ANTIBODY SPECIFICALLY BINDING TO GRP94 OR ANTIGEN-BINDING FRAGMENT THEREOF, AND USES THEREOF

Abstract

The present disclosure relates to an antibody or an antigen-binding fragment thereof specifically binding to GRP94, and uses thereof. A GRP94 antibody or antigen-binding fragment thereof according to the present disclosure has very high specificity and affinity to GRP94, and excellent growth-inhibiting, infiltration-inhibiting, and angiogenesis-inhibiting effects on colorectal cancer cell lines, and thus can be useful as a composition for treating cancer, inhibiting cancer metastasis, and inhibiting angiogenesis.

Claims

1.-13. (canceled)

14. An antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof binds specifically to glucose-regulated protein 94 (GRP94) and comprises: a heavy chain variable region comprising heavy chain CDR1 (HCDR1) including an amino acid sequence represented by general formula 1, HCDR2 including an amino acid sequence represented by general formula 2, and HCDR3; and a light chain variable region comprising light chain CDR1 (LCDR1) including an amino acid sequence represented by general formula 3, LCDR2 including an amino acid sequence represented by general formula 4, and LCDR3 including an amino acid sequence represented by general formula 5: TABLE-US-00002   General Formula 1 GFTFSX.sub.6YX.sub.8MS General Formula 2 X.sub.1IX.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9YYADSVKG General Formula 3 X.sub.1GSX.sub.4SNIGX.sub.9NX.sub.11VX.sub.13 General Formula 4 X.sub.1X.sub.2X.sub.3X.sub.4RPS General Formula 5 X.sub.1X.sub.2WDX.sub.5SLX.sub.8X.sub.9 wherein, i) X.sub.6X.sub.8 in general formula 1 stand for NY, respectively, X.sub.1X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9 in general formula 2 stand for GYPNSGST, respectively, and HCDR3 includes the amino acid sequence of SEQ ID NO: 3, X.sub.1X.sub.4X.sub.9X.sub.11R.sub.13 in general formula 3 stand for TSNAS, respectively, X.sub.1X.sub.2X.sub.3X.sub.4 in general formula 4 stand for ADSH, respectively, and X.sub.1X.sub.2X.sub.5X.sub.8X.sub.9 in general formula 5 stands for GAANA; ii) X.sub.6X.sub.8 in general formula 1 stand for NA, respectively, X.sub.1X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9 in general formula 2 stand for GSSSSGST, respectively, and HCDR3 includes the amino acid sequence of SEQ ID NO: 11, X.sub.1X.sub.4X.sub.9X.sub.11R.sub.13 in general formula 3 stand for SPSTT, respectively, X.sub.1X.sub.2X.sub.3X.sub.4 in general formula 4 stand for ADSH, respectively, and X.sub.1X.sub.2X.sub.5X.sub.8X.sub.9 in in general formula 5 stand for ASDNG, respectively; or iii) X.sub.6X.sub.8 in general formula 1 stand for GA, respectively, X.sub.1X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9 in general formula 2 stand for ASHGGSSK, respectively, and HCDR3 includes the amino acid sequence of SEQ ID NO: 19, X.sub.1X.sub.4X.sub.9X.sub.11R.sub.13 in general formula 3 stand for SSSTS, respectively, X.sub.1X.sub.2X.sub.3X.sub.4 in general formula 4 stand for ADNN, respectively, X.sub.1X.sub.2X.sub.5X.sub.8X.sub.9 in general formula 5 stand for ASDNA, respectively.

15. The antibody or the antigen-binding fragment thereof according to claim 14, wherein the antibody or the antigen-binding fragment thereof comprises any one of i) to iii): i) HCDR1, HCDR2, and HCDR3 including amino acid sequences of SEQ ID NOS: 1 to 3, respectively, and LCDR1, LCDR2, and LCDR3 including amino acid sequences of SEQ ID NOS: 4 to 6, respectively; ii) HCDR1, HCDR2, and HCDR3 including amino acid sequences of SEQ ID NOS: 9 to 11, respectively, and LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NOS: 12 to 14, respectively; or iii) HCDR1, HCDR2, and HCDR3 including amino acid sequences of SEQ ID NOS: 17 to 19, respectively, and LCDR1, LCDR2, and LCDR3 including amino acid sequences of SEQ ID NOS: 20 to 22, respectively.

16. The antibody or the antigen-binding fragment thereof according to claim 14, wherein the antibody or the antigen-binding fragment thereof includes any one of i) to iii): i) a heavy chain variable region including the amino acid sequence of SEQ ID NO: 7 and a light chain variable region including the amino acid sequence of SEQ ID NO: 8; ii) a heavy chain variable region including the amino acid sequence of SEQ ID NO: 15 and a light chain variable region including the amino acid sequence of SEQ ID NO: 16; or iii) a heavy chain variable region including the amino acid sequence of SEQ ID NO: 23 and a light chain variable region including the amino acid sequence of SEQ ID NO: 24.

17. The antibody or the antigen-binding fragment thereof according to claim 14, wherein the antibody or the antigen-binding fragment thereof has a dissociation constant K.sub.D of 10.sup.−8 M or less.

18. The antibody or the antigen-binding fragment thereof according to claim 14, wherein the antibody or the antigen-binding fragment thereof has an activity of inhibiting the growth of at least one tumor cell line selected from the group consisting of HCT-8, HT-29, LoVo, HCT-116, and Caco-2, in vitro and in vivo.

19. A nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof according to claim 14.

20. A recombinant vector carrying the nucleic acid of claim 19.

21. A host cell comprising the recombinant vector of claim 20.

22. A pharmaceutical composition comprising: i) the antibody or the antigen-binding fragment thereof according to claim 14; and ii) a pharmaceutically acceptable carrier.

23. A kit for detecting GRP94, the kit comprising the antibody or the antigen-binding fragment thereof according to claim 14.

24. A method for treating cancer in a subject in need thereof, wherein the method comprising administering the antibody or the antigen-binding fragment thereof according to claim 14 to the subject.

25. The method of claim 24, wherein the cancer is solid cancer or blood cancer.

26. The method of claim 25, wherein the solid cancer is selected from the group consisting of gastric cancer, rectal cancer, colon cancer, colorectal cancer, inflammation-related colon cancer, liver cancer, lung cancer, ovarian cancer, melanoma, pancreatic cancer, uterine cancer, testicular cancer, and breast cancer.

27. The method of claim 25, wherein the blood cancer is selected from the group consisting of acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, acute monocytic leukemia, multiple myeloma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma.

28. The method of claim 24, wherein the antibody or the antigen-binding fragment is administrated with cetuximab.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0136] FIG. 1 is a photographic image of the recombinant human GRP94 protein produced using Expi293 cells after SDS-PAGE and Coomassie staining.

[0137] FIG. 2 is a photographic image of GRP94 IgG antibodies expressed in Expi293 cells after SDS-PAGE and Coomassie staining.

[0138] FIG. 3 is a graph showing outputs of the GRP94-specific IgGs of the present disclosure, as measured by Nano Drop after expression in Expi293 cells and purification.

[0139] FIG. 4 is a graph showing endotoxin levels of the GRP94 antibodies of the present disclosure.

[0140] FIG. 5 is a graph showing protein aggregation indices of the GRP94 antibodies of the present disclosure.

[0141] FIG. 6 and FIG. 7 are plots of equilibrium dissociation constant (K.sub.D) values of the four GRP94 IgGs (B5, E5, 2H5, and 3G7) of the present disclosure.

[0142] FIG. 8 shows graphs illustrating the specificity of the four GRP94 IgGs (B5, E5, 2H5, and 3G7) of the present disclosure for the antigens recombinant human GRP94, recombinant rat GRP94, and recombinant monkey GRP94.

[0143] FIG. 9 and FIG. 10 are views illustrating the tube formation of the four GRP94 antibodies (B5, E5, 2H5, and 3G7) of the present disclosure, compared to cetuximab, as assayed in vitro using HUVEC cells.

[0144] FIG. 11 and FIG. 12 are views illustrating the transwell invasion of the four GRP94 antibodies (B5, E5, 2H5, and 3G7) of the present disclosure as assayed using the colorectal cancer cell line HCT-8.

[0145] FIG. 13 and FIG. 14 are views illustrating the transwell invasion of the four GRP94 antibodies (B5, E5, 2H5, and 3G7) of the present disclosure as assayed using the colorectal cancer cell line HCT-116.

[0146] FIG. 15 and FIG. 16 are views illustrating the transwell invasion of the four GRP94 antibodies (B5, E5, 2H5, and 3G7) of the present disclosure as assayed using the colorectal cancer cell line LoVo.

[0147] FIGS. 17 to 20 are views illustrating effects of the anti-GPR94 antibodies of the present disclosure on cell growth of the colorectal cell lines HCT8, HCT116, HT29, and LoVo.

[0148] FIG. 21a is a schematic view of the structures of GRP94 proteins. FIG. 21b is a photographic view showing rhGRP94 whole fragment (part 1) and rhGRP94 CTD fragment (part 4) after expression and purification thereof.

[0149] FIG. 22 is a graph showing epitope mapping results of the anti-GRP94 antibodies of the present disclosure as analyzed by ELISA.

[0150] FIG. 23 is a graph showing vascular toxicity of the four antibodies of the present disclosure in terms of the viability of HUVEC cells treated with the four antibodies.

[0151] FIG. 24 shows views illustrating whether the four antibodies of the present disclosure activate vascular endothelial cells to induce the expression of the cell adhesion molecules VCAM-1 and ICAM-1.

[0152] FIG. 25 shows views illustrating the mechanism of action (i.e. GRP94 internalization) of the four antibodies of the present disclosure as analyzed by examining whether the four antibodies of the present disclosure bind to cell surface GRP94 antigen depending on the fixation of the HCT116 cell line with paraformaldehyde (PFA).

[0153] FIGS. 26a to 26d are plots illustrating in vivo efficacies of the four antibodies of the present disclosure in the colorectal cancer xenograft mouse model.

[0154] FIG. 26e is a plot illustrating the effects of the antibodies 2H5 and 3G7 of the present disclosure upon co-administration together with conventional anticancer agents (5-FU+Cetuximab).

[0155] FIG. 27a is a plot of body weights in the xenograft mouse models injected with the four antibodies of the present disclosure. FIG. 27b is a graph illustrating in vivo toxicity upon administration of the four antibodies of the present disclosure in terms of serobiochemical indices (GOT, GPT, TBIL, BUN, and CREA) in the xenograft mouse model.

BEST MODE FOR CARRYING OUT THE INVENTION

[0156] A better understanding of the present disclosure may be obtained through the following examples which are set forth to illustrate, but are not to be construed to limit, the present disclosure.

EXAMPLES

[0157] Throughout this specification, “%” used to indicate the concentration of a specific substance is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and (volume/volume) % for liquid/liquid.

[0158] Materials and Methods

[0159] Cell Culture

[0160] Human CRC cell lines (HCT116, HT-29, LoVo, HCT-8, and Caco-2) were purchased from the Korean Cell Line Bank (Seoul, Korea). HCT116, HT-29, LoVo, and HCT-8 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 media (Gibco, Grand Island, N.Y., USA) supplemented with 10% (v/v) fetal bovine serum (Gibco) and 1% (v/v) penicillin/streptomycin (Gibco). Caco-2 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) with the same supplements. Human umbilical vein endothelial cells (HUVECs; Lonza, Allendale, N.J., USA) were cultured in endothelial growth medium-2 (EGM-2; Lonza). All cells were maintained at 37° C. in a humidified incubator with 5% CO.sub.2. Expi293 cells were cultured in Expi293 expression medium (Invitrogen, Carlsbad, Calif., USA) in a humidified CO.sub.2 incubator shaker (N-BIOTEK, S. Korea) at 37° C. with 8% CO.sub.2.

[0161] Transfection

[0162] For antibody and rhGP94 overproduction, the expression vectors pcDNA 3.1 and pcDNA 3.4 (Invitrogen) carrying GRP94 antibody and rhGRP94, respectively, were transfected into Expi293 cells, using an ExpiFectamine transfection kit (Invitrogen) according to the manufacturer's instructions.

[0163] Enzyme-Linked Immunosorbent Assay (ELISA)

[0164] Each well of 96-well plates was coated with 0.1 μg of recombinant human GRP94 (rhGRP94), blocked with 3% (w/v) bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 2 hours at 37° C., and incubated with 100 μL of GRP94 targeting IgG (20 μg/mL) for 2 hours at 37° C. The plates were washed thrice with 0.05% PBS-T, and 100 μL of a 500:1 dilution of the secondary antibody anti-human Fc Ab (Sigma-Aldrich) was added to each well. Incubation at 37° C. for 1 hour was followed by three rounds of washing with 0.05% PBS-T. Then, 100 μL of 3,3′,5,5′-tetramethylbenzidine (1-step ultra TMB) substrate solution was added to each well. Finally, optical densities were measured at 450 nm using a microplate reader (Synergy H1, BioTek).

Example 1: Selection of High-Affinity Antibodies that Bind to rhGRP94 Using Phage Display Technology

[0165] Prior to production of the scFv binding specifically to rhGRP94, the rhGRP94 protein was expressed in Expi293 cells and isolated by affinity chromatography using Ni-NTA sepharose. The isolated protein was predicted for purify by SDS-PAGE and Coomassie Brilliant Blue staining. As shown in FIG. 1, the purify of the rhGRP94 protein produced and isolated by the present inventors was observed to be equivalent to or higher than that of the commercially available one.

[0166] Subsequently, the human synthetic scFv library was reamplified. Through four rounds of biopanning, a scFv showing highly affinity for rhGRP94 was selected using rhGRP94-conjugated Epoxy-270 dynabead (Invitrogen).

[0167] In order to generate an IgG antibody, after the DNA sequencing of the selected scFv, each variable heavy and light chain gene of the selected scFv clone was cloned into the bicistronic mammalian expression vector pCDNA3.1 (Invitrogen). Using the Expi293 expression system (Invitrogen), the IgG antibody (GRP94 IgG) was produced and purified.

[0168] The GRP94 IgG antibody expressed in Expi293 cells was isolated by affinity chromatography using protein A sepharose. The isolated antibody was predicted for purify by SDS-PAGE and Coomassie Brilliant Blue staining. In addition, the final production of antibody was confirmed using the NanoDrop. As shown in FIG. 2, the GRP94-specific IgG antibodies produced and isolated by the present inventors was observed to be high in purity. All of the antibodies (B5, E5, 2H5, and 3G7) of the present disclosure exhibited a production efficiency of 100 mg/L or higher as shown in FIG. 3.

Example 2: Measurement of Endotoxin Levels Using PTS

[0169] The produced antibodies were measured for endotoxin level, using Endosafe PTS™ composed of a spectrophotometer, a reader, and an LAL reagent cartridge. First, the produced GRP94 antibodies were loaded into the LAL reagent cartridge (Charles River) and measured for endotoxin level according to the manufacturer's instructions.

[0170] As can be seen in FIG. 4, all of the GRP94 antibodies of the present disclosure exhibited an endotoxin level of less than 0.005 EU/μg, which is the FDA safe limit.

Example 3: Protein Aggregation Index

[0171] To evaluate the production the antibodies (B5, E5, 2H5, and 3G7) of the present disclosure and the antibody aggregation after purification, each of the antibodies was added to PBS and absorbance was read at 280 nm and 340 nm using NanoDrop 2000 (Thermo Fisher Scientific). The protein aggregation index was calculated from UV absorbance according to the following equation. As a control, the GRP94 antigen that was induced by treatment with HCl to aggregate was used.

Protein Aggregation Index=100×(Abs.sub.340/[Abs.sub.280-Abs.sub.340])  Equation 1

[0172] Antibody aggregation could be triggered by partial unfolding of some domains of the antibody, leading to monomer-monomer association followed by nucleation and growth. Thus, antibody aggregation continues to be a major issue in the developability thereof. As can be seen in FIG. 5, the antibodies (B5, E5, 2H5, and 3G7) of the present disclosure were low in protein aggregation index and thus were observed to be suitable for development.

Example 4: K.SUB.D .Value Measurement of Antibody and Antigen Interactions (ELISA)

[0173] The four GRP94 IgGs (B5, E5, 2H5, and 3G7) of the present disclosure were measured for equilibrium dissociation constant (K.sub.D). In this regard, each well of 96-well plates was coated with 100 ng of rhGRP94 and blocked with 3% (w/v) bovine serum albumin (BSA) in PBS at 37° C. for 2 hours. Next, the plates were incubated with a concentration gradient of 0 nM to 500 nM of each clone for the GRP94 IgGs at 37° C. for 2 hours. The use of a secondary antibody and the measurement of optical density were conducted as described for the ELISA.

[0174] After 96-well plates were coated with BSA, recombinant human GRP94, recombinant rat GRP94, and recombinant monkey GRP94, their binding affinity for each antibody (B5, E5, 2H5, or 3G7) was measured by ELISA. As shown in FIG. 8, the antibodies (B5, E5, 2H5, and 3G7) of the present disclosure were each observed to bind to the recombinant human, rat, and monkey GRP94 antigens, with high specificity for human and rat recombinant GRP94 antigens.

Example 5: In Vitro Tube Formation Assays

[0175] Each well of 48-well plates was coated with 150 μL of Matrigel (Corning) and incubated at 37° C. for 30 minutes. To examine the effects of the four GRP94 IgGs of the present disclosure on tube formation, HUVEC cells cultured in EGM-2 were seeded at a density of 1×10.sup.5 cells/well into the Matrigel-coated plates and incubated in the presence/absence of anti-GRP94 IgG or with 20 μg/mL cetuximab. Images were acquired using the IncuCyte FLR live content imaging system (Essen Bioscience, Ann Arbor, Mich., USA), and tube branches were counted to quantitate tube formation. The results are shown in FIGS. 9 and 10. As seen in FIG. 9 and FIG. 10, the four antibodies of the present disclosure were highly inhibitory of tube formation. Hence, the four antibodies of the present disclosure can find advantageous applications as angiogenesis inhibitors.

Example 6: Transwell Invasion Assay

[0176] Two transwell invasion chambers coated with Matrigel (0.1 mg/ml) (Corning) were used for in-vitro transwell invasion assays for HCT-8, HCT-116, or LoVo cells. First, 200 μl of serum-free medium containing 1×10.sup.5 cells were added to each well in the upper chamber while 0.8 ml of a medium containing 10% FBS was applied to the lower chamber. After incubation at 37° C. for 24 to 48 hours, the non-invasive cells on the membrane in the upper chamber were removed with a cotton swab. The migrated or invasive cells were fixed and then stained with diff quick staining Kit (Sysmex). Cells were counted using ImageJ software, and four random fields selected in each well were photographed under an inverted microscope. Each experiment was independently performed in biplicate.

[0177] As shown in FIGS. 11 to 16, the four antibodies of the present disclosure exhibited inhibitory effects on the invasive activity of colorectal cancer cell lines such as HCT-8, HCT-116, and LoVo, with dominant superiority to cetuximab. Therefore, the four antibodies of the present disclosure can be advantageously used as cancer metastasis inhibitors.

Example 7: In Vitro CRC Proliferation Assay

[0178] The four anti-GRP94 human IgG antibodies B5, E5, 2H5, and 3G7 of the present disclosure were examined in vitro for effect on CRC cell proliferation. For use in the efficacy assay, the four cetuximab-resistant colorectal cancer cell lines HCT116, HT29, LoVo, and HCT-8 were each grown in a culture medium (HCT116, HCT8, HT29, and LoVo: RPMI1640+10% FBS+1% Penicillin-Streptomycin) at 37° C. in T-75 flask in a 5% CO.sub.2 incubator until reaching about 80-90% confluency. The culture medium was removed by suction and the cells were washed with 10 mL of phosphate buffered saline (hereinafter PBS). After removal of PBS by suction, the cells were treated 2 mL of TrypLE Express Enzyme at 37° C. for 3 minutes in a 5% CO.sub.2 incubator. Thereafter, the cells were added with 8 mL of the culture medium, transferred to 15-mL tube, and centrifuged at 500×g for 5 minutes. After removal of the supernatant, the cells were suspended in the culture medium. From a small portion of the cell suspension, the cells were counted using the Countess II Automated Cell Counter. Next, the cells were seeded at a final density of 5×10.sup.3 cells/well into 96-well plates and treated with 20 μg/mL of each the four test materials anti-GRP94 human antibodies B5, E5, 2H5, and 3G7 and cetuximab (CTX). The 96-well plates were mounted on the Incucyte Zoom system. With the microscope magnification set at 4×, images were collected at intervals of 2 hours for 72 hours. From the collected images, cell growth was monitored using the IncuCyte FLR live content imaging system (Essen Bioscience, Ann Arbor, Mich., USA). The results are shown in FIGS. 17 to 20.

[0179] 1) The four GRP94 human antibodies (B5, E5, 2H5, and 3G7) for HCT8 cells were measured to have the following cell indices at the end point: MOCK: 4.8±0.25, B5: 3.15±0.21, E5: 3.33±0.31, 2H5: 3.98±0.09, 3G7: 3.40±0.16, and CTX: 4.84±0.28.

[0180] 2) The four GRP94 human antibodies (B5, E5, 2H5, and 3G7) for HCT116 cells were measured to have the following cell indices at the end point: MOCK: 6.10±0.51, B5: 3.53±0.70, E5: 3.57±0.20, 2H5: 4.60±0.40, 3G7: 3.53±0.32, and CTX:6.57±0.77.

[0181] 3) The four GRP94 human antibodies (B5, E5, 2H5, and 3G7) for HT29 cells were measured to have the following cell indices at the end point: MOCK: 2.94±0.04, B5: 2.32±0.02, E5: 2.32±0.08, 2H5: 2.69±0.02, 3G7: 2.17±0.06, and CTX: 3.27±0.12.

[0182] 4) The four GRP94 human antibodies (B5, E5, 2H5, and 3G7) for LoVo cells were measured to have the following cell indices at the end point: MOCK: 3.76±0.19, B5: 2.69±0.04, E5: 2.73±0.22, 2H5: 3.54±0.03, 3G7: 2.87±0.08, and CTX: 3.43±0.14.

[0183] 1) The cell growth inhibitory ability of the four test substances against HCT8 cells was observed in the order of B5>E5>3G7>2H5. All of the four test substances were statistically significant relative to the control PBS.

[0184] 2) The cell growth inhibitory ability of the four test substances against HCT116 cells was observed in the order of B5>3G7>E5>2H5. The test substances E5, 3G7, and B5 were statistically significant relative to the control PBS, but with no statistical significance for 2H5 relative to the control PBS.

[0185] 3) The cell growth inhibitory ability of the four test substances against HT29 cells was observed in the order of 3G7>E5>B5>2H5. A II of the test substances B5, E5, 2H5, and 3G7 were statistically significant relative to the control PBS.

[0186] 4) The cell growth inhibitory ability of the four test substances against LoVo cells was observed in the order of B5>E5>3G7>2H5. The test substances B5, E5, and 3G7 were statistically significant relative to the control PBS, but with no statistical significance for 2H5 relative to the control PBS.

[0187] In conclusion, the assay of the four test substances for inhibitory activity against CTX-resistant colorectal cell proliferation revealed that the test antibodies B5, E5, and 3G7 exhibited excellent inhibitory activity against growth of the four cell lines (HCT8, HCT116, HT29, and LoVo) as measured at the end point.

Example 8: Epitope Comparison of Developed Antibodies

[0188] The GRP94 protein includes NTD (N-terminal domain), Ca.sup.2+ binding domain (charger linker domain), MD (middle domain), CTD (C-terminal domain), and ER retention sequence in that order in the direction from the N-terminus to the C-terminus (see FIG. 21a).

[0189] To determine which portion (or domain) of the GRP94 protein is bound by the developed antibodies, GRP94 fragments part. 1 and part. 4 were constructed wherein part. 1 was composed of the N-terminal domain (NTD), the Ca.sup.2+ binding domain, the middle domain (MD), and the C-terminal domain (CTD) while part. 4 was derived from part. 1 by cutting off the NTD, the Ca.sup.2+ binding domain, and the MD. In brief, Part. 1 (Asp 22-Glu 799) and Part. 4 (Glu 602-Glu 799) of the human GRP94 gene (Gene ID: 7184) were synthesized and subcloned into pcDNA 3.4 (Invitrogen) mammalian expression vector to finally recombinant DNAs. Using the Expi293 expression system (Invitrogen), the proteins were expressed and purified. The purified antigens part. 1 and part. 4 were analyzed for molecular weight by SDS-PAGE. The results are shown in FIG. 21b.

[0190] Next, ELISA was carried out to examine whether the four antibodies of the present disclosure bind to part 1 and part 4. First, 100 ng of each of the rhGRP94 fragment parts 1 and 4 was fixed to a high binding plate by incubation at 4° C. for 14 hours (overnight binding, 14 hrs). The same amount of BSA was used as a negative control. Then, the plate was blocked with 3% BSA, followed by incubation at 37° C. for 2 hours with 100 μL containing each of the anti-GRP94 antibodies (B5, E5, 2H5, and 3G7) of the present disclosure at a density of 1 μg/mL. After three rounds of washing with PBS-T, the plate was treated at 37° C. for 1 hour with a 5000:1 dilution of anti-Fc-HRP (Catalog #31423 Invitrogen). Color development was induced using 1-step-ultra TMB (Invitrogen) for 10 minutes and then terminated with H2504 (2N). Absorbance at 450 nm was read. The results are shown in FIG. 22.

[0191] As can be seen in FIG. 22, any of the four antibodies of the present disclosure (B5, E5, 2H5, and 3G7) did not bind to part 4 (Glu 602-Glu 799).

Example 9: In Vitro Assay of Developed Antibodies for Vascular Toxicity

[0192] In Vitro Assay for Viability of Vascular Endothelial Cell (HUVEC Viability Assay)

[0193] Examination was made to see whether the developed antibodies could cause toxicity to vascular endothelial cells. In this regard, the vascular endothelial cell line HUVEC (human umbilical vein endothelial cell) was treated with 20 μg/mL of each of the four antibodies of the present disclosure (B5, E5, 2H5, and 3G7) and 36 μg/mL of the conventional anticancer agent 5-FU (5-fluorouracil) as a control and then analyzed for viability using CCK-8 (cell counting kit-8). The results are shown in FIG. 23.

[0194] As shown in FIG. 23, all of the four antibodies of the present disclosure did not degrade the viability of HUVEC, compared to the control (5-FU). From the data, it could be understood that the four antibodies of the present disclosure are unlikely to cause toxicity to vascular endothelial cells.

[0195] Assay for Cell Adhesion Molecule Expression of Vascular Endothelial Cell

[0196] To examine whether the four antibodies of the present disclosure induce endothelial cell activation, the four antibodies were applied for 24 hours to HUVEC which was then analyzed for the expression of VCAM-1 and ICAM-1, which are representative biomarkers accounting for cell adhesion molecule expression, through FACS. As a positive control, a recombinant human TNF-alpha, which is a representative pro-inflammatory cytokine, was used. The results are shown in FIG. 24.

[0197] As can be seen in FIG. 24, all of the four antibodies of the present disclosure were observed to almost little induce the expression of VCAM-1 and ICAM-1, compared to the TNF-alpha-treated positive control. Thus, the four antibodies of the present disclosure were very unlikely to induce activation of vascular endothelial cells.

Example 10: Assay for Mechanism of Action of Developed Antibodies (Grp94 Internalization)

[0198] With the assumption on the mechanism that the anti-GRP94 antibodies of the present disclosure bind to GRP94 antigen and then internalized into cells, the following experiment was carried out. Briefly, the four anti-GRP94 antibodies of the present disclosure were applied to the colorectal cell line HCT116 fixed with 4% paraformaldehyde (PFA) or unfixed cells and then analyzed for binding affinity for GRP94 on the surface of HCT116 cells by FACS. When the colorectal cell line HCT116 is fixed with PFA, its cell activity is halted so that the internalization does not proceed. Thus, the interaction between the antibodies and the GRP94 antigen exposed on the cell surface can be more explicitly detected. The results are depicted in FIG. 25.

[0199] As seen in FIG. 25, the binding of the four antibodies to the cell surface GRP94 was detected in the PFA-fixed cells, but not in non-fixed cells at all. This, assumption could be made of the mechanism of action wherein the four antibodies bind to cell surface GRP94 and then internalized into the cells, with the consequent down-regulation of the cell surface GRP94 level.

Example 11: In Vivo Assay for Inhibitory Activity Against Tumor Growth in Colorectal Xenograft Model

[0200] Assay for Tumor Growth Inhibition by Sole Administration of Antibody

[0201] For use in assaying in-vivo efficacy of the four antibodies of the present disclosure, a subcutaneous xenograft mouse model was established by injecting the metastatic colorectal cancer cell line HCT116 (5×10.sup.6 cells) into 6-week-old Balb/c nude mice. When the tumor size reached about 50-100 mm.sup.3, PBS (negative control), hIgG (negative control), cetuximab, and the four antibodies (B5, E5, 2H5, and 3G7) were each intravenously injected at a dose of 10 mg/kg twice per week for six weeks while monitoring the efficacies. The results are depicted in FIGS. 26a to 26d.

[0202] As shown in FIGS. 26a to 26d, cetuximab was not significantly different from the controls PBS and hIgG in terms of inhibitory activity against tumor growth of colorectal cancer cell line HCT116. E5 did not exhibit inhibitory activity against tumor growth, either, compared to the controls. In contrast, noticeable tumor growth inhibition efficacies were observed in mice injected with B5, 2H5, and 3G7 antibodies. B5, 2H5, and 3G7 antibodies inhibited tumor growth by 30% (p<0.001), 45% (p<0.01), and 33% (p<0.001), respectively, compared to PBS (n=9, statistical analysis was made using two-way ANOVA).

[0203] Assay for Tumor Growth Inhibition by Administration of Antibody and Conventional Anticancer Agent in Combination

[0204] Combined administration of the developed antibodies of the present disclosure and conventional anticancer agents was assayed for in vivo efficacy. In this regard, the 2H5 and 3G7 antibodies, which exhibited the highest efficacy as measured in the sole administration assay, and the conventional colorectal cancer chemotherapeutic agents 5-FU and cetuximab were administered in combination. Briefly, the metastatic colorectal cancer cell line HCT116 (5×10.sup.6 cells) was injected to 6-week-old Balb/c nude mice to establish a subcutaneous xenograft mouse model. When the tumor size reached about 50-100 mm.sup.3, PBS (negative control), hIgG (negative control), 5-FU+cetuximab, 5-FU+cetuximab+2H5, or 5-FU+cetuximab+3G7 was injected twice a week for six weeks. Injection was made of 5-FU at a dose of 30 mg/kg through an intraperitoneal route and cetuximab and 2H5 and 3G7 antibodies at a dose of 10 mg/kg through intravenous route. The results are depicted in FIG. 26e.

[0205] As shown in FIG. 26e, 5-FU+cetuximab was observed to have no significant inhibitory effects on the growth of the HCT116 colorectal tumor, compared to the controls PBS and hIgG. In contrast, noticeable synergistic inhibitory effects on tumor growth were detected from treatment with 5-FU+cetuximab+2H5 and 5-FU+cetuximab+3G7. The combinations of 5-FU+cetuximab+2H5, and 5-FU+cetuximab+3G7 inhibited tumor growth by 47% (p<0.001) and 67% (p<0.001), respectively, compared to PBS (n=10, statistical analysis was made using two-way ANOVA). Particularly, 3G7 antibody exhibited very high synergistic inhibitory effects on colorectal tumor growth when administered in combination with 5-FU and cetuximab.

Example 12: In Vivo Assay for Hepatotoxicity and Nephrotoxicity in Colorectal Xenograft Model

[0206] To assay the four antibodies of the present disclosure for in vivo hepatotoxicity and nephrotoxicity, the same xenograft model as in Example 11 weighed every week. After six weeks of the experiment, GOT (glutamic oxaloacetic transaminase), GPT (glutamate pyruvate transaminase), and TBIL (total bilirubin) were measured for hepatotoxicity assay while BUN (blood urea nitrogen) and CRE (creatinine) were measured for nephrotoxicity assay. The results are depicted in FIGS. 27a and 27b.

[0207] As shown in FIG. 27a, PBS, hIgG, cetuximab, and the four selected antibodies (B5, E5, 2H5, and 3G7) were all observed to cause no significant changes in body weight. In addition, as shown in FIG. 27b, no significant changes in serum levels of liver and kidney functional indices (GOT, GPT, TBIL, BUN, and CREA). From the data of the serobiochemical toxicity assay, it could be understood that the four selected antibodies (B5, E5, 2H5, and 3G7) of the present disclosure are unlikely to cause toxicity in vivo.