ANTI-DR5 ANTIBODY AND USE THEREOF

Abstract

The present invention relates to an antibody that specifically binds to death receptor 5 (DR5) and has a function of killing cancer cells. Specifically, provided are an anti-DR5 antibody or antigen-binding fragment thereof, and a use of the antibody or antigen-binding fragment for preventing or treating cancer. The present invention is characterized in that the anti-DR5 antibody or antigen-binding fragment thereof is improved in terms of affinity to DR5, stability, and an effect of killing cancer cells.

Claims

1. An anti-death receptor 5 (DR5) antibody or an antigen-binding fragment thereof, the antibody comprising: a polypeptide having the amino acid sequence of SEQ ID NO: 1 (V.sub.H-CDR1), a polypeptide having the amino acid sequence of SEQ ID NO: 2 (V.sub.H-CDR2), a polypeptide having the amino acid sequence of SEQ ID NO: 3 (V.sub.H-CDR3), a polypeptide having the amino acid sequence of SEQ ID NO: 4 (V.sub.L-CDR1), a polypeptide having the amino acid sequence of SEQ ID NO: 5 (V.sub.L-CDR2), and a polypeptide having the amino acid sequence of SEQ ID NO: 6 (V.sub.L-CDR3).

2. The anti-DR5 antibody or the antigen-binding fragment thereof according to claim 1, wherein the polypeptide (V.sub.H-CDR3) has an amino acid sequence selected from the group consisting of SEQ ID NOS: 7, 8, and 9.

3. The anti-DR5 antibody or the antigen-binding fragment thereof according to claim 1, wherein the polypeptide(V.sub.L-CDR2) has an amino acid sequence selected from the group consisting of SEQ ID NOS: 10, 11, and 12.

4. The anti-DR5 antibody or the antigen-binding fragment thereof according to claim 1, wherein the polypeptide (V.sub.L-CDR3) has an amino acid sequence selected from the group consisting of SEQ ID NOS: 13, 14, 15, and 16.

5. The anti-DR5 antibody or the antigen-binding fragment thereof according to claim 1, wherein the anti-DR5 antibody or the antigen-binding fragment thereof comprises: a heavy-chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 17 to SEQ ID NO: 37; and a light-chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 38 to SEQ ID NO: 49.

6. The anti-DR5 antibody or the antigen-binding fragment thereof according to claim 1, wherein the anti-DR5 antibody or the antigen-binding fragment thereof has activity of inducing apoptosis in TRAIL (TNF-related apoptosis-inducing ligand)-sensitive cancer cells that expresses DR5 or TRAIL-resistant cancer cells that expresses DR5.

7. The anti-DR5 antibody or the antigen-binding fragment thereof according to claim 1, wherein the anti-DR5 antibody is a monoclonal antibody.

8. The anti-DR5 antibody or the antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment is selected from the group consisting of scFv, (scFv)2, Fab, Fab′, and F(ab′)2 of the anti-DR5 antibody.

9. The anti-DR5 antibody or the antigen-binding fragment thereof according to claim 1, wherein the anti-DR5 antibody is a mouse-derived antibody, a mouse-human chimeric antibody, a humanized antibody, or a human antibody.

10-15. (canceled)

16. A polynucleotide molecule coding for the anti-DR5 antibody or the antigen-binding fragment according to claim 1.

17. A recombinant vector carrying the polynucleotide molecule of claim 16.

18. A recombinant cell harboring the recombinant vector of claim 17.

19. A method of producing an anti-DR5 antibody or an antigen-binding fragment thereof, the method comprising a step of expressing the polynucleotide molecule of claim 16.

20. A pharmaceutical composition comprising the anti-DR5 antibody or the antigen-binding fragment thereof according to claim 1.

21. A method of prevention or treatment of cancer, the method comprising a step of administering the anti-DR5 antibody or the antigen-binding fragment thereof according to claim 1 to a subject in need thereof.

22. The method according to claim 21, wherein the cancer is selected from the group consisting of blood cancer, lung cancer, stomach cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin melanoma, uterine cancer, ovarian cancer, rectal cancer, colorectal cancer, colon cancer, breast cancer, uterine sarcoma, fallopian tube carcinoma, endometrial carcinoma, uterine cervical carcinoma, vaginal carcinoma, vulva carcinoma, esophageal cancer, laryngeal cancer, small-intestine cancer, thyroid cancer, parathyroid cancer, soft-tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, solid tumors in juvenile stage, differentiated lymphoma, bladder cancer, renal cancer, renal cell carcinoma, renal pelvic carcinoma, primary central nervous system lymphoma, spinal axis tumors, brain stem glioma, and pituitary adenoma.

23. The method according to claim 21, further comprising a step of administering TNF-related apoptosis-inducing ligand (TRAIL) to the subject.

24. The method according to claim 21, further comprising a step of administering at least one anticancer agent selected from the group consisting of an alkylating anticancer agent, a metabolism antagonist-based anticancer agent, an anthracycline-based anticancer agent; and a proteasome inhibitor-based anticancer agent to the subject.

25. The method according to claim 24, wherein the anticancer agent is at least one selected from the group consisting of carboplatin, paclitaxel, gemcitabine, doxorubicin and bortezomib.

26. The method according to claim 23, further comprising a step of administering at least one anticancer agent selected from the group consisting of an alkylating anticancer agent, a metabolism antagonist-based anticancer agent, an anthracycline-based anticancer agent; and a proteasome inhibitor-based anticancer agent to the subject.

27. The method according to claim 26, wherein the anticancer agent is selected from the group consisting of carboplatin, paclitaxel, gemcitabine, doxorubicin, and bortezomib.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0128] FIG. 1 is an association and dissociation sensorgram depicting association and dissociation rates of the antibody D0.

[0129] FIGS. 2a to 2f are graphs comparing antibodies D0 and DA1 to DA35 with the control antibody AP with regard to binding activity for DR5.

[0130] FIGS. 3a and 3b exhibit SDS-polyacrylamide gel electrophoresis (SDS-PAGE) results of representative antibodies according to the present disclosure.

[0131] FIGS. 4a to 4h are size exclusion chromatograms (SEC) of representative antibodies according to the present disclosure.

[0132] FIG. 5 is shows the pharmacokinetic profile of the antibodies according to the present disclosure.

[0133] FIGS. 6a to 6r demonstrate that the antibodies according to the present disclosure have cytotoxic activity in tumor cells.

[0134] FIGS. 7a and 7b show the results of tumor growth inhibition of the antibodies according to the present disclosure in xenograft animal models.

[0135] FIGS. 8a and 8b demonstrate that the antibodies according to the present disclosure do not compete with TRAIL for binding to DR5.

[0136] FIGS. 9a and 9b are graphs delineating synergistic effects of the antibodies according to the present disclosure and TRAIL in assays for inducing cell death.

[0137] FIGS. 10a to 10c are graphs demonstrating that the antibodies according to the present disclosure induce cell death via apoptosis.

[0138] FIGS. 11a and 11b show the cytotoxic activity of the antibodies according to the present disclosure when used in combination with certain concentrations of gemcitabine.

MODE FOR CARRYING OUT THE INVENTION

[0139] Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

EXAMPLE 1

DR5 Immunization and cDNA Library Construction

[0140] In order to select antibodies binding specifically to DR5, animal-immunized antibody libraries were constructed. The libraries were constructed by obtaining mRNA from the immune cells of animals immunized with an antigen, amplifying an antibody gene through PCR using a combination of primers for the antibody gene, and cloning the antibody gene into phage display vectors.

[0141] Briefly, human DR5 (R&D systems, U.S.A.) in mixture with complete Freund's adjuvant and incomplete Freund's adjuvant (Sigma, U.S.A.) was subcutaneously injected to three White Leghorn chickens five times at regular intervals of three weeks. Sera from the immunized animals were diluted at a concentration of 1:100, 1:500, 1:2500, and 1:12500 in PBSB (3% bovine serum albumin in phosphate buffered saline) before being stored. Enzyme-linked immunosorbent assay was conducted to examine whether the sera bound human DR5. ELISA plates were coated overnight with 1 μg/ml human DR5 (R&D systems, U.S.A.) at 4° C., followed by reaction with the diluted sera for 2 hours. The plates were washed three times with PB ST (0.1% Tween-20 in PBS) and then incubated with anti-chicken immunoglobulin-HRP (horse radish peroxidase) (1:3000) for one hour. After three rounds of washing with PBST, color development was made for 20 min with ABTS (Thermo, U.S.A.). Absorbance at 405 nm was read on a microplate reader. Sera before immunization did not bind to DR5. Animals that produced sera with strong binding to human DR5 were selected.

[0142] Five days after the last injection, the bone marrow, spleens, and bursa of Fabricius were collected from the selected chicken. The tissues were mixed with 10 ml of TRI reagent (Molecular research center, U.S.A.) and homogenized with a homogenizer. Addition of 20 ml of TRI reagent was followed by centrifugation. The supernatant thus obtained was mixed with 3 ml of 1-bromo-3-chloropropane (BCP) and centrifuged to obtain supernatant. Total RNA was precipitated by addition of 15 ml of isopropanol. Reverse transcription was conducted using the SuperScript Transcription System (Invitrogen, U.S.A.) with random hexamers as primers (5 min at 65° C.; 5 min at 4° C.; 50 min at 50° C.; 5 min aft 85° C.; and 4° C.). 5 ul (microliters) of the reverse transcription reaction mixture containing the resulting cDNA was loaded onto 1% agarose gel and electrophoresed to detect bands of cDNA of various lengths.

EXAMPLE 2

Construction of Antibody Library

[0143] (2-1) Amplification of Immune Antibody Gene

[0144] In order to amplify the heavy- and light-chain variable regions V.sub.H and V.sub.L of the chicken antibody, PCR was performed as follows. For PCR, the cDNA prepared in Example 1 served as a template and combinations of primers designed for heavy-chain variable regions, light-chain variable regions, and scFv (single chain Fv) linking the heavy- and light-chain variable regions were used as shown in Table 4 below. 0.5 μl of each of the V.sub.H and V.sub.L cDNA libraries, 30 pmoles of the forward primer, 30 pmoles of the reverse primer, 10× PCR buffer, 200 μM dNTPs, and 0.5 μl Taq DNA polymerase was mixed and adjusted to a final volume of 50 pi and subjected to PCR starting with denaturation at 94° C. for 5 min, followed by 30 cycles of 94° C. for 15 sec, 56° C. for 30 sec, and 72° C. for 90 sec. PCR-amplified antibody DNA was separated according to size by 1% agarose gel electrophoresis and purified using a gel extraction kit (Qiagen, U.S.A.).

[0145] For obtaining scFv DNA, 50 ng of each of the purified V.sub.H and V.sub.L DNAs was used as a template and mixed with 30 pmoles of the forward primer, 30 pmoles of the reverse primers, 10× PCR buffer, 200 μM dNTPs, and 0.5 μl Taq DNA polymerase to a final volume of 50 μl. PCR was conducted by denaturation at 94° C. for 5 min, followed by 20 cycles of 94° C. for 30 sec, 56° C. for 30, and 72° C. for 2 min The PCR-amplified DNA was separated according to size on a 1% agarose gel electrophoresis and purified using a gel extraction kit (Qiagen, U.S.A.).

[0146] The primers used in the PCR are summarized in Table 4 below.

TABLE-US-00010 TABLE 4 Primers used in PCR SEQ ID Primer Sequence NO V.sub.H Forward GGT CAG TCC TCT AGA TCT TCC GGC GGT 50 GGTcGGC AGC TCC GGT GGT GGC GGT TCC GCC GTGcACG TTG GAC GAG Reverse CTG GCC GGC CTG GCC ACT AGT GGA GGA 51 GACcGAT GAC TTC GGT CC V.sub.L Forward GTG GCC CAG GCG GCC CTG ACT CAG CCG 52 TCCcTCG GTG TC Reverse GGA AGA TCT AGA GGA CTG ACC TAG GAC 53 GGTcCAGG scFV Forward GAG GAG GAG GAG GAG GAG GTG GCC CAG 54 GCG GCC CTG ACT CAG Reverse GAG GAG GAG GAG GAG GAG GAG CTG GCC 55 GGC CTG GCC ACT AGT GGA GG

[0147] (2-2) Restriction Enzyme Digestion of Antibody DNA

[0148] The scFV prepared above and the phagemid vector pComb3× (the Scripps Research Institute, CA, U.S.A.) were digested with the restriction enzyme SfiI (Roche, U.S.A.). A mixture of 10 μg of the scFv-encoding PCR fragment, 360 units SfiI (Roche, U.S.A.), and 20 μl of 10× buffer was volumetrically adjusted to have a final volume of 200 μl and allowed to react overnight at 50° C. In addition, a mixture of 20 μg of the pComb3× vector, 120 units SfiI, and 20 μl of 10× buffer was volumetrically adjusted to 200 μl and reacted overnight at 50° C. Each of the resulting digests was electrophoresed on agarose gel and purified using a gel extraction kit (Qiagen, U.S.A.).

[0149] (2-3) Ligation of Antibody DNA and Library Construction

[0150] In order to insert scFv fragments into pComb3×, a mixture of 700 ng of the scFV-encoding PCR fragments digested with restriction enzyme SfiI in (2-2) and 1.4 μg of pComb3× was reacted overnight at 16° C. in the presence of T4 DNA ligase (Invitrogen, U.S.A.). The ligation mixture thus obtained was purified by ethanol precipitation and transformed into E. coli ER2738(New England Biolab, U.S.A.) by electroporation. The E. coli were cultured in the presence of 46 μg/ml carbenicillin and 70 ug/ml kanamycin to construct a library having a complexity of 5×10.sup.9.

EXAMPLE 3

Selection of Phage Clone Carrying Anti-DR5 scFv

[0151] From the library obtained in Example 2, having randomized heavy and light chains in the form of scFV, antibodies binding to human DR5 were selected using solid phase immobilized DR5.

[0152] (3-1) Selection of Antibody Binding to DR5

[0153] First, 10 μg of human DR5 (R&D systems, U.S.A.) was conjugated to magnetic beads. An antibody DNA library was constructed by fusing the scFv-type antibodies obtained in Example 2 to phage coat protein PIII to enable expression of the antibodies on the phage surface. After being transformed with the antibody library DNA by electroporation, E. coli ER2738(New England Biolab) was cultured at 37° C. and then incubated overnight with VCSM13 helper phage (Stratagene, U.S.A.) in the presence of 46 μg/ml carbenicillin and 70 μg/ml kanamycin in SB medium (30 g/L Tryptone, 20 g/L yeast extract, and 10 g/L MOPS, pH 7.0). The resulting culture broth containing E. coli and phages was centrifuged to precipitate and remove E. coli. The supernatant was recovered and centrifuged after addition of 40 mg/ml polyethylene glycol 8000 and 30 mg/ml NaCl. The PEG precipitated phages were collected and resuspended in PBS. The phages were reacted with human DR5 conjugated to magnetic beads at room temperature for 2 hours to capture phages having affinity for DR5. Thereafter, the beads were washed with 0.5% Tween 20 in PBS and the bound phages were eluted with 0.1M glycine (pH 2.2) and neutralized with 2M Tris. The eluted phages were allowed to infect E. coli ER2738 and cultured overnight for the next round of panning This panning procedure was repeated four times. The repeated rounds of panning resulted in the accumulation of phages with high binding affinity. Individual clones selected from the plates of the fourth panning were incubated overnight with VCSM13 helper phage (1:1000) at 37° C. in the presence of 100 μg/ml carbenicillin and 70 μg/ml kanamycin in 96-deep well plates to induce the amplification of phages which express the antibody. After centrifugation of the resulting culture broth, the phages in the supernatant were pre-bound with TRAIL and then plated into DR5-coated ELISA plates. Incubation at 37° C. for 2 hours was followed by ELISA using an HRP-conjugated anti-M13 antibody to identify DR5-binding antibodies.

[0154] (3-2) Sequencing of Selected Antibody

[0155] E. coli ER2738 that were shown to harbor DR5-reactive clones under the selection conditions of Example (3-1) were cultured overnight in SB medium and harvested by centrifugation. Plasmid DNA was prepared using a DNA min-prep kit (GeneAll, Korea) and sequenced. For sequencing, the sequencing primers given in Table 5 below were used.

TABLE-US-00011 TABLE 5 Primers used in PCR Primer Sequence SEQ ID NO Forward ACA CTT TAT GCT TCC GGC TC 56 Reverse CAA AAT CAC CGG AAC CAG AG 57

EXAMPLE 4

Antibody Optimization—Humanization and Affinity Improvement

[0156] Among the antibody clones obtained from the animal immunized antibody libraries, a parental clone D0 (chimeric antibody) with high affinity and activity was selected. The framework of the antibody was switched to a human antibody framework (Nishibori et al., Molecular Immunology, 43 (2006)). For affinity improvement, a new phage library was constructed with random mutations in the CDR sequences of the heavy and light chain regions. The phage library was obtained in the same manner as in Example 2, reacted for 2 hours at room temperature with 10 μg of human DR5 immobilized onto magnetic beads, and washed five times with 0.5% Tween 20 in PBS. Thereafter, the bound phages were eluted with 0.1 M glycine (pH 2.2) and then neutralized with 2M Tris solution. To increase the selection pressure, 1 μg of the human DR5 was immobilized to magnetic beads and washed 10 times with Tween 20 in PBS in the second round of panning, while 0.1 μg of human DR5 was immobilized and washed 20 times with Tween 20 in PBS in the third round of panning In addition to the above humanization and affinity improvement methods, deimmunized variants were also constructed by substituting amino acids in the variable region framework sequences of the parental clone s that were predicted to have a high immunogenicity in immune cells. The humanized or deimmunized variants of the parental clone obtained as described above were designated “DA1 to DA35”.

[0157] Sequences of the complementarity determining regions and variable regions obtained from D0 and DA1 to DA35 clones are summarized in Tables 6 to 9 below:

TABLE-US-00012 TABLE 6 Sequences of Heavy Chain CDRs SEQ SEQ SEQ Clone ID ID ID no. NO V.sub.H-CDR1 NO V.sub.H-CDR2 NO V.sub.H-CDR3 D0 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA1 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA2 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA3 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA4 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA5 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA6 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA7 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA8 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA9 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA10 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA11 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA12 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA13 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA14 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA15 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA16 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA17 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA18 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA19 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA20 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA21 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA22 1 GFTFSSF 2 GIGKSDRYTGY 8 DAGSPCGSGGW NML GSAVKG TGACIDT DA23 1 GFTFSSF 2 GIGKSDRYTGY 9 DAGSPCGKGGW NML GSAVKG TGACIDT DA24 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA25 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA26 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA27 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA28 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA29 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA30 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA31 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA32 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA33 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA34 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT DA35 1 GFTFSSF 2 GIGKSDRYTGY 7 DAGSGCGSGGW NML GSAVKG TGACIDT

TABLE-US-00013 TABLE 7 Sequences of Light Chain CDRs SEQ SEQ SEQ Clone ID ID ID no. NO V.sub.L-CDR1 NO V.sub.L-CDR2 NO V.sub.L-CDR3 D0 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA1 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA2 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA3 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA4 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA5 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA6 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA7 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA8 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA9 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA10 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA11 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA12 4 SGGDSYAGSY 11 NNNNL 13 GSRDSSY YYG MP VGI DA13 4 SGGDSYAGSY 10 NNNNR 14 GSRDSAG YYG PS MGA DA14 4 SGGDSYAGSY 12 NNNNK 13 GSRDSSY YYG AK VGI DA15 4 SGGDSYAGSY 10 NNNNR 15 GSRDSDG YYG PS GGR DA16 4 SGGDSYAGSY 10 NNNNR 16 GSRDSSG YYG PS AGG DA17 4 SGGDSYAGSY 12 NNNNK 13 GSRDSSY YYG AK VGI DA18 4 SGGDSYAGSY 10 NNNNR 15 GSRDSDG YYG PS GGR DA19 4 SGGDSYAGSY 11 NNNNL 13 GSRDSSY YYG MP VGI DA20 4 SGGDSYAGSY 10 NNNNR 14 GSRDSAG YYG PS MGA DA21 4 SGGDSYAGSY 10 NNNNR 16 GSRDSSG YYG PS AGG DA22 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA23 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA24 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA25 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA26 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA27 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA28 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA29 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA30 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA31 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA32 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA33 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA34 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI DA35 4 SGGDSYAGSY 10 NNNNR 13 GSRDSSY YYG PS VGI

TABLE-US-00014 TABLE 8 Sequences of Heavy Chain Variable Regions SEQ VH- VH- VH- VH- VH- ID Clone VH-FW1 CDR1 FW2 CDR2 VH-FW3 CDR3 FW4 NO D0 AVTLD GFT WV GIGK RATISRDD DAGS WGH 17 ESGGG FSS RQA SDRY GQSTVRL GCGS GTEV LQTPG FN PGK TGY QLNNLRA GGWT IVSS GGLSL ML GLE GSA EDTGTYY GACID VCKGS WV VKG CVK T A DA1 EVQLV GFT WV GIGK RFTISRDD DAGS WGQ 18 ESGGG FSS RQA SDRY SKSTVYL GCGS GTLV LVQPG FN PGK TGY QMNSLRA GGWT TVSS GSLRLS ML GLE GSA EDTAVYY GACID CAAS WV VKG CVR T A DA2 EVQLV GFT WV GIGK RFTISRDD DAGS WGQ 18 ESGGG FSS RQA SDRY SKSTVYL GCGS GTLV LVQPG FN PGK TGY QMNSLRA GGWT TVSS GSLRLS ML GLE GSA EDTAVYY GACID CAAS WV VKG CVR T A DA3 EVQLV GFT WV GIGK RFTISRDT DAGS WGQ 19 ESGGG FSS RQA SDRY SKSTVYL GCGS GTLV LVQPG FN PGK TGY QMNSLRA GGWT TVSS GSLRLS ML GLE GSA EDTAVYY GACID CAAS WV VKG CVR T A DA4 EVQLV GFT WV GIGK RFTISRDD DAGS WGQ 20 ESGGG FSS RQA SDRY SKNTVYL GCGS GTLV LVQPG FN PGK TGY QMNSLRA GGWT TVSS GSLRLS ML GLE GSA EDTAVYY GACID CAAS WV VKG CVR T A DA5 EVQLV GFT WV GIGK RFTISRDD DAGS WGQ 21 ESGGG FSS RQA SDRY SKSTAYL GCGS GTLV LVQPG FN PGK TGY QMNSLRA GGWT TVSS GSLRLS ML GLE GSA EDTAVYY GACID CAAS WV VKG CVR T A DA6 EVQLV GFT WV GIGK RFTISRDD DAGS WGQ 22 ESGGG FSS RQA SDRY SKSTVYL GCGS GTLV LVQPG FN PGK TGY QMNSLRA GGWT TVSS GSLRLS ML GLE GSA EDTAVYY GACID CAAS WV VKG CAR T A DA7 EVQLV GFT WV GIGK RFTISRDD DAGS WGQ 23 ESGGG FSS RQA SDRY SKSTVYL GCGS GTLV LVQPG FN PGK TGY QMNSLRA GGWT TVSS GSLRLS ML GLE GSA EDTAVYY GACID CAAS WV VKG CSR T A DA8 EVQLV GFT WV GIGK RFTISRDD DAGS WGQ 18 ESGGG FSS RQA SDRY SKSTVYL GCGS GTLV LVQPG FN PGK TGY QMNSLRA GGWT TVSS GSLRLS ML GLE GSA EDTAVYY GACID CAAS WV VKG CVR T A DA9 EVQLV GFT WV GIGK RFTISRDD DAGS WGQ 24 ESGGG FSS RQA SDRY SKSTLYL GCGS GTLV LVQPG FN PGK TGY QMNSLRA GGWT TVSS GSLRLS ML GLE GSA EDTAVYY GACID CAAS WV VKG CVR T A DA10 EVQLV GFT WV GIGK RFTISRDT DAGS WGQ 25 ESGGG FSS RQA SDRY SKNTAYL GCGS GTLV LVQPG FN PGK TGY QMNSLRA GGWT TVSS GSLRLS ML GLE GSA EDTAVYY GACID CAAS WV VKG CSR T A DA11 EVQLV GFT WV GIGK RFTISRDT DAGS WGQ 26 ESGGG FSS RQA SDRY SKNTAYL GCGS GTLV LVQPG FN PGK TGY QMNSLRA GGWT TVSS GSLRLS ML GLE GSA EDTAVYY GACID CAAS WV VKG CAR T A DA12 EVQLV GFT WV GIGK RFTISRDT DAGS WGQ 27 ESGGG FSS RQA SDRY SKNTAYL GCGS GTLV LVQPG FN PGK TGY QMNSLRA GGWT TVSS GSLRLS ML GLE GSA EDTAVYY GACID CAAS WV VKG CSR T A DA13 EVQLV GFT WV GIGK RFTISRDT DAGS WGQ 27 ESGGG FSS RQA SDRY SKNTAYL GCGS GTLV LVQPG FN PGK TGY QMNSLRA GGWT TVSS GSLRLS ML GLE GSA EDTAVYY GACID CAAS WV VKG CSR T A DA14 EVQLV GFT WV GIGK RFTISRDT DAGS WGQ 27 ESGGG FSS RQA SDRY SKNTAYL GCGS GTLV LVQPG FN PGK TGY QMNSLRA GGWT TVSS GSLRLS ML GLE GSA EDTAVYY GACID CAAS WV VKG CSR T A DA15 EVQLV GFT WV GIGK RFTISRDT DAGS WGQ 27 ESGGG FSS RQA SDRY SKNTAYL GCGS GTLV LVQPG FN PGK TGY QMNSLRA GGWT TVSS GSLRLS ML GLE GSA EDTAVYY GACID CAAS WV VKG CSR T A DA16 EVQLV GFT WV GIGK RFTISRDT DAGS WGQ 27 ESGGG FSS RQA SDRY SKNTAYL GCGS GTLV LVQPG FN PGK TGY QMNSLRA GGWT TVSS GSLRLS ML GLE GSA EDTAVYY GACID CAAS WV VKG CSR T A DA17 EVQLV GFT WV GIGK RFTISRDD DAGS WGQ 18 ESGGG FSS RQA SDRY SKSTVYL GCGS GTLV LVQPG FN PGK TGY QMNSLRA GGWT TVSS GSLRLS ML GLE GSA EDTAVYY GACID CAAS WV VKG CVR T A DA18 EVQLV GFT WV GIGK RFTISRDD DAGS WGQ 18 ESGGG FSS RQA SDRY SKSTVYL GCGS GTLV LVQPG FN PGK TGY QMNSLRA GGWT TVSS GSLRLS ML GLE GSA EDTAVYY GACID CAAS WV VKG CVR T A DA19 EVQLV GFT WV GIGK RFTISRDD DAGS WGQ 18 ESGGG FSS RQA SDRY SKSTVYL GCGS GTLV LVQPG FN PGK TGY QMNSLRA GGWT TVSS GSLRLS ML GLE GSA EDTAVYY GACID CAAS WV VKG CVR T A DA20 EVQLV GFT WV GIGK RFTISRDD DAGS WGQ 18 ESGGG FSS RQA SDRY SKSTVYL GCGS GTLV LVQPG FN PGK TGY QMNSLRA GGWT TVSS GSLRLS ML GLE GSA EDTAVYY GACID CAAS WV VKG CVR T A DA21 EVQLV GFT WV GIGK RFTISRDD DAGS WGQ 18 ESGGG FSS RQA SDRY SKSTVYL GCGS GTLV LVQPG FN PGK TGY QMNSLRA GGWT TVSS GSLRLS ML GLE GSA EDTAVYY GACID CAAS WV VKG CVR T A DA22 EVQLV GFT WV GIGK RFTISRDD DAGSP WGQ 28 ESGGG FSS RQA SDRY SKSTVYL CGSG GTLV LVQPG FN PGK TGY QMNSLRA GWTG TVSS GSLRLS ML GLE GSA EDTAVYY ACIDT CAAS WV VKG CVR A DA23 EVQLV GFT WV GIGK RFTISRDD DAGSP WGQ 29 ESGGG FSS RQA SDRY SKSTVYL CGKG GTLV LVQPG FN PGK TGY QMNSLRA GWTG TVSS GSLRLS ML GLE GSA EDTAVYY ACIDT CAAS WV VKG CVR A DA24 AVTLD GFT WV GIGK RATISRDD DAGS WGH 17 ESGGG FSS RQA SDRY GQSTVRL GCGS GTEV LQTPG FN PGK TGY QLNNLRA GGWT IVSS GGLSL ML GLE GSA EDTGTYY GACID VCKGS WV VKG CVK T A DA2 5AVTLD GFT WV GIGK RATISRDD DAGS WGH 30 ESGGG FSS RQA SDRY GQSTVYL GCGS GTEV LQTPG FN PGK TGY QMNSLRA GGWT IVSS GGLSL ML GLE GSA EDTGTYY GACID VCKGS WV VKG CVK T A DA26 AVTLD GFT WV GIGK RATISRDD DAGS WGH 30 ESGGG FSS RQA SDRY GQSTVYL GCGS GTEV LQTPG FN PGK TGY QMNSLRA GGWT IVSS GGLSL ML GLE GSA EDTGTYY GACID VCKGS WV VKG CVK T A DA27 AVTLD GFT WV GIGK RATISRDD DAGS WGH 17 ESGGG FSS RQA SDRY GQSTVRL GCGS GTEV LQTPG FN PGK TGY QLNNLRA GGWT IVSS GGLSL ML GLE GSA EDTGTYY GACID VCKGS WV VKG CVK T A DA28 AVTLD GFT WV GIGK RATISRDD DAGS WGH 31 ESGGG FSS RQA SDRY GQSTARL GCGS GTEV LQTPG FN PGK TGY QLNNLRA GGWT IVSS GGLSL ML GLE GSA EDTGTYY GACID VCKGS WV VKG CVK T A DA29 AVTLD GFT WV GIGK RATISRDD DAGS WGH 31 ESGGG FSS RQA SDRY GQSTARL GCGS GTEV LQTPG FN PGK TGY QLNNLRA GGWT IVSS GGLSL ML GLE GSA EDTGTYY GACID VCKGS WV VKG CVK T A DA30 AVTLD GFT WV GIGK RATISRDD DAGS WGH 32 ESGGG FSS RQA SDRY GQSTAYL GCGS GTEV LQTPG FN PGK TGY QMNSLRA GGWT IVSS GGLSL ML GLE GSA EDTGTYY GACID VCKGS WV VKG CVK T A DA31 AVTLD GFT WV GIGK RATISRDD DAGS WGH 33 ESGGG FSS RQA SDRY GQSTARL GCGS GTEV LQTPG FN PGK TGY QMNSLRA GGWT IVSS GGLSL ML GLE GSA EDTGTYY GACID VCKGS WV VKG CVK T A DA32 AVTLD GFT WV GIGK RATISRDD DAGS WGH 34 ESGGG FSS RQA SDRY GQSTVRL GCGS GTEV LQTPG FN PGK TGY QLNNLRA GGWT IVSS GGLSL ML GLE GSA EDTAVYY GACID VCKGS WV VKG CVK T A DA33 AVTLD GFT WV GIGK RATISRDD DAGS WGH 35 ESGGG FSS RQA SDRY GQSTARL GCGS GTEV LQTPG FN PGK TGY QLNNLRA GGWT IVSS GGLSL ML GLE GSA EDTAVYY GACID VCKGS WV VKG CVK T A DA34 AVTLD GFT WV GIGK RATISRDD DAGS WGH 36 ESGGG FSS RQA SDRY GQSTAYL GCGS GTEV LQTPG FN PGK TGY QMNSLRA GGWT IVSS GGLSL ML GLE GSA EDTAVYY GACID VCKGS WV VKG CVK T A DA35 AVTLD GFT WV GIGK RATISRDD DAGS WGH 37 ESGGG FSS RQA SDRY GQSTARL GCGS GTEV LQTPG FN PGK TGY QMNSLRA GGWT IVSS GGLSL ML GLE GSA EDTAVYY GACID VCKGS WV VKG CVK T A

[0158] (In Table 8 above, VH-FW1, VH-FW2, and VH-FW3 represent frameworks of the heavy-chain variable region)

TABLE-US-00015 TABLE 9 Sequences of Light Chain Variable Regions VL- SEQ VL- VL- CDR VL- VL- ID Clone VL-FW1 CDR1 FW2 2 VL-FW3 CDR3 FW4 NO D0 LTQPSS SGG WYQ NN NIPSRFSG GSR FGA 38 VSANL DSY QKA NNR STSGSTAT DSS GTT GGTVEI AGS PGSA PS LTITGVQA YV LTV TC YY PVTV EDEAVYF GI L YG IY C DA1 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 39 QSPSSL DSY QKP NNR STSGTDFT DSS GTK SASVG AGS GKA PS LTISSLQP YV VEI DRVTIT YY PKTL EDFATYY GI K C YG IY C DA2 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 40 QSPSSL DSY QKP NNR SRSGTDFT DSS GTK SASVG AGS GKA PS LTISSLQP YV VEI DRVTIT YY PKTL EDFATYY GI K C YG IY C DA3 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 39 QSPSSL DSY QKP NNR STSGTDFT DSS GTK SASVG AGS GKA PS LTISSLQP YV VEI DRVTIT YY PKTL EDFATYY GI K C YG IY C DA4 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 39 QSPSSL DSY QKP NNR STSGTDFT DSS GTK SASVG AGS GKA PS LTISSLQP YV VEI DRVTIT YY PKTL EDFATYY GI K C YG IY C DA5 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 39 QSPSSL DSY QKP NNR STSGTDFT DSS GTK SASVG AGS GKA PS LTISSLQP YV VEI DRVTIT YY PKTL EDFATYY GI K C YG IY C DA6 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 39 QSPSSL DSY QKP NNR STSGTDFT DSS GTK SASVG AGS GKA PS LTISSLQP YV VEI DRVTIT YY PKTL EDFATYY GI K C YG IY C DA7 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 39 QSPSSL DSY QKP NNR STSGTDFT DSS GTK SASVG AGS GKA PS LTISSLQP YV VEI DRVTIT YY PKTL EDFATYY GI K C YG IY C DA8 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 41 QSPSSL DSY QKP NNR SGSGTDFT DSS GTK SASVG AGS GKA PS LTISSLQP YV VEI DRVTIT YY PKTL EDFATYY GI K C YG IY C DA9 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 40 QSPSSL DSY QKP NNR SRSGTDFT DSS GTK SASVG AGS GKA PS LTISSLQP YV VEI DRVTIT YY PKTL EDFATYY GI K C YG IY C DA10 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 40 QSPSSL DSY QKP NNR SRSGTDFT DSS GTK SASVG AGS GKA PS LTISSLQP YV VEI DRVTIT YY PKTL EDFATYY GI K C YG IY C DA11 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 40 QSPSSL DSY QKP NNR SRSGTDFT DSS GTK SASVG AGS GKA PS LTISSLQP YV VEI DRVTIT YY PKTL EDFATYY GI K C YG IY C DA12 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 42 QSPSSL DSY QKP NNL SRSGTDFT DSS GTK SASVG AGS GKA MP LTISSLQP YV VEI DRVTIT YY PKTL EDFATYY GI K C YG IY C DA13 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 43 QSPSSL DSY QKP NNR SRSGTDFT DSA GTK SASVG AGS GKA PS LTISSLQP GM VEI DRVTIT YY PKTL EDFATYY GA K C YG IY C DA14 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 44 QSPSSL DSY QKP NN SRSGTDFT DSS GTK SASVG AGS GKA KA LTISSLQP YV VEI DRVTIT YY PKTL K EDFATYY GI K C YG IY C DA15 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 45 QSPSSL DSY QKP NNR SRSGTDFT DSD GTK SASVG AGS GKA PS LTISSLQP GG VEI DRVTIT YY PKTL EDFATYY GR K C YG IY C DA16 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 46 QSPSSL DSY QKP NNR SRSGTDFT DSS GTK SASVG AGS GKA PS LTISSLQP GA VEI DRVTIT YY PKTL EDFATYY GG K C YG IY C DA17 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 44 QSPSSL DSY QKP NN SRSGTDFT DSS GTK SASVG AGS GKA KA LTISSLQP YV VEI DRVTIT YY PKTL K EDFATYY GI K C YG IY C DA18 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 45 QSPSSL DSY QKP NNR SRSGTDFT DSD GTK SASVG AGS GKA PS LTISSLQP GG VEI DRVTIT YY PKTL EDFATYY GR K C YG IY C DA19 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 42 QSPSSL DSY QKP NNL SRSGTDFT DSS GTK SASVG AGS GKA MP LTISSLQP YV VEI DRVTIT YY PKTL EDFATYY GI K C YG IY C DA20 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 43 QSPSSL DSY QKP NNR SRSGTDFT DSA GTK SASVG AGS GKA PS LTISSLQP GM VEI DRVTIT YY PKTL EDFATYY GA K C YG IY C DA21 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 46 QSPSSL DSY QKP NNR SRSGTDFT DSS GTK SASVG AGS GKA PS LTISSLQP GA VEI DRVTIT YY PKTL EDFATYY GG K C YG IY C DA22 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 47 QSPSSL DSY QKP NNR STSGTDFT DSS GTK SASVG AGS GKA PS LTISSLQP YV VEI DRVTIT YY PKTL EDFATYY GI K C YG IY C DA23 DIQMT SGG WYQ NN GVPSRFSG GSR FGQ 47 QSPSSL DSY QKP NNR STSGTDFT DSS GTK SASVG AGS GKA PS LTISSLQP YV VEI DRVTIT YY PKTL EDFATYY GI K C YG IY C DA24 LTQPSS SGG WYQ NN NIPSRFSG GSR FGA 48 VSANL DSY QKA NNR STSGSTAT DSS GTT GGTVEI AGS PGSA PS LTITGVQA YV LTV TC YY PVTV EDEATYY GI L YG IY C DA25 LTQPSS SGG WYQ NN NIPSRFSG GSR FGA 38 VSANL DSY QKA NNR STSGSTAT DSS GTT GGTVEI AGS PGSA PS LTITGVQA YV LTV TC YY PVTV EDEAVYF GI L YG IY C DA26 LTQPSS SGG WYQ NN NIPSRFSG GSR FGA 48 VSANL DSY QKA NNR STSGSTAT DSS GTT GGTVEI AGS PGSA PS LTITGVQA YV LTV TC YY PVTV EDEATYY GI L YG IY C DA27 LTQPSS SGG WYQ NN NIPSRFSG GSR FGA 49 VSANL DSY QKA NNR STSGSTAT DSS GTT GGTVEI AGS PGSA PS LTITGVQA YV LTV TC YY PVTV EDEATYF GI L YG IY C DA28 LTQPSS SGG WYQ NN NIPSRFSG GSR FGA 38 VSANL DSY QKA NNR STSGSTAT DSS GTT GGTVEI AGS PGSA PS LTITGVQA YV LTV TC YY PVTV EDEAVYF GI L YG IY C DA29 LTQPSS SGG WYQ NN NIPSRFSG GSR FGA 49 VSANL DSY QKA NNR STSGSTAT DSS GTT GGTVEI AGS PGSA PS LTITGVQA YV LTV TC YY PVTV EDEATYF GI L YG IY C DA30 LTQPSS SGG WYQ NN NIPSRFSG GSR FGA 48 VSANL DSY QKA NNR STSGSTAT DSS GTT GGTVEI AGS PGSA PS LTITGVQA YV LTV TC YY PVTV EDEATYY GI L YG IY C DA31 LTQPSS SGG WYQ NN NIPSRFSG GSR FGA 49 VSANL DSY QKA NNR STSGSTAT DSS GTT GGTVEI AGS PGSA PS LTITGVQA YV LTV TC YY PVTV EDEATYF GI L YG IY C DA32 LTQPSS SGG WYQ NN NIPSRFSG GSR FGA 38 VSANL DSY QKA NNR STSGSTAT DSS GTT GGTVEI AGS PGSA PS LTITGVQA YV LTV TC YY PVTV EDEAVYF GI L YG IY C DA33 LTQPSS SGG WYQ NN NIPSRFSG GSR FGA 49 VSANL DSY QKA NNR STSGSTAT DSS GTT GGTVEI AGS PGSA PS LTITGVQA YV LTV TC YY PVTV EDEATYF GI L YG IY C DA34 LTQPSS SGG WYQ NN NIPSRFSG GSR FGA 48 VSANL DSY QKA NNR STSGSTAT DSS GTT GGTVEI AGS PGSA PS LTITGVQA YV LTV TC YY PVTV EDEATYY GI L YG IY C DA35 LTQPSS SGG WYQ NN NIPSRFSG GSR FGA 49 VSANL DSY QKA NNR STSGSTAT DSS GTT GGTVEI AGS PGSA PS LTITGVQA YV LTV TC YY PVTV EDEATYF GI L YG IY C

[0159] (In Table 9, VL-FW1, VL-FW2, and VL-FW3 represent frameworks of the light-chain variable region)

EXAMPLE 5

Production of Antibody

[0160] Immunoglobulin (IgG) proteins were produced for use in assaying the affinity and activity of the antibodies obtained above. From pComb3× carrying the scFv nucleotide, variable and constant region fragments of the heavy and light chains were obtained by PCR using the primer combinations shown in Table 10 and the condition used in Example 2.

[0161] Genes of variable regions (V.sub.H to V.sub.L) and constant regions (C.sub.H to C.sub.k) in the heavy and light chains were amplified by PCR using the combinations of HC and LC primers in Table 10 and inserted into mammalian cell expression plasmids using the pcDNATM3.3-TOPO® TA cloning kit and pOptiTMVEC-TOPO® TA cloning kit (Invitrogen, U.S.A.). 1 μl of each of the vectors (pcDNATM3.3-TOPO® vector and pOptiTM VEC-TOPO® vector) and fragments were added to a buffer containing 200 mM NaCl and 10 mM MgCl.sub.2 to a final volume of 6 μl and reacted at room temperature for 5 min. The vectors were transformed into DH5α E. coli competent cells by heat shock. The resultant colonies were mass cultured to obtain plasmids. The plasmids prepared above were transfected into HEK293F cells (Invitrogen, U.S.A.), which were than cultured for 7 days to express the antibodies. The antibodies were purified using a protein A column (GE, U.S.A.). Cell culture supernatants were loaded onto the column to bind the antibodies (IgG) to protein A, followed by elution with 20 mM sodium citrate buffer (pH 3.0). The antibodies were confirmed by SDS-PAGE to have high purity and light chain and heavy chain molecular weights in accordance with the theoretical size.

TABLE-US-00016 TABLE 10 SEQ ID Primer Sequence NO V.sub.H Forward GCT AGC CGC CAC CAT GGG C 58 Reverse AGG GGC CCT TGG TGG AGG CCT GGC 59 CGG CCT GGC CAC T C.sub.H Forward GCC TCC ACC AAG GGC CCC TC 60 Reverse CGG GAT CCC TTG CCG GCC GT 61 HC Forward GCT AGC CGC CAC CAT GGG C 62 (Heavy Reverse CGG GAT CCC TTG CCG GCC GT 63 Chain) V.sub.L Forward AAG CTT GCC GCC ACC ATG 64 Reverse AGG GGG CGG CCA CGG TCC GGG AAG 65 ATC TAG AGG ACT G c.sub.k Forward CGG ACC GTG GCC GCC CCC TC 66 Reverse GCT CTA GAC TAG CAC TCG C 67 LC Forward AAG CTT GCC GCC ACC ATG 68 (Light Reverse GCT CTA GAC TAG CAC TCG C 69 Chain)

EXAMPLE 6

Assay of Binding Affinity of Antibody to Human DR5

[0162] In order to measure affinity, human DR5 was coupled to carboxylated dextran biosensor chips (CM5, GE) according to the manufacturer's instruction. Association and dissociation rates were assessed by injecting IgG proteins that were 2-fold serially diluted to 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, 0.313 nM, and 0.156 nM.

[0163] Association and dissociation rates were depicted in association and dissociation sensorgrams and calculated using a simple 1:1 Langmuir binding model (BIAcore X100 Evaluation Software, ver. 2.0). The equilibrium dissociation constants (KD), which is the ratio of dissociation constant (Kd) to association constant (Ka), were confirmed to be in the sub-nanomolar range. Measurements of the binding of antibody D0 and DA1 to DA35 of the present disclosure to DR5 are given in Table 11 below (antibody names are given as the names of the clones from which the antibodies were derived). A representative sensorgram for D0 is depicted in FIG. 1.

TABLE-US-00017 TABLE 11 Binding Antibody Molecule Ka(1/Ms) Kd(1/s) KD(M) D0 Human DR5 1.49 × 10{circumflex over ( )}6  2.26 × 10{circumflex over ( )}−5 1.52 × 10{circumflex over ( )}−11 DA1 Human DR5 4.90 × 10{circumflex over ( )}6  1.80 × 10{circumflex over ( )}−9 3.80 × 10{circumflex over ( )}−16 DA2 Human DR5 1.90 × 10{circumflex over ( )}6   4.90 × 10{circumflex over ( )}−10 2.10 × 10{circumflex over ( )}−16 DA3 Human DR5 4.90 × 10{circumflex over ( )}6  1.80 × 10{circumflex over ( )}−9 3.80 × 10{circumflex over ( )}−16 DA4 Human DR5 1.20 × 10{circumflex over ( )}6  1.20 × 10{circumflex over ( )}−7 1.00 × 10{circumflex over ( )}−13 DA5 Human DR5 1.20 × 10{circumflex over ( )}6  2.60 × 10{circumflex over ( )}−9 2.10 × 10{circumflex over ( )}−15 DA6 Human DR5 7.80 × 10{circumflex over ( )}6  4.20 × 10{circumflex over ( )}−6 5.30 × 10{circumflex over ( )}−13 DA7 Human DR5 1.40 × 10{circumflex over ( )}6  2.90 × 10{circumflex over ( )}−6 1.90 × 10{circumflex over ( )}−12 DA8 Human DR5 1.60 × 10{circumflex over ( )}6  2.20 × 10{circumflex over ( )}−8 1.30 × 10{circumflex over ( )}−14 DA9 Human DR5 5.60 × 10{circumflex over ( )}6  4.00 × 10{circumflex over ( )}−7 7.10 × 10{circumflex over ( )}−14 DA10 Human DR5 2.30 × 10{circumflex over ( )}6  1.60 × 10{circumflex over ( )}−5 7.00 × 10{circumflex over ( )}−12 DA12 Human DR5 2.37 × 10{circumflex over ( )}06 2.99 × 10{circumflex over ( )}−5 1.27 × 10{circumflex over ( )}−11 DA13 Human DR5 2.62 × 10{circumflex over ( )}06 2.94 × 10{circumflex over ( )}−5 1.12 × 10{circumflex over ( )}−11 DA14 Human DR5 4.27 × 10{circumflex over ( )}06 1.00 × 10{circumflex over ( )}−4 2.35 × 10{circumflex over ( )}−11 DA15 Human DR5 2.56 × 10{circumflex over ( )}06 2.78 × 10{circumflex over ( )}−6 1.09 × 10{circumflex over ( )}−12 DA16 Human DR5 2.11 × 10{circumflex over ( )}06 5.53 × 10{circumflex over ( )}−5 2.61 × 10{circumflex over ( )}−11 DA17 Human DR5 2.54 × 10{circumflex over ( )}06 5.97 × 10{circumflex over ( )}−5 2.35 × 10{circumflex over ( )}−11 DA18 Human DR5 1.33 × 10{circumflex over ( )}06 3.67 × 10{circumflex over ( )}−5 2.77 × 10{circumflex over ( )}−11 DA20 Human DR5 1.00 × 10{circumflex over ( )}06 1.21 × 10{circumflex over ( )}−5 1.20 × 10{circumflex over ( )}−11 DA21 Human DR5 1.11 × 10{circumflex over ( )}06 7.17 × 10{circumflex over ( )}−6 6.43 × 10{circumflex over ( )}−12 DA22 Human DR5 9.32 × 10{circumflex over ( )}05 3.19 × 10{circumflex over ( )}−5 3.42 × 10{circumflex over ( )}−11 DA23 Human DR5 6.96 × 10{circumflex over ( )}05 2.80 × 10{circumflex over ( )}−5 4.03 × 10{circumflex over ( )}−11 DA24 Human DR5 1.20 × 10{circumflex over ( )}06 1.23 × 10{circumflex over ( )}−6 1.02 × 10{circumflex over ( )}−12 DA25 Human DR5 1.05 × 10{circumflex over ( )}06 1.71 × 10{circumflex over ( )}−6 1.62 × 10{circumflex over ( )}−12 DA26 Human DR5 1.09 × 10{circumflex over ( )}06 1.10 × 10{circumflex over ( )}−6 1.01 × 10{circumflex over ( )}−12 DA27 Human DR5 1.17 × 10{circumflex over ( )}06 2.24 × 10{circumflex over ( )}−6 1.92 × 10{circumflex over ( )}−12 DA28 Human DR5 1.16 × 10{circumflex over ( )}06 4.52 × 10{circumflex over ( )}−6 3.89 × 10{circumflex over ( )}−12 DA29 Human DR5 1.21 × 10{circumflex over ( )}06 8.47 × 10{circumflex over ( )}−5 6.98 × 10{circumflex over ( )}−11 DA30 Human DR5 1.03 × 10{circumflex over ( )}06 7.97 × 10{circumflex over ( )}−5 7.74 × 10{circumflex over ( )}−11 DA31 Human DR5 1.24 × 10{circumflex over ( )}06 8.79 × 10{circumflex over ( )}−5 7.06 × 10{circumflex over ( )}−11 DA32 Human DR5 1.14 × 10{circumflex over ( )}06 8.59 × 10{circumflex over ( )}−5 7.51 × 10{circumflex over ( )}−11 DA33 Human DR5 1.24 × 10{circumflex over ( )}06 9.77 × 10{circumflex over ( )}−5 7.88 × 10{circumflex over ( )}−11 DA34 Human DR5 1.00 × 10{circumflex over ( )}06 9.50 × 10{circumflex over ( )}−5 9.42 × 10{circumflex over ( )}−11 DA35 Human DR5 1.13 × 10{circumflex over ( )}06  9.12 × 10{circumflex over ( )}−05 8.01 × 10{circumflex over ( )}−11

EXAMPLE 7

Assay of Binding Activity of Antibodies for Human DR5

[0164] The binding activities of the antibodies of the present disclosure were for human DR5 were confirmed by enzyme-linked immunosorbent assay. Human DR5 protein at a concentration of 10 ng/ml was plated at a volume of 150 μl per well onto 96-well immune plates (Nunc, U.S.A.) and adsorbed at room temperature for one hour. After the plates were washed three times with a buffer solution, serial dilutions (0.1-2000 ng/ml) of the antibodies were added to the wells at 150 μl per well and incubated at room temperature for 1-2 hours. The plates were washed again with a buffer solution. Then, HRP (horseradish peroxidase)-conjugated antibody (Serotec, U.S.A.) against anti-human immunoglobulin Fc was 1:20,000 diluted and plated at a volume of 150 μl per well, followed by incubation at room temperature for 1 hour. After washing, a 3,3′,5,5′-tetramethylbenzidine (TMB; Sigma, U.S.A.) solution was added at an amount of 100 μl per well and incubated for 3 to 10 min for color development. When the color development reached a certain level, the reaction was terminated with 1 N sulfuric acid (H.sub.2SO.sub.4). Absorbance at 450 nm was read using a spectrophotometer (Molecular Device, U.S.A.) and the results are depicted in FIGS. 2a to 2f. In this assay, the antibody AP was synthesized according to the anti-DR5 antibody sequence disclosed in PCT/US2006/03577 (WO 2006/083971) and used as a control antibody. As can be seen in FIGS. 2a to 2f, antibodies D0 and DA1 to DA35 were observed to have higher antigen binding activity compared to the control antibody AP.

EXAMPLE 8

Analysis of Physicochemical Properties

[0165] [8-1] Confirmation of Antibody Size

[0166] The sizes of the antibodies according to the present disclosure were analyzed by SDS-PAGE using NuPAGE 4-12% Bis-Tris gel (Invitrogen, U.S.A.). The prepared anti-DR5 antibodies (D0, DA1, DA4, DA16, DA18, DA20, DA23, DA26, and DA29) were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at reduced conditions after treatment with DTT (Invitrogen, U.S.A.) to remove disulfide bonds, and at non-reduced conditions without DTT treatment, to identify the presence of light and heavy chains of the complete antibody. The results are depicted in FIGS. 3a (non-reduced condition) and 3b (reduced condition). As shown in FIG. 3a, for samples analyzed at non-reduced conditions, a band was detected at a position between a 98 kDa size marker and a 188 kDa size marker, which corresponds to the size of the complete antibody. As shown in FIG. 3b, for samples analyzed at reduced conditions, two bands were detected at vicinities of a 49 kDa size marker and a 28 kDa size marker, which correspond to the heavy and light chain of the antibodies, respectively. Thus, bands corresponding to the entire antibody, the heavy chain, and the light chain were each detected by SDS-polyacrylamide gel electrophoresis.

[0167] [8-2] Confirmation of Antibody Purity

[0168] Antibody purity and (soluble) aggregates were analyzed by size exclusion chromatography (SEC) using a TSKgel G3000SWx1 column (Tosoh, Japan). The results are depicted in FIGS. 4a (control antibody AP), 4b (D0), 4c (DA1), 4d (DA4), 4e (DA15), 4f (DA23), 4g (DA26), and 4h (DA29). SEC was operated with isocratic elution using 100 mM phosphate buffer (pH 6.6) as the mobile phase and measurement of absorbance at 280 nm. In general, a high purity of over 94% was observed, with monomers accounting for 94% or more of the total peak area and aggregate peaks accounting for less than 6%.

EXAMPLE 9

Pharmacokinetics of Antibody

[0169] In order to examine in vivo kinetics of the antibodies constructed above, blood half-life was evaluated in mice. Antibodies were intravenously injected at doses of 1-10 mg/kg into nude mice. Blood was sampled from the orbital vein from 5 min to 14 days after injection. The blood collection was conducted using heparinized capillary tubes. Plasma obtained by centrifugation of the blood samples was diluted at certain ratios and used as samples for ELISA evaluation. Human DR5 diluted to a concentration of 10 ng/ml was plated at 100 μl per well onto 96-well immune plates (Nunc, U.S.A.) and adsorbed to the wells for 12-14 hours. The plates were washed three times with buffer solution containing 0.1% Tween 20 and blocked with buffer solution containing bovine serum albumin (Sigma, U.S.A.) at room temperature for 1 hour. After three rounds of washing with buffer solution, the plates were incubated with each of the mouse plasma dilutions and a serial dilution of standard substance at room temperature for 2 to 3 hours. After three washes with buffer solution, a 1:20000 dilution of an HRP-conjugated anti-human immunoglobulin Fc (Fc-HRP) antibody (Bethyl, U.S.A.) was added at a volume of 100 μl per well and incubated at room temperature for 1 hour. After washing three times, 100 μl of a TMB solution (Sigma, U.S.A.) was added to each well and allowed to react. When the color development reached a certain level, the reaction was terminated with 0.2 N sulfuric acid solution. Absorbance at 450 nm was measured using a spectrophotometer (Molecular Device, U.S.A.). The antibody concentration in each plasma sample was calculated from the optical density (OD) measurements, and pharmacokinetic parameters were estimated using WinNonLin ver. 6.2.0.495. The results are shown in FIG. 5 and Table 12 below.

TABLE-US-00018 TABLE 12 D0 DA22 DA23 10 10 10 Unit mg/kg mg/kg mg/kg Cmax ug/ml 286.9 227.9 195.8 AUCinf hr*ug/ml 914.1 1025.3 685.3 t½ initial (−1 d) day 0.57 0.70 0.58 Terminal (2 d~14 d) day 6.68 8.37 7.97 Rsq initial (−1 d) 1.000 1.000 1.000 Terminal (2 d~14 d) 1.000 0.988 0.990

[0170] As shown in FIG. 5 and Table 12, the antibodies (D0, DA22, and DA23) showed blood half-lifes of 6.68 to 8.37 days in the mice employed.

EXAMPLE 10

Confirmation of Cytotoxicity of Antibody in Tumor Cells

[0171] The biological activity of the prepared antibodies was determined using in vitro cell death assays. For this assays, the human colorectal cancer cell line Colo205 (ATCC, U.S.A.), the human pancreatic cancer cell line Miapaca-2(ATCC, U.S.A.), and the human lung cancer cell line H2122 (ATCC, U.S.A.) were employed.

[0172] Each tumor cell line was suspended at a density of 1×10.sup.5 cells/ml, plated at 50 μl per well into 96-well cell culture plates, and cultured for 16-24 hours in a constant temperature and humidity chamber. The antibodies having certain concentrations (0.06-66,000 μM) were applied alone (Ab alone) or in combination with a crosslinking antibody (anti-human Fc) (Serotec, U.S.A.) (Ab linked) and then incubated for 48 hours. 10 μl of resazurin solution (Invitrogen, U.S.A.) was added to each well and incubated for 1 to 2 hours in a constant temperature and humidity chamber. Thereafter, fluorescence was read at 590 nm using a spectrophotometer (Molecular Device). Cell viability was calculated as the percentage of relative fluorescence units (RFU) of test substance-treated group (treated RFU) to relative fluorescence units of a medium-treated group (untreated RFU) (Cell viability (%)=treated RFU/Untreated RFU×100). The results are depicted in FIGS. 6a to 6r. For comparison, the same assay as described above was performed on the control antibody AP.

[0173] As can be seen in FIGS. 6a to 6r, the antibodies constructed above showed excellent cell death activity in all the three human cancer cell lines.

EXAMPLE 11

Evaluation of Anti-Tumor Activity in Xenograft Models

[0174] Anti-tumor activity was evaluated by injecting the antibodies i nude mice to which human cancer cell lines had been implanted. For human DR5-expressing cancer cell lines, the human lung cancer cell line H2122(ATCC, U.S.A.) and the human pancreatic cancer cell line Miapaca-2(ATCC, U.S.A.) were employed. Two to five million tumor cells were implanted to lateral subcutaneous sites of female BALB/c nude mice 6-8 weeks old (OrientBio), and then tumor volumes were measured using calipers. When the tumors grew to an average volume of 100 to 300 mm.sup.3, the mice were separated into groups, and the above constructed antibodies (control antibody AP and antibody D0) were injected to the assigned groups. The antibodies were given as three weekly intraperitoneal injections (on the day of group separation, on day 7 and on day 14) at doses of 0.05-2 mg/kg. Tumor volumes were measured twice a week over a period of 28 days to 50 days following initial injection. Buffer solution (PBS) was injected as the negative control.

[0175] The results are depicted in FIGS. 7a (H2122 implanted mice) and 7b (Miapaca-2-implanted mice). As shown in FIGS. 7a and 7b, the antibody D0 showed anti-tumoral activity superior to the control antibody AP.

EXAMPLE 12

TRAIL-Uncompetitive Binding

[0176] In order to examine antigen-binding characteristics of the antibodies selected in Example 3, a TRAIL competitive enzyme-linked immunosorbent assay was conducted. ELISA plates were coated with 0.1 μg/ml human DR5 (R&D systems, U.S.A.) at 4° C. Thereafter, the plates were incubated with 0.01 nM-1440 nM of antibodies (D0, DA20, DA21, DA21, DA22, and DA23) together with 0.01-400 nM of TRAIL protein for 2 hours. After the plates were washed three times with PBST (0.1% bovine serum albumin and Tween-20 in PBS), HRP (horse radish peroxidase)-conjugated anti-human immunoglobulin Fc was added to each well and incubated for 1 hour. After three washes with PBST, color development was performed for 20 min with ABTS (Thermo, U.S.A.). When the color development reached to a certain level, the reaction was terminated with 1 N sulfuric acid. Absorbance at 405 nm was measured using a spectrophotometer (Molecular Device, U.S.A.) and is depicted in FIGS. 8a and 8b. The antibody AP disclosed in Example (7-1) was used as a control.

[0177] In FIGS. 8a and 8b, antigen binding of the control antibody AP decreased in a TRAIL dose-dependent manner whereas the antibodies D0, DA20, DA21, DA21, DA22, and DA23 maintained a certain level of antigen binding irrespective of TRAIL concentrations, implying that the antibodies of the present invention do not compete with TRAIL for binding to DR5.

EXAMPLE 13

Effect of Antibody in Combination with TRAIL

[0178] Antibody properties according to epitopes were analyzed in terms of cell death activity upon co-treatment with the constructed antibodies and TRAIL. In this test, the human colorectal cancer cell line Colo205 (ATCC, U.S.A.) and the human pancreatic cell line Miapaca-2 (ATCC, U.S.A.) were employed. The tumor cells were suspended at a concentration of 1×10.sup.5 cells/ml and the suspensions were plated at 50 μl per well into 96-well culture plates, followed by incubation for 16-24 hours in a constant temperature and humidity chamber. Serial dilutions of the antibodies (0.1-10000 ng/ml) and TRAIL protein at determined concentrations (0.5, 1, 10 ng/ml) were applied in combination to the 96-well plates. After incubation for 48 to 72 hours at a constant temperature in a humidified condition, a resazurin solution (Invitrogen, U.S.A.) was added at an amount of 10 μl to per well. When the reaction reached a certain level after 1 to 2 hours, fluorescence was read at 590 nm using a spectrophotometer (Molecular Device). Cell viability was calculated as the percentage of relative fluorescence units (RFU) of a test substance-treated group (treated RFU) to the relative fluorescence units of a culture medium-treated group (untreated RFU) (Cell viability (%)=treated RFU/Untreated RFU×100).

[0179] Representative results are depicted in FIGS. 9a (Colo205) and 9b (Miapaca-2). For comparison, the above described antibody AP was used as a control, based on the observation in literature (Cell Death and Differentiation, 15, 751-761, 2008) that this antibody AP has a similar binding site to that of TRAIL.

[0180] As can be seen in FIGS. 9a and 9b, higher cell death activity was detected when the antibody D0 or DA23 at concentrations as low as 10 ng/ml were used in combination with TRAIL at certain concentrations, compared to antibody alone, whereas no such increase in activity was observed for the control antibody AP.

EXAMPLE 14

Assay for Intracellular Caspase Activity

[0181] In order to investigate the cell death mechanism thereof, the constructed antibodies were assayed for caspase activity. The activity of caspase-3/7 was measured using Apo-ONE® Homogeneous Caspase-3/7 Assay kit (Promega, U.S.A.). In this assay, the human colorectal cancer cell line Colo205(ATCC, U.S.A.), the human pancreatic cancer cell line Miapaca-2 (ATCC, U.S.A.), and the lung cancer cell line H2122 (ATCC, U.S.A.) were employed. A suspension of each tumor cell line having a concentration of 1×10.sup.5 cells/ml was plated at a volume of 50 μl per well into 96-well cell culture plates and cultured for 16-24 hours in a constant temperature and humidity chamber. The cells were treated with predetermined concentrations (0.01-10000 ng/ml) of the antibody alone (Ab alone) or in combination with a crosslinking antibody (anti-human Fc) (Serotec, U.S.A.) (Ab linked) and then cultured for 4 hours. Subsequently, the cells were incubated for 3 to 16 hours with 100 μl of Apo-ONE® Caspase-3/7 Reagent (Promega, U.S.A.) per well in a constant temperature and humidity chamber. When the reaction reached a predetermined level, fluorescence was read at 520 nm using a spectrophotometer (Molecular Device). Caspase activity was calculated as a fold increase of relative fluorescence units (RFU) of a test substance-treated group (treated RFU) to over the relative fluorescence units of a culture medium-treated group (untreated RFU) (Caspase activity=treated RFU/Untreated RFU).

[0182] Representative results are depicted in FIGS. 10a (Colo205), 10b (H2122), and 10c (Miapaca-2). For comparison, the antibody AP was treated in the same manner

[0183] As can be seen in FIGS. 10a and 10c, the application of the antibody D0 or DA29 to the cancer cell lines elicited an increase in the activity of casapse-3/7, implying that the cell death mechanism of the antibody of the present disclosure involves the apoptosic pathway.

EXAMPLE 15

Combination Effect of Antibody and Drug

[0184] Effects obtained from co-treatment with the above constructed antibody and gemcitabine were evaluated using in vitro tumor cell death assay. This assay was based on the observation in the literature (J Gastrointest Surg. 2006 November; 10(9):1291-300) wherein the co-treatment of pancreatic cancer cells with gemcitabine and an anti-DR5 antibody resulted in greater tumor cell death and caspase activity.

[0185] The human pancreatic cancer cell lines Miapaca-2 (ATCC, U.S.A.) and Panc-1 (ATCC, U.S.A.) were each suspended at a density of 1×10.sup.5 cells/ml. The suspensions were added in an amount of 50 μl to each well of 96-well cell culture plates and incubated for 16-24 hours in a constant temperature and humidity chamber. The cancer cell lines were treated with the anti-DR5 antibodies of the present disclosure including D0 in combination with certain concentrations (3 nM-300 nM) of gemcitabine and cultured for 48 to 72 hours. The cells were further incubated for 1 to 3 hours after addition of 10 μl of a resazurin solution (Invitrogen, U.S.A.) per well. When the reaction proceeded to a predetermined level, fluorescence was read at 590 nm using a spectrophotometer (Molecular Device). Cell viability was calculated as the percentage of relative fluorescence units (RFU) of a test substance-treated group (treated RFU) to relative fluorescence units of a culture medium-treated group (untreated RFU) (Cell viability (%)=treated RFU/Untreated RFU×100). The results are depicted in FIGS. 6a to 6r. Representative results are depicted in FIGS. 11a (Miapaca-2) and 11b (Panc-1). For comparison, the control antibody AP was treated in the same manner

[0186] As can be seen in FIGS. 11a and 11b, increased apoptotic activity was detected upon co-treatment with the antibody D0 or DA29 and gemcitabine relative to treatment with each individual substance.

[0187] Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.