensitive and TRAIL-resistant cancers, such as breast cancer, colon cancer, brain tumor, glioma, ovarian cancer, endometrial cancer, bone sarcoma, cervix cancer, prostatic cancer, lung cancer, synovial cancer, pancreatic cancer and sarcoma.
  • the present invention further provides a composition for preventing or treating cancer comprising the inventive antibody as an active ingredient.
  • the inventive composition for preventing or treating cancer may additionally comprise one or more pharmaceutically acceptable excipients, carriers, diluents and so on.
  • Example of the carrier employed in the present invention is a slowly metabolized macromolecule such as protein, polypeptide, liposome, polysaccharide, polylactic acid, polyglycolic acid, polymeric amino acid, amino acid polymer and inactive viral particle.
  • a pharmaceutically acceptable salt such as a salt of inorganic acid (e.g., hydrochloride, hydrobromide, phosphate and sulfate), and a salt of organic acid (e.g., acetate, propionate, malonate and benzoate); a liquid such as water, saline solution, glycerol and ethanol; and an auxiliary material such as a wetting agent, an emulsifier and a pH buffer may be used.
  • inorganic acid e.g., hydrochloride, hydrobromide, phosphate and sulfate
  • organic acid e.g., acetate, propionate, malonate and benzoate
  • a liquid such as water, saline
  • the pharmaceutically acceptable carrier is described in [Remington's Pharmaceutical Sciences, Mack Publishing Company, 1991].
  • composition may be formulated to a unit dosage form suitable for administering to a patient, preferably for administering a protein drug, by conventional methods in the pharmaceutical field, and it can be administered in accordance with any conventional method in the art through oral route or parenteral route such as intravenous, intramuscular, intraarterial, intramedullar, intrathecal, intraventricular, intrapulmonary, intracutaneous, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or intrarectal route without limitation.
  • oral route or parenteral route such as intravenous, intramuscular, intraarterial, intramedullar, intrathecal, intraventricular, intrapulmonary, intracutaneous, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or intrarectal route without limitation.
  • Example of the formulations suitable for such purposes may include oral formulations such as tablet, pill, dragee, powder, capsule, syrup, solution, gel, suspension, emulsion and microemulsion; and parenteral formulations such as injection formulation (e.g., injection ampule), infusion formulation, and spray formulation (e.g., hypospray).
  • injection formulation or infusion formulation may be in the form of suspension, solution or emulsion, and include formulation aids such as suspending agents, preservatives, stabilizers and/or dispersing agents.
  • the antibody molecule may be formulated in the form of a dried formulation, which can be readjusted to a suitable sterile solution before use.
  • composition of the present invention comprises an antibody molecule easily degraded in the gastrointestine
  • composition for gastrointestinal administration is preferred to include an agent, which protects the antibody from degradation and is absorbed into the gastrointestine after releasing the antibody.
  • the present invention further provides a method of preventing or treating a cancer comprising a step of administering the inventive antibody to an animal, preferably a mammal, more preferably a human, in accordance with any methods described above.
  • composition or pharmaceutical formulation may be used solely or in combination with other anticancer agents, for instance, TRAIL or an anticancer agent conventionally used in the art.
  • inventive antibody may be administered by gene therapy.
  • DNAs encoding heavy and light chains of the inventive antibody or an expression vector thereof should be introduced into a patient by any of the conventional methods known in the art so that the heavy and light chains are combined in situ to form the antibody molecule.
  • inventive antibody as an active ingredient of the inventive composition or pharmaceutical formulation may be administered in a dose ranging from about 0.01 to 50 mg/kg (body weight), preferably 0.1 to 20 mg/kg (body weight) per day in case of a mammal including a human being.
  • inventive composition or pharmaceutical composition may be administered in a single dose or in divided doses.
  • the amount of the active ingredient actually administered ought to be determined in light of various relevant factors including the disease to be treated, the condition to be treated, the severity of the patient's symptom, the chosen route of administration, and the body weight, age and sex of the individual patient, drug combination, reaction sensitivity, and tolerance/reactivity to treatment; and, therefore, the above dose should not be intended to limit the scope of the invention in any way.
  • Example 1 Selection of a serum having a high antibody titer against DR5
  • each well of a 96-well ELISA plate (Nunc, Invitrogen) was coated with 10 ⁇ g/mC of a test antigen dissolved in TBS (50 mM Tris-HCl, pH 7.5, 50 mM NaCl) at 25 °C for 1 hour, washed with TBS containing 0.05% tween 20 (TBST) 3 times, and blocked with TBS containing 3% BSA.
  • TBS 50 mM Tris-HCl, pH 7.5, 50 mM NaCl
  • TBS 0.05% tween 20
  • the selected 8 serums each showed antibody titers against DR5 and DcRl which are more than 2 times higher than that of BSA as a control.
  • V H heavy chain variable region
  • V ⁇ and V ⁇ light chain variable region
  • the primers used were those of Little et al. (J. Immunol. Methods, 231 :3-9, 1999) with the following slight modifications. Specifically, in order to generate a linker sequence between V H and V L gene, the sequence 5 '-CGA GCC CCC GCC ACC CGA ACC GCC CCC ACC TCT-3' (SEQ ID NO: 17) was added to the 5' end of the reverse primer of V H and the sequence 5 '-GGT TCG GGT GGC GGG GGC TCG GGC GGG GGT GGC TCA GAT CT-3 ' (SEQ ID NO: 18) was added to the 5' end of the forward primers of V ⁇ and Vj 1 .
  • the sequence 5 '- AGT GGT GGT GGT GGT TCT GGT GGT GGT GGT GGT TCT GGT GGT GGT GGT GGT TCT GCT AGC-3' was added to the 5' end of the forward primer of V H and the sequence 5 ' -TCA GAT CTC GAG CTA TTA CAA GTC CTC TTC AGA AAT AAG CTT TTG TTC GGA TCC-3' (SEQ ID NO: 20) was added to the 5' end of the reverse primers of V ⁇ and V ⁇ .
  • the amplified V H and V L genes were subjected to 1% agarose gel electrophoresis and purified. Equal amounts of the purified V H and V L genes were mixed together, and overlap extension PCR was conducted to prepare scFv gene repertoires. The PCR products were purified as described above. Each PCR condition was 30 cycles of 2 min at 95 "C , 1 min at 55 ° C and 1 min at 72 ° C .
  • the transformants were pooled and propagated directly in liquid selective SD-CAA medium (-ura, -trp), which contained 20 g/£ glucose, 6.7 g/t yeast nitrogen base without amino acids (Difco, USA), 5.4 g/t Na 2 HPO 4 , 8.6 g/£ Na 2 HPO 4 H 2 O and 5 g/£ casamino acid, and then the libraries which induced cell surface expression of scFv were selected using a selective SG-CAA medium, which contained the same composition as SD-CAA, except that glucose was replaced with galactose.
  • the cells containing the libraries were each serially diluted 10-fold with the selective SD-CAA medium, and the library size was determined by plating each diluted cells on the selective SD-CAA agar plate.
  • Example 3 Selection of an anti-DR5 scFv antibody library
  • the cells containing the selected libraries were incubated with anti-c-myc 9E10 mAb (1:100 dilution, Ig Therapy) and 1 ⁇ M of biotin-labeled DR5 by EZ-LINKTM Sulfo-NHS-LC- Biotinylation kit (Pierce, USA) at 25 ° C for 30 min in 0.2 mH of PBSB (phosphate-buffered saline, pH 7.4, containing 1 mg/m£ of BSA).
  • PBSB phosphate-buffered saline, pH 7.4, containing 1 mg/m£ of BSA
  • the cells were washed with ice-cold PBSB, and labeled with FITC-labeled anti-mouse IgG (1 :25 dilution) and streptavidin-R-phycoerythrin conjugate (SA-PE, 1 :100 dilution; Molecular Probes, USA) as a secondary antibody, respectively.
  • FITC-labeled anti-mouse IgG 1 :25 dilution
  • SA-PE streptavidin-R-phycoerythrin conjugate
  • the labeled cells were washed, resuspended in PBSB, and then one round of MACS (magnetic activated cell sorting) was performed using biotin-labeled DR5 to float the scFv library having the binding affinity to DR5, followed by performing FACS (fluorescence activated cell sorting) to select HWl and HW6 bound to the antigen in the anti-DR5 scFv antibody libraries expressed on yeast cell surface.
  • MACS magnetic activated cell sorting
  • FACS fluorescence activated cell sorting
  • the nucleotide sequences of the selected HWl and HW6 were analyzed using a forward primer 5'-GTT CCA GAC TAC GCT CTG CAG G-3' (SEQ ID NO: 1
  • the isolated antibodies HWl and HW6 of Example 3 were subcloned in-frame into a bacterial expression vector using restriction enzymes Nhel and BamHl.
  • the bacterial expression vector was expression vector pKJl designed to encode T7 promoter-PelB periplasm targeting sequence-human scFv-Flag tag-6 ⁇ His tag (Fig. 4). This vector was prepared by inserting PeIB nucleotide sequence and restriction enzyme site Nhel/ BamHl based on pET21b vector (Novagen) and fusing Flag tag and 6> ⁇ His tag on the C-terminus.
  • the expression vector was transformed into E.coli BL21 (DE3) (Novagen), and the transformed cells were cultured at 37 ° C until the OD at 600 ran reached 0.6 to 0.8, followed by induction with 0.5 mM IPTG at 25 ° C for 20 hours.
  • the periplasm fraction or supernatant of the cultured bacteria was subjected to Talon resin (Clontech) to purify the expressed antibody, and the size and the purity of the purified antibody were analyzed by SDS-PAGE and western blotting using anti-His tag, respectively.
  • the antibodies each having size of about 29 kDa (Fig. 5A), and a purity of more than 98% were obtained (Fig. 5B).
  • Test Example 1 Confirmation of anti-DR5 scFv antibody form
  • Example 4 In order to identify whether the HWl and HW6 purified in Example 4 were in the monomeric or oligomeric form in solution, size exclusion HPLC (SEC), reducing SDS-PAGE and non-reducing SDS-PAGE were performed, respectively.
  • SEC size exclusion HPLC
  • HWl and HW6 were each eluted as a monomeric form in solution at concentrations of 10 mg/m£ as determined by size-exclusion chromatography. Further, there were no antibodies of the oligomeric form, which have non-native disulfide bonds, as determined by reducing SDS-PAGE
  • the inventive antibody exists in the monomeric form even at a very high concentration.
  • Test Example 2 Analysis of the binding affinity and cross reactivity of anti-DR5 scFv antibody
  • the binding affinity of the anti-DR5 scFv antibody to DR5, and the cross-reactivities of the antibody with other antigens, DcRl, DcR2, DR4, TNFRl, TNFR2 and CD95 were determined by Biacore 2000 SPR (surface plasmon resonance) biosensor (Pharmacia, Sweden), respectively.
  • CM5 chip carboxymethylated dextran surface chip; Pharmacia
  • HWl 200-3,200 nM
  • HW6 25-1,000 nM
  • the surface of the chip was regenerated with 25 mM NaCl/50 mM NaOH, and the kinetic rate constants (k on and k Off ) as well as the equilibrium dissociation constant (K D ) were determined by using BIA evaluation software ver. 3.2. The results are shown in Table 1, and the results of cross-reactivity are shown in Figs. 8A to 8D.
  • HWl and HW6 bind to DR5 with high affinities (Figs. 8A and 8C), but they showed no affinity to DcRl, DcR2, DR4, TNFRl, TNFR2 and CD95 even at a very high concentration (up to 10 ⁇ M) (Figs. 8B and 8D). Accordingly, it was confirmed that the inventive HWl and HW6 specifically bind only to DR5.
  • Test Example 3 Confirmation of the binding of anti-DR5 scFv antibody to DR5 expressed on a cell
  • plasmid TOlO expressing protein DR5 ⁇ CDYFP which is a fusion protein of extracellular domain of DR and YFP (yellow fluorescent protein)
  • plasmid T30 expressing protein DcR2 ⁇ CDYFP which is a fusion protein of extracellular domain of DcR2 and YFP
  • HCTl 16 colon cancer cell (CCL-247, ATCC (American Type Culture Collection)) by electroporation to overexpress each of the proteins.
  • the transformed cells were seeded at a concentration of 5 ⁇ lO 4 cells
  • HWl and TRAIL were stained with red fluorescence using Alexa633 labeling kit (Molecular Probes, USA), and added to the cultured transformed cells, followed by reacting the cells at 4 " C for 30 min.
  • Each well was washed 3 times with PBS containing 2% FCS and 2% paraformaldehyde, and observed with a fluorescent microscope (LMS510 model laser scanning confocal fluorescence microscope, Carl Zeiss). The results are shown in Figs. 9 A and 9B.
  • HWl specifically binds to
  • HWl antibody specifically binds to DR5 expressed on the cell membrane as well as to DR5 in solution, without any cross-reactivity.
  • Test Example 4 Identification of an epitope of DR5 for anti-DR5 scFv antibody
  • HWl, HWl+TRAIL, HW6 and HW6+TRAIL having varying concentrations of the HWl or HW6 in the range of I x 10 "4 to I x 10 3 ⁇ gM and 20 ⁇ g/mt of TRAIL were added to each well, and the plate was incubated at 37 °C for 1 hour and washed.
  • alkaline phosphatase-conjugated anti- mouse IgG Fc specific antibody Sigma
  • a secondary antibody was added to each well and the plate was incubated at 37 ° C for 1 hour, followed by washing the plate 3 times.
  • 50 ⁇ l of /?-NPP substrate was added to each well and the plate was incubated for 100 min, and the absorbance at 405 ran was measured.
  • the binding of both HWl and HW6 to DR5 was enhanced with the concentration of either of the antibodies regardless of the presence of TRAIL.
  • the binding of HWl and HW6 to DR5 was not influenced by the treatment with a high concentration up to 500 ng/m£ of TRAIL.
  • HWl and HW6 bind to epitopes of DR5 that does not overlap with the TRAIL binding sites.
  • Test Example 5 Test of cancer cell death induced by anti-DR5 scFv antibody
  • HWl and HW6 were each evaluated using various cancer cells (ATCC), i.e., HL60 (human acute myeloid leukemia cell), HCTl 16 (human colon cancer cell line) and Du 145 (human prostate cancer cell) for TRAIL-sensitive cells; and HepG2 and Huh7 (human hepatocellular carcinoma cancer cell), U87MG (human astrocyte cancer cell) as well as Molt-4 (Human T-lymphoblastoid leukemia) for TRAIL-resistant cells.
  • ATCC i.e., HL60 (human acute myeloid leukemia cell), HCTl 16 (human colon cancer cell line) and Du 145 (human prostate cancer cell) for TRAIL-sensitive cells
  • HepG2 and Huh7 human hepatocellular carcinoma cancer cell
  • U87MG human astrocyte cancer cell
  • Molt-4 Human T-lymphoblastoid leukemia
  • the adherent cells such as HCTl 16, Dul45, HepG2, Huh7 and U87MG were each incubated in DMEM medium supplemented with 10% (v/v) fetal calf serum (FCS) (Gibco Invitrogen), and the non-adherent cells such as HL60 and Molt-4 were incubated in RPMI 1640 medium supplemented with 10% FCS, 100 unitsM penicillin, and 100 ⁇ g/mt of streptomycin. The incubation was conducted at 37 ° C under 5% CO 2 .
  • FCS fetal calf serum
  • U87MG cells were each treated with 1 mi of TE buffer (Trypsin-EDTA), followed by adding 5 m € of DMEM medium containing 10% FBS to stop the reaction of TE buffer. The resulting cells were isolated by centrifugation at
  • HL60 and Molt-4 cells were directly recovered from the culture flask, and isolated by centrifugation at 1,000 rpm for 5 min.
  • the cells obtained above were resuspended in the above culture medium, seeded at a density of Ix IO 4 (100 ⁇ Jl) cells/well in a 96-well plate, and cultured for 24 hour for use in an MTT assay.
  • TRAIL Purified TRAIL (0.001 to 1.0 //g/m «), HWl and HW6 antibodies (0.05 to 50 ⁇ g/vd) were each added to a predetermined well of a 96-well plate, and MTT assay (see Muhlenbeck et al., J Biol Chem., 275:32208-32213, 2000) was conducted to measure the degree of cell death induced by the inventive antibodies of TRAIL-sensitive HL60, HCTl 16 and Dul45 cells (Figs. HA and HB).
  • a MTT solution (5 mg/m- ⁇ , Sigma) dissolved in 100 mi of PBS was added to each well of the plate, and the plate was incubated for 4 to 5 hours, followed by removing the culture medium and MTT solution. 200 mi of DMSO was added thereto to dissolve MTT-formazan crystals, and the absorbance thereof was read at 570 ran.
  • the plate was centrifuged to precipitate the cells before removing the culture medium and MTT solution, and then the supernatant was removed before measuring said absorbance.
  • TRAIL-sensitive cells treated with TRAIL such as HCTl 16, HL60 and Du 145 showed effective cell death with the concentration of TRAIL (Fig. HA), in contrast to the untreated control cells. Further, HWl and HW6 as well as TRAIL induced cell death with the concentration of the antibodies (Fig. 1 IB).
  • MTT assay was repeated as described in (5-2) except for using TRAIL- resistant cells, HepG2, U87MG, Molt-4 and Huh7 cells. The results are shown in Figs. 12A to 12D, and 13A and 13B.
  • TRAIL treated TRAIL-resistant cells such as HepG2, U87MG and Molt-4 did not show any cell death (Fig. 12A and 12C), while HWl or HW6-treated cells showed significant cell death (Figs. 12B and 12D).
  • Anti-DR5 IgG mAb showing cytotoxicity in TRAIL-sensitive cancer cells has recently been reported, but an antibody showing cytotoxicity toward TRAIL-resistant cancer cells has not yet been reported.
  • HW6 (25 ⁇ g/mi) was used to treat TRAIL-resistant HepG2 cells (Fig. 13A) and
  • Huh7 cells (Fig. 13B) for 20 hours, cell death was observed, the dead cells being detached from culture plates, but TRAIL (500 ng/m£)-treated cells remained unharmed.
  • the inventive antibody of the monomeric form induces death of cancer cells, especially TRAIL-resistant cells.
  • Figs. 14A and 14B when human hepatocytes (Fig. 14A) and mammary epithelial cells (Fig. 14B) were treated with HWl or TRAIL at varying concentration for 30 hours, HWl as well as TRAIL induced about 30% cell death at a high concentration, but did not induce cell death at a lower concentration. Accordingly, it was confirmed that HWl exerted lesser cytotoxicity in normal cells.
  • HWl 40 ⁇ g/m£
  • HW6 40 ⁇ g/M
  • the inventive antibodies induce cell death only in cancer cells, but exhibit no cytotoxicity in normal cells.
  • Test Example 6 Test of cancer cell death through DR5 induced by anti- DR5 scFv antibody
  • HWl induces cell death through DR5 expressed on cell surface
  • various concentration of a soluble competitor of DR5-Fc conjugated protein (DR5 fused to antibody constant region Fc) was added to TRAIL (100 ngM) or HWl (5 ⁇ glmt), and cell death inhibition was assessed while incubating the mixture for 30 hours. The result is shown in Fig. 16.
  • HCTl 16 cancer cells were incubated, and treated with sulforaphane (10 ⁇ M) for 9 hours to overexpress DR5 (see Kim et al., Cancer Res., 66: 1740-1750, 2006). 0.01 to 10 ⁇ g/mi of TRAIL or HWl was added to the cells, incubated for 30 hours, and MTT assay was performed to analyze the degree of cancer cell death. The result is shown in Fig. 17.
  • both TRAIL and HWl showed enhanced cell death activity for sulforaphane-treated cells, compared with the untreated control cells. Accordingly, it was proved that HWl induces cancer cell death through cell death signaling transduction through the specific binding to DR5.
  • Test Example 7 Evaluation of influence of a cross-linker on cancer cell death induced by anti-DR5 scFv antibody
  • the molecule was added to TRAIL-sensitive HCTl 16 cells and TRAIL-resistant HepG2 cells, and the cells were incubated for 30 min. MTT assay was conducted as described in (5-2) of Test Example 5, and the result is shown in Fig. 18.
  • HWl induces cell death of cancer cells as a monomeric form.
  • Test Example 8 Study of cell death mechanism of anti-DR5 scFv antibody
  • TRAIL-sensitive HCTl 16 and Dul45 cells were incubated with TRAIL (0.2 ⁇ glmi) and HWl (25 ⁇ g/m-C) for 5 hours and 30 hours, respectively, and prefixed with Karnovsky's solution (2% glutaraldehyde and 1% paraformaldehyde in 100 mmol/£ sodium cacodylate buffer, pH 7.4) at 25 ° C for 2 hours.
  • Karnovsky's solution 2% glutaraldehyde and 1% paraformaldehyde in 100 mmol/£ sodium cacodylate buffer, pH 7.4
  • the cells were washed with 100 mmol/X sodium cacodylate buffer containing 2% glutaraldehyde and 1% paraformaldehyde (pH 7.4), postfixed with 1% osmium tetroxide and 1.5% potassium ferrocyanide for 1 hour, dehydrated by a graded series of ethanol (50- 100%), embedded on Poly Bed 812 resin (Pelco, Canada), cut into ultrathin slices using a ultramicrotome (Reichert Ultracut E microtome), and observed under an electron microscope (EM 902A, Carl Zeiss, Germany). The results are shown in Figs. 19A to 19D.
  • TRAIL-sensitive cancer cells treated with TRAIL showed typical characteristics of apoptotic cell deaths such as chromatic condensation and membrane blebbing.
  • both TRAIL- sensitive and -resistant cells treated with HWl exhibited numerous autophagic vacuoles in the cells and damaged cellular organelles such as mitochondria (Fig. 19A).
  • Autophagic cell death can be confirmed by using Iysotracker-red (DND- 99, Molecular probes), which specifically stains autophagic vacuoles ⁇ see Kondo et al., Nat. Rev. Cancer, 5:726-734, 2005; and Tsujimoto et al., Cell Death Differ., 2: 1528-1534, 2005).
  • DND- 99 Molecular probes
  • TRAIL-sensitive HCTl 16 cells and TRAIL-resistant U87MG cells were treated with HWl (25 ⁇ glvd) for 20 hours, fixed, stained with Iysotracker-red, and observed under a fluorescence microscope. The results are shown in Figs. 2OA and 2OB.
  • HWl induces autophagic cell death of cancer cells.

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Abstract

The present invention provides an antibody specifically binding to death receptor 5 (DR5), which is selected from the group consisting of: an antibody comprising a heavy chain variable region (VH) having the amino acid sequences of SEQ ID NOs: 1 to 3 at complementary determining regions (CDRs) and a light chain variable region (VL) having the amino acid sequences of SEQ ID NOs: 4 to 6 at CDRs; and an antibody comprising a (VH) having the amino acid sequences of SEQ ID NOs: 7 to 9 at CDRs and a (VL) having the amino acid sequences of SEQ ID NOs: 10 to 12 at CDRs, and a composition for preventing or treating a cancer comprising the same. The antibody of the present invention can be effectively used for the prevention or treatment of various cancers, through inducing autophagic cell death of TRAIL-sensitive cancer cells as well as TRAIL-resistant cancer cells by specific binding to DR5.

Description

ANTIBODY SPECIFICALLY BINDING TO DR5
AND COMPOSITION FOR PREVENTING OR TREATING
CANCERS COMPRISING THE SAME
Field of the Invention
The present invention relates to an antibody specifically binding to death receptor 5 (DR5), and a composition for preventing or treating cancers comprising the same.
Background of the Invention
Among the apoptotic pathways involving p53-independent tumor necrosis factor receptors (TNFRs), cell death through the death receptor 5 (DR5, TRAIL receptor 2, TRICK2) or the death receptor 4 (DR4, TRAIL receptor 1) pathway activated by TNF-related apoptosis inducing ligand (TRAIL) has been a very attractive target for cancer therapy as they can specifically induce cancer cell death with little adverse side effects on normal cells (Ashkenazi et al., J. Clin. Invest, 104:155-162, 1999; and Ashkenazi, Nat. Rev. Cancer, 2:420-430, 2002).
Currently, there exist several methods to develop caner cell-specific therapeutic agents which target DR4 or DR5 such as a method using a recombinant TRAIL (for example, 114th to 281st amino acid residues of TRAIL) as a ligand of the death receptor and a method of developing an agonistic antibody among mouse- or human-derived complete antibodies (e.g.: mAb or IgG) specific to the death receptor (Pollack et al., Clin. Cancer Res., 7: 1362- 1369, 2001; Jo et al., Nat. Med., 6:564-567, 2000; Ichikawa et al., Nat. Med., 7:954-960, 2001; and Walczak et al., Nat. Med., 5: 157-161, 1999). However, the recombinant TRAIL is very unstable and tends to form a soluble oligomer, whose apoptotic activity is about 20 to 100 times lower, and it causes side effects such as cytotoxicity and immune reaction on normal cells such as astrocytes, hepatocytes and keratinocytes (Jo et al., Nat. Med., 6:564-567, 2000). Further, TRAIL is not capable of inducing more than 50% cell death of malignant tumor cells (Zhang et al., Cancer Gene Ther., 12:228-237, 2005). Hence, a cancer cell that can be killed by TRAIL is designated a TRAIL- sensitive cancer cell, and a cancer cell not killed by TRAIL, a TRAIL-resistant cancer cell.
There have been developed antibodies, which have the specific binding affinity to DR5 to induce the cell death: humanized antibodies developed from mouse-derived monoclonal antibodies such as TRA-8 (mouse IgG) (Walczak et al, Nat. Med., 5: 157-161) and AD5-10 (mouse IgG) (Guo et al., J. Biol. Chem., 280:41940-41952, 2005), as well as human-derived monoclonal antibodies such as HGS-ETR2 (human IgGl) (Georgakis et al., Br. J. Haematol, 130:501-510, 2005) and KMTR2 (human IgG4) (Motoki et al., CHn. Cancer Res., 11 :3126- 3135, 2005).
However, the above antibodies induce apoptotic cell death of TRAIL- sensitive cancer cells but not TRAIL-resistant cancer cells. Further, the antibodies in the form of an antigen-binding fragment (Fab) or a single chain variable fragment (scFv) having a monovalent antigen binding site does not induce cell death of cancer cells (e.g.: KMTR2), while antibodies in the form of IgG having divalent antigen binding site (e.g.: HGS-ETR2 and AD5-10) show cytotoxicity or induced cell death when an IgG is used as a cross-linker (Chuntharapai et al., J. Immunol, 166:4891-4898, 2001; Motoki et al., Clin. Cancer Res., 11 :3126-3135, 2005; and Wajant et al., Oncogene, 20:4101-4106, 2001). But, it has not been reported that an anti-DR5 antibody in the form of a scFv and a Fab induces cancer cell death. Currently, there remains a question as to whether or not autophagy is the mechanism responsible for inducing cancer cell death (Kondo et al., Nat. Rev. Cancer, 5:726-734, 2005), and it has been reported that only specific compounds can kill cancer cells by an autophagic cell death pathway (Yu et al., Science, 304: 1500-1502, 2004). However, there have been no reports of anti- DR5 mAbs which induce autophagic cell death.
Summary of the Invention Accordingly, it is an object of the present invention to provide an antibody inducing autophagic cell death of both TRAIL-sensitive and TRAIL- resistant cancer cells through specifically binding to DR5. It is another object of the present invention to provide a DNA encoding the antibody.
It is a further object of the present invention to provide a cell transformed with the DNA or an expression vector comprising the DNA.
It is a still further object of the present invention to provide a composition for preventing or treating cancers comprising the antibody.
It is a still further object of the present invention to provide a method of preventing or treating cancers by using the antibody.
In accordance with one aspect of the present invention, there is provided an antibody specifically binding to death receptor 5 (DR5), which is selected from the group consisting of: an antibody comprising a heavy chain variable region (VH) having the amino acid sequences of SEQ ID NOs: 1 to 3 at complementary determining regions (CDRs) and a light chain variable region
(VL) having the amino acid sequences of SEQ ID NOs: 4 to 6 at CDRs; and an antibody comprising a VH having the amino acid sequences of SEQ ID NOs: 7 to 9 at CDRs and a VL having the amino acid sequences of SEQ ID NOs: 10 to
12 at CDRs.
In accordance with another aspect of the present invention, there is provided a DNA encoding the antibody.
In accordance with a further aspect of the present invention, there is provided a cell transformed with the DNA or an expression vector comprising the DNA.
In accordance with a still further aspect of the present invention, there is provided a composition for preventing or treating a cancer comprising the antibody as an active ingredient. In accordance with a still further aspect of the present invention, there is provided a method of preventing or treating a cancer comprising administering the antibody to a subject. Brief Description of Drawings
The above and other objects and features of the present invention will become apparent from the following description of the invention taken in conjunction with the following accompanying drawings, which respectively show:
Figs. IA and IB: amino acid sequences of HWl and HW6, and DNAs encoding thereof as anti-DR5 scFv antibodies specifically binding to DR5, respectively (wherein, CDRl to CDR3 of a heavy chain variable region, a linker oligopeptide and CDRl to CDR3 of a light chain variable region of each antibody are underlined in order);
Fig. 2: the result of ELISA quantifying the antibody titer against DR5, DcRl and BSA in 8 serums showing high antibody titers against DR5; Fig. 3: the result of FACS (fluorescence activated cell sorting) isolating an anti-DR5 scFv antibody library expressed on cell surface by using biotin- labeled DR5 as an antigen;
Fig. 4: a diagram of an E. coli expression vector prepared by cloning HWl and HW6 to pKJl vector, respectively; Figs. 5 A and 5B: the results of SDS-PAGE and western blotting, respectively, obtained for HWl and HW6 expressed in E. coli and purified;
Fig. 6: the result of size-exclusion chromatography of purified HWl and HW6;
Figs. 7 A and 7B: the results of reducing SDS-PAGE and non-reducing SDS-PAGE of purified HWl and HW6;
Figs. 8A to 8D: the results of SPR (surface plasmon resonance) showing the cross reactivity of HWl (Figs. 8A and 8B) and HW6 (Figs. 8C and 8D) to antigens, DR5, and DcRl, DcR2, DR4, TNFRl, TNFR2 and CD95, respectively; Figs. 9A and 9B: images showing the binding of Alexa633-labeled HWl
(Fig. 9A) or TRAIL (Fig. 9B) to YFP (yellow fluorescence protein)-labeled DR5 (DR5ΔCDYFP) and DcR2 (DcR2ΔCDYFP) in HCTl 16 cells (human colon cancer cell); Figs. 1OA and 1OB: the results of competitive ELISA examining whether or not the binding sites of HWl and HW6 to DR5 are identical to those of TRAIL;
Figs. 1 IA and 1 IB: the results of MTT assay showing the degree of cell death induced in TRAIL-sensitive HCTl 16, HL60 and DU 145 cells as function of the concentration of TRAIL (Fig. 1 IA) and HWl and HW6 (Fig. 1 IB);
Figs. 12A to 12D: the results of MTT assay showing the degree of cell death induced in TRAIL-resistant HepG2, U87MG and Molt-4 cells as function of the concentration of TRAIL (Figs. 12A and 12C) and HWl and HW6 (Figs. 12B and 12D);
Figs. 13A and 13B: microscopic images showing the cell death induced in TRAIL-resistant HepG2 cells (Fig. 13A) and Huh7 cell (Fig. 13B) by TRAIL, HWl and HWό;
Figs. 14A and 14B: the results of cytotoxicity test showing that no cytotoxicity to normal human cells, hepatocytes (Fig. 14A) and mammary epithelial cells (Fig. 14B) was caused by increased concentration of TRAIL and HWl;
Fig. 15: microscopic images showing that no cytotoxicity to normal human brain astrocytes was caused by HWl or HW6; Fig. 16: the result of MTT assay to quantify the cytotoxicity of varying concentration of HWl and TRAIL in HCTl 16 cells, after treating cell membrane-expressed DR5 and soluble competitor DR5-Fc;
Fig. 17: the result of MTT assay to quantify cell death by TRAIL and HWl of TRAIL-sensitive HCTl 16 cells in the presence or absence of sulforaphane;
Fig. 18: the result of MTT assay to quantify the cytotoxicity of HWl for TRAIL-sensitive HCTl 16 cells and TRAIL-resistant HepG2 cells in the presence or absence of a cross-linker (CL);
Figs. 19A to 19D: microscopic images showing that cell death of TRAIL-sensitive cells (HCTl 16 and Dul45) and TRAIL-resistant cells (HepG2 and U87MG) induced by HWl is autophagic cell death (wherein, Fig. 19A is
TEM images, Figs. 19B to 19D are high-magnification images showing multiple-membrane-bound autophagic vacuoles, double-membrane-bound autophagic vacuoles, and autophagic vacuole induction step by fusing empty vacuoles with vacuoles containing cellular organelles, respectively); and
Figs. 2OA and 2OB: fluorescent microscopic images obtained using lysotracker-red DND-99 which specifically stains cellular autophagic vacuoles, showing autophagic cell death induced by HWl on TRAIL-sensitive HCTl 16 cells (Fig. 20A) and TRAIL-resistant U87MG cells (Fig. 20B).
Detailed Description of the Invention
In the present invention, the term "death receptor 5 (DR5) protein" means a receptor as a member of tumor necrosis factor (TNF) receptor family, which binds to TRAIL and has a C-terminal cytoplasmic death domain (Pan et al., Science, 277:815-818, 1997). When DR5 binds to TRAIL, apoptosis is induced in TRAIL-sensitive cancer cells, but not in normal cells.
In the present invention, the term "DR5" includes any protein having the above characteristics, e.g., a protein having the amino acid sequences described in United States Patent No. 6,872,568.
In the present invention, the term "antibody" may be a whole antibody or a fragment thereof. The whole antibody can be a monomer selected from the group consisting of IgG, IgM, IgA, IgD and IgE, or a multimer formed by combining monomers. Further, the term "a functional fragment of an antibody" means an antibody having heavy chain and light chain variable regions of a whole antibody actually recognizing the same epitope recognized by the whole antibody. The functional fragment of an antibody may be a single chain variable fragment (scFv), (ScFv)2, Fab, Fab', F(ab')2 or scFv-Fc, and it is preferably scFv.
Further, in the present invention, the term "single chain variable fragment (scFv)" means an antibody fragment of a single chain polypeptide form having a heavy chain variable region and a light chain variable region linked through a linker peptide.
The antibody may be produced by any of the conventional methods known to those in the art, such as the phage display method or yeast cell surface expression system. scFv may be prepared by any of the conventional methods known to those in the art, e.g., the method described in United States Patent No.
4,946,778 or 5,258,498, and Fab, Fab1 and F(ab')2 fragments may be prepared by recombinant antibody technology, e.g., the method described in International
Patent Publication No. WO 92/22324.
The inventive antibody may be derived from any animal which may be mammals excluding human, birds and so on. Preferably, the antibody may be derived from a human, mouse, donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken. The antibody derived from a human is an antibody having amino acid sequences of human immunoglobulin, and may include an antibody isolated from human immunoglobulin libraries, or an antibody isolated from animals, which are transformed to produce one or more human immunoglobulins and incapable of expressing endogenous immunoglobulin (see United States Patent No. 5,939,598)
The inventive antibody may be conjugated with a marker including, but not limited to, an enzyme, fluorescent material, radioactive material, protein and so on. The methods to conjugate the materials are well known in the art.
The inventive antibody specifically binds to DR5 protein. In the present invention, the term "specifically bind" means that the inventive antibody does not bind to DR5-like TNFR family receptors such as DcRl (death decoy receptor 1), DcR2, DR4, TNFRl, TNFR2 and CD95.
In the inventive antibody, the amino acid sequences of SEQ ID NOs: 1 to 3 or 7 to 9 are CDRl, CDR2 and CDR3, respectively, of the heavy chain variable region of an antibody specifically binding to DR5, while the amino acid sequences of SEQ ID NOs: 4 to 6 or 10 to 12 are CDRl, CDR2 and CDR3, respectively, of the light chain variable region of an antibody specifically binding to DR5. Preferable examples of the inventive antibody are scFv antibodies, HWl and HW6, which have the amino acid sequences of SEQ ID NOs: 13 and 14, respectively, and the antibodies comprise CDRl to CDR3 of a heavy chain variable region, linker-oligopeptide and CDRl to CDR3 of a light chain variable region in order {see Figs. IA and IB).
HWl and HW6 specifically bind to DR5, and have binding affinities
(KD) of about 2.02χ l0'7 M, and 5.45χ lO"8 M, respectively. Further, the antibodies in the form of monovalent scFv induce autophagic cell death of both TRAIL-sensitive and -resistant cancer cells in the absence of a cross linker, but they are not toxic to normal cells.
Accordingly, the inventive antibody is believed to induce autophagic cell death of various cancer cells including TRAIL-resistant cancer cells through specific binding to epitopes (binding sites) of DR5 which do not overlap with the TRAIL-binding epitopes.
The present invention further provides a DNA encoding the inventive antibody.
The DNA is preferably a DNA encoding the scFv having the amino acid sequence of SEQ ID NO: 13 or 14, and more preferably, a DNA having the nucleotide sequence of SEQ ID NO: 15 or 16.
The DNA sequence encoding the inventive antibody can be obtained by any of the conventional methods known in the art. For example, based on the DNA sequence encoding the antibody heavy or light chain, a part thereof, or the corresponding amino acid sequence, an appropriate DNA sequence can be synthesized by the well known oligonucleotide synthesis method, e.g., site- directed mutagenesis and polymerase chain reaction (PCR).
Further, the present invention provides a cell transformed with the inventive DNA or an expression vector comprising the DNA.
The inventive DNA or the expression vector comprising the DNA may be transferred to a host cell by any of the conventional methods, e.g., viral transfection or non-viral method. Such introduction of the DNA or the expression vector may be conducted in accordance with any of the methods known to those in the art including adenoviral transformation, gene gun, liposome-mediated transformation, retrovirus or lentivirus-mediated transformation, plasmid or adeno-associated virus. Further, the transformed cell may be transplanted together with carriers having gene delivery system, which can release or deliver the designed gene to the cells of a subject for a long period of time.
The present invention further provides a method of producing the inventive antibody molecule comprising the steps of: 1) expressing the antibody by culturing a host cell comprising the inventive DNA sequence or an expression vector comprising the DNA under a suitable condition; and 2) isolating the expressed antibody.
The antibody molecule may be accumulated in the cell cytoplasm, or in the periplasm or extracellular medium (supernatant) using a proper signal sequence, and the accumulation thereof in the periplasm or extracellular medium is preferred. Further, the produced antibody molecule may be refolded to confer a functional conformation thereto using any of the conventional methods known in the art. In order to produce only a heavy chain or a light chain polypeptide of the antibody molecule, a single vector comprising a heavy chain or light chain polypeptide may be transformed into a host cell. In order to produce both heavy chain and light chain polypeptides of the antibody molecule, both the first vector encoding a light chain polypeptide and the second vector encoding a heavy chain polypeptide, or a single vector comprising both heavy chain and light chain polypeptides may be transformed into a host cell.
As described above, the inventive antibody, which induces autophagic cell death of DR5 -expressed TRAIL-sensitive and TRAIL-resistant cancer cells by specific binding to DR5, can be used to prevent or treat various cancers. Such cancers may be any cancer expressing DR5 and include TRAIL-sensitive and TRAIL-resistant cancers, such as breast cancer, colon cancer, brain tumor, glioma, ovarian cancer, endometrial cancer, bone sarcoma, cervix cancer, prostatic cancer, lung cancer, synovial cancer, pancreatic cancer and sarcoma.
Accordingly, the present invention further provides a composition for preventing or treating cancer comprising the inventive antibody as an active ingredient.
The inventive composition for preventing or treating cancer may additionally comprise one or more pharmaceutically acceptable excipients, carriers, diluents and so on.
Example of the carrier employed in the present invention is a slowly metabolized macromolecule such as protein, polypeptide, liposome, polysaccharide, polylactic acid, polyglycolic acid, polymeric amino acid, amino acid polymer and inactive viral particle. For example, a pharmaceutically acceptable salt, such as a salt of inorganic acid (e.g., hydrochloride, hydrobromide, phosphate and sulfate), and a salt of organic acid (e.g., acetate, propionate, malonate and benzoate); a liquid such as water, saline solution, glycerol and ethanol; and an auxiliary material such as a wetting agent, an emulsifier and a pH buffer may be used.
The pharmaceutically acceptable carrier is described in [Remington's Pharmaceutical Sciences, Mack Publishing Company, 1991].
Further, the composition may be formulated to a unit dosage form suitable for administering to a patient, preferably for administering a protein drug, by conventional methods in the pharmaceutical field, and it can be administered in accordance with any conventional method in the art through oral route or parenteral route such as intravenous, intramuscular, intraarterial, intramedullar, intrathecal, intraventricular, intrapulmonary, intracutaneous, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or intrarectal route without limitation.
Example of the formulations suitable for such purposes may include oral formulations such as tablet, pill, dragee, powder, capsule, syrup, solution, gel, suspension, emulsion and microemulsion; and parenteral formulations such as injection formulation (e.g., injection ampule), infusion formulation, and spray formulation (e.g., hypospray). Injection formulation or infusion formulation may be in the form of suspension, solution or emulsion, and include formulation aids such as suspending agents, preservatives, stabilizers and/or dispersing agents. Further, the antibody molecule may be formulated in the form of a dried formulation, which can be readjusted to a suitable sterile solution before use.
Because the composition of the present invention comprises an antibody molecule easily degraded in the gastrointestine, the composition for gastrointestinal administration is preferred to include an agent, which protects the antibody from degradation and is absorbed into the gastrointestine after releasing the antibody.
The present invention further provides a method of preventing or treating a cancer comprising a step of administering the inventive antibody to an animal, preferably a mammal, more preferably a human, in accordance with any methods described above.
In the inventive method of preventing or treating a cancer, the composition or pharmaceutical formulation may be used solely or in combination with other anticancer agents, for instance, TRAIL or an anticancer agent conventionally used in the art.
Further, the inventive antibody may be administered by gene therapy. For this purpose, DNAs encoding heavy and light chains of the inventive antibody or an expression vector thereof should be introduced into a patient by any of the conventional methods known in the art so that the heavy and light chains are combined in situ to form the antibody molecule.
The inventive antibody as an active ingredient of the inventive composition or pharmaceutical formulation may be administered in a dose ranging from about 0.01 to 50 mg/kg (body weight), preferably 0.1 to 20 mg/kg (body weight) per day in case of a mammal including a human being. The inventive composition or pharmaceutical composition may be administered in a single dose or in divided doses. However, it should be understood that the amount of the active ingredient actually administered ought to be determined in light of various relevant factors including the disease to be treated, the condition to be treated, the severity of the patient's symptom, the chosen route of administration, and the body weight, age and sex of the individual patient, drug combination, reaction sensitivity, and tolerance/reactivity to treatment; and, therefore, the above dose should not be intended to limit the scope of the invention in any way.
The following Examples are intended to further illustrate the present invention without limiting its scope. Example 1: Selection of a serum having a high antibody titer against DR5
In order to construct human antibody libraries using a serum having a high antibody titer against death receptor DR5 and death decoy receptor 1 (DcRl), antibody titers against DR5 and DcRl of blood serums of 60 normal persons were measured by ELISA. At this time, bovine serum albumin (BSA) was used as a control antigen.
Specifically, each well of a 96-well ELISA plate (Nunc, Invitrogen) was coated with 10 μg/mC of a test antigen dissolved in TBS (50 mM Tris-HCl, pH 7.5, 50 mM NaCl) at 25 °C for 1 hour, washed with TBS containing 0.05% tween 20 (TBST) 3 times, and blocked with TBS containing 3% BSA. Each diluted serum sample (1 :100) was added to each well and incubated at 25 °C for about 1 hour, and the well was washed with TBST. 100 fd of alkaline phosphatase-conjugated anti-human IgG/IgA (2.5 /ig/m-C, Pierce) was added to each well, incubated at 25 °C for about 1 hour, and washed with TBST. Then, 100 μi of /7-NPP (p-nitrophenyl phosphate, 1 mg/mC) was added to each well, allowed to react for 4 hours, and the absorbance was read at 405 inn with a microplate reader to quantify the antibody titer against DR5 or DcRl antigen. The results for 8 serums having high antibody titers against DR5 and DcRl antigen are shown in Fig. 2.
As shown in Fig. 2, the selected 8 serums each showed antibody titers against DR5 and DcRl which are more than 2 times higher than that of BSA as a control.
Example 2: Construction of a scFv antibody library
(2-1) Amplification of a heavy chain and a light chain of the antibody
Total RNA was extracted from peripheral blood lymphocytes of the selected 8 blood samples of Example 1 using TRIzol reagent (Invitrogen, USA), and mRNA was separated by using oligotex mRNA kit (Qiagen). Then, single strand cDNA libraries of a human scFv antibody were amplified by reverse transcription with random hexamers as primers (Amersham Pharmacia Biosciences) using AccuPower RT PreMix (Bioneer, Korea).
In order to amplify the heavy chain variable region (VH) of human antibody IgG (γ) and IgM (μ), and the light chain variable region (Vκ and Vλ) of human antibody, a total of 71 PCRs (28, 16 and 27 reactions for VH, Vκ and Vλ, respectively) were performed using each primer set. Each PCR condition was
30 cycles of 2 min at 95 °C , 1 min at 55 °C and 1 min at 72 °C .
The primers used were those of Little et al. (J. Immunol. Methods, 231 :3-9, 1999) with the following slight modifications. Specifically, in order to generate a linker sequence between VH and VL gene, the sequence 5 '-CGA GCC CCC GCC ACC CGA ACC GCC CCC ACC TCT-3' (SEQ ID NO: 17) was added to the 5' end of the reverse primer of VH and the sequence 5 '-GGT TCG GGT GGC GGG GGC TCG GGC GGG GGT GGC TCA GAT CT-3 ' (SEQ ID NO: 18) was added to the 5' end of the forward primers of Vκ and Vj1.
Further, in order to allow transforming the amplified scFv libraries into the yeast surface display vector by homologous recombination, the sequence 5 '- AGT GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GCT AGC-3' (SEQ ID NO: 19) was added to the 5' end of the forward primer of VH and the sequence 5 '-TCA GAT CTC GAG CTA TTA CAA GTC CTC TTC AGA AAT AAG CTT TTG TTC GGA TCC-3' (SEQ ID NO: 20) was added to the 5' end of the reverse primers of Vκ and V^.
(2-2) Amplification of a scFv antibody gene
The amplified VH and VL genes were subjected to 1% agarose gel electrophoresis and purified. Equal amounts of the purified VH and VL genes were mixed together, and overlap extension PCR was conducted to prepare scFv gene repertoires. The PCR products were purified as described above. Each PCR condition was 30 cycles of 2 min at 95 "C , 1 min at 55 °C and 1 min at 72 °C .
(2-3) Construction of a scFv antibody gene library 10 μglμl of the obtained scFv antibody gene libraries were mixed with 1 μg/μi of a scFv yeast surface display vector (pCTCON; Colby et al., Methods Enzymol, 388:348-358, 2004), and the mixture was transformed into a yeast EBY 100 strain (Invitrogen) by electrophoration. The transformants were pooled and propagated directly in liquid selective SD-CAA medium (-ura, -trp), which contained 20 g/£ glucose, 6.7 g/t yeast nitrogen base without amino acids (Difco, USA), 5.4 g/t Na2HPO4, 8.6 g/£ Na2HPO4 H2O and 5 g/£ casamino acid, and then the libraries which induced cell surface expression of scFv were selected using a selective SG-CAA medium, which contained the same composition as SD-CAA, except that glucose was replaced with galactose. The cells containing the libraries were each serially diluted 10-fold with the selective SD-CAA medium, and the library size was determined by plating each diluted cells on the selective SD-CAA agar plate.
As a result, approximately 2 x 106 ScFv antibody libraries, so called a pseudo-immune library, were constructed.
Example 3: Selection of an anti-DR5 scFv antibody library
In order to select scFv antibodies having high specific affinity to DR5 among the scFv antibody libraries obtained in Example 2, the cells containing the selected libraries were incubated with anti-c-myc 9E10 mAb (1:100 dilution, Ig Therapy) and 1 μM of biotin-labeled DR5 by EZ-LINK™ Sulfo-NHS-LC- Biotinylation kit (Pierce, USA) at 25 °C for 30 min in 0.2 mH of PBSB (phosphate-buffered saline, pH 7.4, containing 1 mg/m£ of BSA). The cells were washed with ice-cold PBSB, and labeled with FITC-labeled anti-mouse IgG (1 :25 dilution) and streptavidin-R-phycoerythrin conjugate (SA-PE, 1 :100 dilution; Molecular Probes, USA) as a secondary antibody, respectively. The labeled cells were washed, resuspended in PBSB, and then one round of MACS (magnetic activated cell sorting) was performed using biotin-labeled DR5 to float the scFv library having the binding affinity to DR5, followed by performing FACS (fluorescence activated cell sorting) to select HWl and HW6 bound to the antigen in the anti-DR5 scFv antibody libraries expressed on yeast cell surface. The results are shown in Fig. 3.
The nucleotide sequences of the selected HWl and HW6 were analyzed using a forward primer 5'-GTT CCA GAC TAC GCT CTG CAG G-3' (SEQ ID
NO: 21) and a reverse primer 5'-GAT TTT GTT ACA TCT ACA CTG TTG-3' (SEQ ID NO: 22). The amino acid sequences thereof were then deduced from the analyzed nucleotide sequences.
Example 4: Anti-DR5 scFv antibody expression and purification
The isolated antibodies HWl and HW6 of Example 3 were subcloned in-frame into a bacterial expression vector using restriction enzymes Nhel and BamHl. The bacterial expression vector was expression vector pKJl designed to encode T7 promoter-PelB periplasm targeting sequence-human scFv-Flag tag-6χHis tag (Fig. 4). This vector was prepared by inserting PeIB nucleotide sequence and restriction enzyme site Nhel/ BamHl based on pET21b vector (Novagen) and fusing Flag tag and 6><His tag on the C-terminus.
The expression vector was transformed into E.coli BL21 (DE3) (Novagen), and the transformed cells were cultured at 37 °C until the OD at 600 ran reached 0.6 to 0.8, followed by induction with 0.5 mM IPTG at 25 °C for 20 hours. The periplasm fraction or supernatant of the cultured bacteria was subjected to Talon resin (Clontech) to purify the expressed antibody, and the size and the purity of the purified antibody were analyzed by SDS-PAGE and western blotting using anti-His tag, respectively. As a result, the antibodies each having size of about 29 kDa (Fig. 5A), and a purity of more than 98% were obtained (Fig. 5B).
Test Example 1: Confirmation of anti-DR5 scFv antibody form
In order to identify whether the HWl and HW6 purified in Example 4 were in the monomeric or oligomeric form in solution, size exclusion HPLC (SEC), reducing SDS-PAGE and non-reducing SDS-PAGE were performed, respectively.
SEC was performed using PBS (50 mM phosphate, pH 7.4, 150 mM NaCl) as a elution buffer, Sephadex 25 size exclusion column (Pharmacia) and Agilent 1100 HPLC system at a flow rate of 0.7 m£/min, and the absorbance was read at 280 lim. The results are shown in Fig. 6.
Reducing SDS-PAGE was performed using 10% gel in sample buffer supplemented with 1 mM DTT, and non-reducing SDS-PAGE was performed using 10% gel in sample buffer without 1 mM DTT (see Laemmli UK, Nature, 227:680-685, 1970). The results are shown in Fig. 7.
As shown in Fig. 6, HWl and HW6 were each eluted as a monomeric form in solution at concentrations of 10 mg/m£ as determined by size-exclusion chromatography. Further, there were no antibodies of the oligomeric form, which have non-native disulfide bonds, as determined by reducing SDS-PAGE
(Fig. 7A) and non-reducing SDS-PAGE (Fig. 7B).
Accordingly, it was confirmed that the inventive antibody exists in the monomeric form even at a very high concentration.
Test Example 2: Analysis of the binding affinity and cross reactivity of anti-DR5 scFv antibody
The binding affinity of the anti-DR5 scFv antibody to DR5, and the cross-reactivities of the antibody with other antigens, DcRl, DcR2, DR4, TNFRl, TNFR2 and CD95 were determined by Biacore 2000 SPR (surface plasmon resonance) biosensor (Pharmacia, Sweden), respectively.
Specifically, about 0.5-1.0 mg/m« of each antigen (DR5, DcRl, DcR2, DR4, TNFRl, TNFR2 and CD95 (R&D Systems) was immobilized on a CM5 chip (carboxymethylated dextran surface chip; Pharmacia) at a level of about 2,000 to 4,000 response units in accordance with the manufacturer's instruction. Then, HWl (200-3,200 nM) and HW6 (25-1,000 nM) each diluted with PBS (pH 7.4) was injected on the chip at 25°C at a flow rate of 30 μ£/min to measure the degrees of interactions between the antibodies and the antigens. The surface of the chip was regenerated with 25 mM NaCl/50 mM NaOH, and the kinetic rate constants (kon and kOff) as well as the equilibrium dissociation constant (KD) were determined by using BIA evaluation software ver. 3.2. The results are shown in Table 1, and the results of cross-reactivity are shown in Figs. 8A to 8D.
Table 1
Figure imgf000019_0001
As can be seen in Figs. 8 A to 8D, HWl and HW6 bind to DR5 with high affinities (Figs. 8A and 8C), but they showed no affinity to DcRl, DcR2, DR4, TNFRl, TNFR2 and CD95 even at a very high concentration (up to 10 μM) (Figs. 8B and 8D). Accordingly, it was confirmed that the inventive HWl and HW6 specifically bind only to DR5.
Test Example 3: Confirmation of the binding of anti-DR5 scFv antibody to DR5 expressed on a cell
In order to examine whether HWl specifically binds to DR5 expressed on cell surface, plasmid TOlO expressing protein DR5ΔCDYFP, which is a fusion protein of extracellular domain of DR and YFP (yellow fluorescent protein), and plasmid T30 expressing protein DcR2ΔCDYFP, which is a fusion protein of extracellular domain of DcR2 and YFP, were kindly provided by Prof. Chan (U. of Massachusetts, MA) (see Clancy et al., Proc. Natl. Acad. Sci. USA, 102:18099-18104, 2005), and transformed into HCTl 16 colon cancer cell (CCL-247, ATCC (American Type Culture Collection)) by electroporation to overexpress each of the proteins. The transformed cells were seeded at a concentration of 5χ lO4 cells
/well to a 24-well plate, and cultured for 30 hours in an incubator under 5% CO2. HWl and TRAIL (KOMA Biotech, Korea) were stained with red fluorescence using Alexa633 labeling kit (Molecular Probes, USA), and added to the cultured transformed cells, followed by reacting the cells at 4 "C for 30 min. Each well was washed 3 times with PBS containing 2% FCS and 2% paraformaldehyde, and observed with a fluorescent microscope (LMS510 model laser scanning confocal fluorescence microscope, Carl Zeiss). The results are shown in Figs. 9 A and 9B.
As shown in Figs. 9A and 9B, HWl (red) specifically binds to
DR5ΔCDYFP (green) to reveal a colocalization pattern (orange), but not to
DcR2ΔCDYFP (Fig. 9A). Further, TRAIL, which binds well to DR5 and
DcR2, were indeed found to be bound to both DR5ΔCDYFP and DcR2ΔCDYFP (Fig. 9B).
Accordingly, it was demonstrated that HWl antibody specifically binds to DR5 expressed on the cell membrane as well as to DR5 in solution, without any cross-reactivity.
Test Example 4: Identification of an epitope of DR5 for anti-DR5 scFv antibody
In order to identify the binding sites (epitopes) of HWl and HW6 to DR5, competitive ELISA with TRAIL was performed. Specifically, each well of a 96-well plate for ELISA was coated with 50 μl of DR5 (5-20 μg/mt), followed by incubating the plate at 37 °C for 1 hour. Each well was washed 3 times with PBS, PBS containing 1% BSA was added thereto, and the plate was incubated at 37 °C for 1 hour. Then, HWl, HWl+TRAIL, HW6 and HW6+TRAIL having varying concentrations of the HWl or HW6 in the range of I x 10"4 to I x 103 μgM and 20 μg/mt of TRAIL were added to each well, and the plate was incubated at 37 °C for 1 hour and washed. Anti-Flag M2 (Sigma) or anti E-tag (Amersham), a primary antibody, was added to each well and the plate was incubated at 37 °C for 1 hour, followed by washing the plate 3 times. Then, alkaline phosphatase-conjugated anti- mouse IgG Fc specific antibody (Sigma), a secondary antibody, was added to each well and the plate was incubated at 37 °C for 1 hour, followed by washing the plate 3 times. 50 μl of /?-NPP substrate was added to each well and the plate was incubated for 100 min, and the absorbance at 405 ran was measured. As shown in Fig. 1OA, the binding of both HWl and HW6 to DR5 was enhanced with the concentration of either of the antibodies regardless of the presence of TRAIL.
Further, preincubated HWl (5 μg/vd) and HW6 (16 μg/m£) were each bound to a DR5-fixed plate, and TRAIL (0.01 βg/mi to 1,000 μg/mi) was added thereto to examine whether or not the binding of HWl and HW6 to DR5 was changed. The result is shown in Fig. 1OB.
As shown in Fig. 1OB, the binding of HWl and HW6 to DR5 was not influenced by the treatment with a high concentration up to 500 ng/m£ of TRAIL.
Accordingly, it was confirmed that HWl and HW6 bind to epitopes of DR5 that does not overlap with the TRAIL binding sites.
Test Example 5: Test of cancer cell death induced by anti-DR5 scFv antibody
The possibility of inducing cell death of anti-DR5 human scFv antibody,
HWl and HW6 were each evaluated using various cancer cells (ATCC), i.e., HL60 (human acute myeloid leukemia cell), HCTl 16 (human colon cancer cell line) and Du 145 (human prostate cancer cell) for TRAIL-sensitive cells; and HepG2 and Huh7 (human hepatocellular carcinoma cancer cell), U87MG (human astrocyte cancer cell) as well as Molt-4 (Human T-lymphoblastoid leukemia) for TRAIL-resistant cells. The adherent cells such as HCTl 16, Dul45, HepG2, Huh7 and U87MG were each incubated in DMEM medium supplemented with 10% (v/v) fetal calf serum (FCS) (Gibco Invitrogen), and the non-adherent cells such as HL60 and Molt-4 were incubated in RPMI 1640 medium supplemented with 10% FCS, 100 unitsM penicillin, and 100 μg/mt of streptomycin. The incubation was conducted at 37 °C under 5% CO2.
(5-1) Cell preparation The cells stored in a liquid nitrogen tank was quickly thawed at 37 °C, and centrifuged to remove the medium. The pellets thus obtained were mixed with the culture medium, poured in a culture flask, and cultured for 2 to 3 days. When the cell culture proceeded normally, HCTl 16, HepG2, Dul45 and
U87MG cells were each treated with 1 mi of TE buffer (Trypsin-EDTA), followed by adding 5 m€ of DMEM medium containing 10% FBS to stop the reaction of TE buffer. The resulting cells were isolated by centrifugation at
1,000 rpm for 5 min. Further, HL60 and Molt-4 cells were directly recovered from the culture flask, and isolated by centrifugation at 1,000 rpm for 5 min.
Then, the cells obtained above were resuspended in the above culture medium, seeded at a density of Ix IO4 (100 μJl) cells/well in a 96-well plate, and cultured for 24 hour for use in an MTT assay.
(5-2) Assay for cell death induction in TRAIL-sensitive cancer cell
Purified TRAIL (0.001 to 1.0 //g/m«), HWl and HW6 antibodies (0.05 to 50 μg/vd) were each added to a predetermined well of a 96-well plate, and MTT assay (see Muhlenbeck et al., J Biol Chem., 275:32208-32213, 2000) was conducted to measure the degree of cell death induced by the inventive antibodies of TRAIL-sensitive HL60, HCTl 16 and Dul45 cells (Figs. HA and HB).
Specifically, a MTT solution (5 mg/m-β, Sigma) dissolved in 100 mi of PBS was added to each well of the plate, and the plate was incubated for 4 to 5 hours, followed by removing the culture medium and MTT solution. 200 mi of DMSO was added thereto to dissolve MTT-formazan crystals, and the absorbance thereof was read at 570 ran. In case of dealing with an anchorage- independent cell line such as HL-60, the plate was centrifuged to precipitate the cells before removing the culture medium and MTT solution, and then the supernatant was removed before measuring said absorbance.
As shown in Figs. HA and HB, TRAIL-sensitive cells treated with TRAIL such as HCTl 16, HL60 and Du 145 showed effective cell death with the concentration of TRAIL (Fig. HA), in contrast to the untreated control cells. Further, HWl and HW6 as well as TRAIL induced cell death with the concentration of the antibodies (Fig. 1 IB).
Accordingly, it was confirmed that the inventive antibody in the form of a monomer alone is particularly effective in inducing cell death, which is consistent with the results of Test Example 1.
(5-3) Assay for cell death induction in TRAIL-resistant cancer cell
MTT assay was repeated as described in (5-2) except for using TRAIL- resistant cells, HepG2, U87MG, Molt-4 and Huh7 cells. The results are shown in Figs. 12A to 12D, and 13A and 13B.
As shown in Figs. 12A to 12D, TRAIL treated TRAIL-resistant cells such as HepG2, U87MG and Molt-4 did not show any cell death (Fig. 12A and 12C), while HWl or HW6-treated cells showed significant cell death (Figs. 12B and 12D).
Anti-DR5 IgG mAb showing cytotoxicity in TRAIL-sensitive cancer cells has recently been reported, but an antibody showing cytotoxicity toward TRAIL-resistant cancer cells has not yet been reported.
Further, as shown in Figs. 13A and 13B, when HWl (25 μg/m£) or
HW6 (25 βg/mi) was used to treat TRAIL-resistant HepG2 cells (Fig. 13A) and
Huh7 cells (Fig. 13B) for 20 hours, cell death was observed, the dead cells being detached from culture plates, but TRAIL (500 ng/m£)-treated cells remained unharmed.
Accordingly, it was confirmed that the inventive antibody of the monomeric form induces death of cancer cells, especially TRAIL-resistant cells.
(5-4) Assay for cell death induction in normal cell
In order to evaluate the cytotoxicities of HWl and HW6 for normal cells, cell culture, MTT assay and cell observation were conducted as described in (5- 1) and (5-2) except for using normal human hepatocytes and mammary epithelial cells (Cambrex BioScience) as well as brain astrocyte (see Kim et al., Oncogene, 24:838-849, 2005). The results are shown in Figs. 14A and 14B, and 15.
As shown in Figs. 14A and 14B, when human hepatocytes (Fig. 14A) and mammary epithelial cells (Fig. 14B) were treated with HWl or TRAIL at varying concentration for 30 hours, HWl as well as TRAIL induced about 30% cell death at a high concentration, but did not induce cell death at a lower concentration. Accordingly, it was confirmed that HWl exerted lesser cytotoxicity in normal cells.
Further, as shown in Fig. 15, the treatment with HWl (40 μg/m£) or HW6 (40 μg/M) of normal brain astrocytes at a high concentration for up to 48 hours did not induce any cell death.
Accordingly, it was demonstrated that the inventive antibodies induce cell death only in cancer cells, but exhibit no cytotoxicity in normal cells.
Test Example 6: Test of cancer cell death through DR5 induced by anti- DR5 scFv antibody
(6-1) Test of cell death in HCTl 16 cell expressing DR5
In order to check whether or not HWl induces cell death through DR5 expressed on cell surface, various concentration of a soluble competitor of DR5-Fc conjugated protein (DR5 fused to antibody constant region Fc) was added to TRAIL (100 ngM) or HWl (5 βglmt), and cell death inhibition was assessed while incubating the mixture for 30 hours. The result is shown in Fig. 16.
As shown in Fig. 16, cell death mediated by either TRAIL or HWl was gradually inhibited with concentrations of DR5-Fc, and cell death was completely inhibited with 10 μg/ml of DR5-Fc. Accordingly, it was confirmed that HWl as well as TRAIL induces cell death by the specific binding to DR5 expressed on cell surface.
(6-2) Test of cell death induced in HCTl 16 cell overexpressing DR5
In order to check whether or not HWl induces cell death of DR5- overexpressed cancer cell, HCTl 16 cancer cells were incubated, and treated with sulforaphane (10 μM) for 9 hours to overexpress DR5 (see Kim et al., Cancer Res., 66: 1740-1750, 2006). 0.01 to 10 μg/mi of TRAIL or HWl was added to the cells, incubated for 30 hours, and MTT assay was performed to analyze the degree of cancer cell death. The result is shown in Fig. 17.
As shown in Fig. 17, both TRAIL and HWl showed enhanced cell death activity for sulforaphane-treated cells, compared with the untreated control cells. Accordingly, it was proved that HWl induces cancer cell death through cell death signaling transduction through the specific binding to DR5.
Test Example 7: Evaluation of influence of a cross-linker on cancer cell death induced by anti-DR5 scFv antibody
Some anti-DR5 antibodies as IgG form having divalent binding sites have induced cell death, but their respective monovalent Fab form could not (see Motoki et al., Clin. Cancer Res., 1 1 :3126-3135, 2005; and Wajant et al., Oncogene, 20:4101-4106, 2001). Therefore, in order to check whether or not the inventive antibody induces cell death as a monomeric form as well as oligomeric form, 6X His tagged HWl was mixed with mouse derived anti-His6 IgG as a cross-linker (1 :1 molar concentration), and the mixture was incubated at 4 °C for 1 hour to form an oligomeric molecule. The molecule was added to TRAIL-sensitive HCTl 16 cells and TRAIL-resistant HepG2 cells, and the cells were incubated for 30 min. MTT assay was conducted as described in (5-2) of Test Example 5, and the result is shown in Fig. 18.
As shown in Fig. 18, both cells induced cell death regardless of treatment of a cross-linker.
Accordingly, it was proved that HWl induces cell death of cancer cells as a monomeric form.
Test Example 8: Study of cell death mechanism of anti-DR5 scFv antibody
In order to identify which cell death pathway is used for HWl -induced cell death of cancer cells, the following experiments were performed.
(8-1) Transmission electron microscopy (TEM)
TRAIL-sensitive HCTl 16 and Dul45 cells, and TRAIL-resistant Hep2 and U87MG cells were incubated with TRAIL (0.2 βglmi) and HWl (25 μg/m-C) for 5 hours and 30 hours, respectively, and prefixed with Karnovsky's solution (2% glutaraldehyde and 1% paraformaldehyde in 100 mmol/£ sodium cacodylate buffer, pH 7.4) at 25 °C for 2 hours. The cells were washed with 100 mmol/X sodium cacodylate buffer containing 2% glutaraldehyde and 1% paraformaldehyde (pH 7.4), postfixed with 1% osmium tetroxide and 1.5% potassium ferrocyanide for 1 hour, dehydrated by a graded series of ethanol (50- 100%), embedded on Poly Bed 812 resin (Pelco, Canada), cut into ultrathin slices using a ultramicrotome (Reichert Ultracut E microtome), and observed under an electron microscope (EM 902A, Carl Zeiss, Germany). The results are shown in Figs. 19A to 19D.
As shown in Figs. 19A to 19D, TRAIL-sensitive cancer cells treated with TRAIL showed typical characteristics of apoptotic cell deaths such as chromatic condensation and membrane blebbing. However, both TRAIL- sensitive and -resistant cells treated with HWl exhibited numerous autophagic vacuoles in the cells and damaged cellular organelles such as mitochondria (Fig. 19A).
Further, in higher magnificated images, multiple- and double- membrane-bound autophagic vacuoles (Figs. 19B and 19C), and autophagic vacuole induction step by fusing empty vacuoles with vacuoles containing cellular organelles (Fig. 19D) were observed in both cells. These characteristics indicated that the cell death was caused by autophagic cell death surely distinguished from apoptosis {see Kondo et al., Nat. Rev. Cancer, 5:726-734, 2005; and Tsujimoto et al., Cell Death Differ., 2:1528-1534, 2005). Accordingly, it was confirmed that cell death of various cancer cells induced by the inventive antibodies is resulted from autophagy. This fact is first reported for anti-DR5 antibody, and supports that a DR5 receptor can kill TRAIL-resistant cancer cells through autophagic cell death as well as apoptosis.
(8-2) Specific autophagic vacuole staining using Iysotracker-red
Autophagic cell death can be confirmed by using Iysotracker-red (DND- 99, Molecular probes), which specifically stains autophagic vacuoles {see Kondo et al., Nat. Rev. Cancer, 5:726-734, 2005; and Tsujimoto et al., Cell Death Differ., 2: 1528-1534, 2005).
Therefore, TRAIL-sensitive HCTl 16 cells and TRAIL-resistant U87MG cells were treated with HWl (25 βglvd) for 20 hours, fixed, stained with Iysotracker-red, and observed under a fluorescence microscope. The results are shown in Figs. 2OA and 2OB.
As shown in Figs. 2OA and 2OB, specifically stained autophagic vacuoles were observed in both HCTl 16 cells (Fig. 20A) and U87MG cells (Fig. 20B) treated with HWl.
Accordingly, it was proved that HWl induces autophagic cell death of cancer cells.
While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made and also fall within the scope of the invention as defined by the claims that follow.

Claims

What is claimed is:
1. An antibody specifically binding to death receptor 5 (DR5), which is selected from the group consisting of: an antibody comprising a heavy chain variable region (VH) having the amino acid sequences of SEQ ID NOs: 1 to 3 at complementary determining regions (CDRs) and a light chain variable region (VL) having the amino acid sequences of SEQ ID NOs: 4 to 6 at CDRs; and an antibody comprising a VH having the amino acid sequences of SEQ ID NOs: 7 to 9 at CDRs and a VL having the amino acid sequences of SEQ ID NOs: 10 to 12 at CDRs.
2. The antibody of claim 1, which induces cell death of TRAIL-sensitive and TRAIL-resistant cancer cells expressing DR5.
3. The antibody of claim 1, which is a whole antibody selected from the group consisting of IgCi, IgM, IgA, IgD and IgE.
4. The antibody of claim 1, which is an antibody fragment selected from the group consisting of single chain variable fragment (scFv), (scFv)2, Fab, Fab1, F(ab')2 and scFv-Fc.
5. The antibody of claim 1, which has an amino acid sequence of SEQ ID NO: 13 or 14.
6. The antibody of claim 1, which is conjugated with a marker selected from the group consisting of an enzyme, a fluorescent material and a radioactive material.
7. A DNA encoding the antibody of claim 1.
8. The DNA of claim 7, which encodes the amino acid sequence of SEQ ID NO: 13 or 14.
9. The DNA of claim 7, which has the nucleotide sequence of SEQ ID NO: 15 or 16.
10. A cell transformed with the DNA of claim 7 or an expression vector comprising the DNA.
11. The cell of claim 10, which expresses an antibody having the amino acid sequence of SEQ ID NO: 13 or 14.
12. A composition for preventing or treating a cancer comprising the antibody of claim 1 as an active ingredient.
13. The composition of claim 12, wherein the antibody is a whole antibody selected from the group consisting of IgG, IgM, IgA, IgD and IgE.
14. The composition of claim 12, wherein the antibody is an antibody fragment selected from the group consisting of scFv, (scFv)2, Fab, Fab1, F(ab')2 and scFv- Fc.
15. The composition of claim 12, wherein the antibody has the amino acid sequence of SEQ ID NO: 13 or 14.
16. The composition of claim 12, wherein the cancer expresses DR5.
17. The composition of claim 16, wherein the cancer is selected from the group consisting of breast cancer, colon cancer, brain tumor, glioma, ovarian cancer, endometrial cancer, bone sarcoma, cervix cancer, prostatic cancer, lung cancer, Synovial cancer, pancreatic cancer and sarcoma.
18. A method of preventing or treating a cancer comprising administering the antibody to a subject.
19. The method of claim 18, wherein the antibody is a whole antibody selected from the group consisting of IgG, IgM, IgA, IgD and IgE.
20. The method of claim 18, wherein the antibody is an antibody fragment selected from the group consisting of scFv, (scFv)2, Fab, Fab', F(ab')2 and scFv- Fc.
21. The method of claim 18, wherein the antibody has the amino acid sequence of SEQ ID NO: 13 or 14.
PCT/KR2007/002644 2006-07-05 2007-05-31 Antibody specifically binding to dr5 and composition for preventing or treating cancers comprising the same WO2008004760A1 (en)

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EP07746791A EP2046836A4 (en) 2006-07-05 2007-05-31 Antibody specifically binding to dr5 and composition for preventing or treating cancers comprising the same
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KR100847010B1 (en) 2008-07-17
JP2009542202A (en) 2009-12-03
EP2046836A4 (en) 2012-11-21
KR20080004339A (en) 2008-01-09
EP2046836A1 (en) 2009-04-15
US20090317396A1 (en) 2009-12-24
US8097704B2 (en) 2012-01-17

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