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 (V.sub.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.sub.L) having the amino acid sequences of SEQ ID NOs: 4 to 6 at CDRs; and an antibody comprising a (V.sub.H) having the amino acid sequences of SEQ ID NOs: 7 to 9 at CDRs and a (V.sub.L) 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 of the Invention

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, 114.sup.th to 281.sup.st 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 IgG1) (Georgakis et al., Br. J. Haematol., 130:501-510, 2005) and KMTR2 (human IgG4) (Motoki et al., Clin. 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 (V.sub.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.sub.L) having the amino acid sequences of SEQ ID NOs: 4 to 6 at CDRs; and an antibody comprising a V.sub.H having the amino acid sequences of SEQ ID NOs: 7 to 9 at CDRs and a V.sub.L 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.

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 U.S. Pat. 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).sub.2, Fab, Fab', F(ab').sub.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 U.S. Pat. No. 4,946,778 or 5,258,498, and Fab, Fab' and F(ab').sub.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 U.S. Pat. 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 DcR1 (death decoy receptor 1), DcR2, DR4, TNFR1, TNFR2 and CD95.

In the inventive antibody, the amino acid sequences of SEQ ID NOs: 1 to 3 or 7 to 9 are CDR1, 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 CDR1, 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, HW1 and HW6, which have the amino acid sequences of SEQ ID NOs: 13 and 14, respectively, and the antibodies comprise CDR1 to CDR3 of a heavy chain variable region, linker-oligopeptide and CDR1 to CDR3 of a light chain variable region in order (see FIGS. 1A and 1B (see Original Patent)).

HW1 and HW6 specifically bind to DR5, and have binding affinities (K.sub.D) of about 2.02.times.10.sup.-7 M, and 5.45.times.10.sup.-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.

Claim 1 of 10 Claims

1. An isolated antibody which specifically binds to death receptor 5 (DR5) selected from the group consisting of: an antibody comprising a heavy chain variable region (V.sub.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.sub.L) having the amino acid sequences of SEQ ID NOs: 4 to 6 at CDRs; and an antibody comprising a V.sub.H having the amino acid sequences of SEQ ID NOs: 7 to 9 at CDRs and a V.sub.L having the amino acid sequences of SEQ ID NOs: 10 to 12 at CDRs.
 

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