ANTIBODY WITH ENHANCED BINDING AFFINITY FOR ENDOTHELIN RECEPTOR TYPE A

Abstract

The present invention relates to an antibody or an antigen-binding fragment thereof that has improved binding affinity for endothelin receptor type A. The present invention also relates to an antibody or an antigen-binding fragment thereof that has improved productivity. The antibody developed in the present invention is suitable for use in the treatment and diagnosis of diseases associated with endothelin receptor type A due to its remarkably improved binding affinity for the antigen and high productivity compared to conventional antibodies.

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region and specifically binding to endothelin receptor type A wherein (a) the heavy chain variable region comprises VH-CDR1 comprising the sequence set forth in SEQ ID NO: 25 and VH-CDR2 comprising the sequence set forth in SEQ ID NO: 26, (b) the light chain variable region comprises VL-CDR1 comprising the sequence set forth in SEQ ID NO: 27, VL-CDR2 comprising the sequence set forth in SEQ ID NO: 28, and VL-CDR3 comprising the sequence set forth in SEQ ID NO: 29, and (c) the heavy chain variable region comprises VH-CDR3 comprising the sequence set forth in SEQ ID NO: 30 or VH-CDR3 comprising a sequence comprising mutations at one or more positions selected from the group consisting of amino acid positions 4, 7, 9, and 12 in the sequence of SEQ ID NO: 30.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the VH-CDR3 of the heavy chain variable region comprises one or more mutations comprising a substitution of the amino acid at position 4 in the sequence of SEQ ID NO: 30 to proline (P), a substitution of the amino acid at position 7 in the sequence of SEQ ID NO: 30 to leucine (L), a substitution of the amino acid at position 9 in the sequence of SEQ ID NO: 30 to valine (V) or a substitution of the amino acid at position 12 in the sequence of SEQ ID NO: 30 to glutamate (E).

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the VH-CDR3 of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of those set forth in SEQ ID NOs: 30, 31, 32, 33, and 34.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises a framework region 1 (VH-FR1) comprising the sequence set forth in SEQ ID NO: 1 or a sequence comprising mutations at one or more positions selected from the group consisting of amino acid positions 16 and 24 in the sequence of SEQ ID NO: 1.

5. The monoclonal antibody or antigen-binding fragment thereof according to claim 4, wherein the VH-FR1 of the heavy chain variable region comprises one or more mutations comprising a substitution of the amino acid at position 16 in the sequence of SEQ ID NO: 1 to arginine (R) or a substitution of the amino acid at position 24 in the sequence of SEQ ID NO: 1 to valine (V).

6. The monoclonal antibody or antigen-binding fragment thereof according to claim 4, wherein the VH-FR1 of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of those set forth in SEQ ID NOs: 1, 2, and 3.

7. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises a framework region 2 (VH-FR2) comprising the sequence set forth in SEQ ID NO: 4 or a sequence comprising mutations at one or more positions selected from the group consisting of amino acid positions 1, 2, 4, 16, and 17 in the sequence of SEQ ID NO: 4.

8. The monoclonal antibody or antigen-binding fragment thereof according to claim 7, wherein the VH-FR2 of the heavy chain variable region comprises one or more mutations selected from the group consisting of mutations comprising a substitution of the amino acid at position 1 in the sequence of SEQ ID NO: 4 to methionine (M), mutations comprising a substitution of the amino acid at position 2 in the sequence of SEQ ID NO: 4 to asparagine (N), mutations comprising a substitution of the amino acid at position 4 in the sequence of SEQ ID NO: 4 to isoleucine (I), mutations comprising a substitution of the amino acid at position 16 in the sequence of SEQ ID NO: 4 to serine (S) or glycine (G), and mutations comprising a substitution of the amino acid at position 17 in the sequence of SEQ ID NO: 4 to alanine (A), tryptophan (W) or serine (S).

9. The monoclonal antibody or antigen-binding fragment thereof according to claim 7, wherein the VH-FR2 of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of those set forth in SEQ ID NOs: 4, 5, 6, and 7.

10. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises a framework region 3 (VH-FR3) comprising the sequence set forth in SEQ ID NO: 8 or a sequence comprising mutations at one or more positions selected from the group consisting of amino acid positions 1, 4, 5, 6, 7, 8, 12, 14, 16, 17, 19, 20, and 21 in the sequence of SEQ ID NO:8.

11. The monoclonal antibody or antigen-binding fragment thereof according to claim 10, wherein the VH-FR3 of the heavy chain variable region comprises one or more mutations selected from the group consisting of mutations comprising a substitution of the amino acid at position 1 in the sequence of SEQ ID NO: 8 to aspartate (D), threonine (T) or asparagine (N), mutations comprising a substitution of the amino acid at position 4 in the sequence of SEQ ID NO: 8 to alanine (A), mutations comprising a substitution of the amino acid at position 5 in the sequence of SEQ ID NO: 8 to aspartate (D), mutations comprising a substitution of the amino acid at position 6 in the sequence of SEQ ID NO: 8 to phenylalanine (F), mutations comprising a substitution of the amino acid at position 7 in the sequence of SEQ ID NO: 8 to glutamate (E), mutations comprising a substitution of the amino acid at position 8 in the sequence of SEQ ID NO: 8 to arginine (R), mutations comprising a substitution of the amino acid at position 12 in the sequence of SEQ ID NO: 8 to phenylalanine (F), mutations comprising a substitution of the amino acid at position 14 in the sequence of SEQ ID NO: 8 to arginine (R) or leucine (L), mutations comprising a substitution of the amino acid at position 16 in the sequence of SEQ ID NO: 8 to asparagine (N) or aspartate (D), mutations comprising a substitution of the amino acid at position 17 in the sequence of SEQ ID NO: 8 to alanine (A), mutations comprising a substitution of the amino acid at position 19 in the sequence of SEQ ID NO: 8 to serine (S), mutations comprising a substitution of the amino acid at position 20 in the sequence of SEQ ID NO: 8 to serine (S), and mutations comprising a substitution of the amino acid at position 21 in the sequence of SEQ ID NO: 8 to leucine (L) or phenylalanine (F).

12. The monoclonal antibody or antigen-binding fragment thereof according to claim 10, wherein the VH-FR3 of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of those set forth in SEQ ID NOs: 8, 9, 10, and 11.

13. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises a framework region 4 (VH-FR4) comprising the sequence set forth in SEQ ID NO: 12.

14. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region comprises a framework region 1 (VL-FR1) comprising the sequence set forth in SEQ ID NO: 13 or a sequence comprising mutations at one or more positions selected from the group consisting of amino acid positions 4 and 24 in the sequence of SEQ ID NO: 13.

15. The monoclonal antibody or antigen-binding fragment thereof according to claim 14, wherein the VL-FR1 of the light chain variable region comprises one or more mutations comprising a substitution of the amino acid at position 4 in the sequence of SEQ ID NO: 13 to leucine (L) or a substitution of the amino acid at position 24 in the sequence of SEQ ID NO: 13 to serine (S).

16. The monoclonal antibody or antigen-binding fragment thereof according to claim 14, wherein the VL-FR1 of the light chain variable region comprises an amino acid sequence selected from the group consisting of those set forth in SEQ ID NOs: 13, 14, and 15.

17. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region comprises a framework region 2 (VL-FR2) comprising the sequence set forth in SEQ ID NO: 16 or a sequence comprising mutations at one or more positions selected from the group consisting of amino acid positions 1, 2, and 14 in the sequence of SEQ ID NO: 16.

18. The monoclonal antibody or antigen-binding fragment thereof according to claim 17, wherein the VL-FR2 of the light chain variable region comprises one or more mutations selected from the group consisting of mutations comprising a substitution of the amino acid at position 1 in the sequence of SEQ ID NO: 16 to leucine (L) or methionine (M), mutations comprising a substitution of the amino acid at position 2 in the sequence of SEQ ID NO: 16 to asparagine (N), and mutations comprising a substitution of the amino acid at position 14 in the sequence of SEQ ID NO: 16 to valine (V).

19. The monoclonal antibody or antigen-binding fragment thereof according to claim 17, wherein the VL-FR2 of the light chain variable region comprises an amino acid sequence selected from the group consisting of those set forth in SEQ ID NOs: 16, 17, 18, and 19.

20. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region comprises a framework region 3 (VL-FR3) comprising the sequence set forth in ID NO: 20 or a sequence comprising mutations at one or more positions selected from the group consisting of amino acid positions 1, 3, 14, and 31 in the sequence of SEQ ID NO: 20.

21. The monoclonal antibody or antigen-binding fragment thereof according to claim 20, wherein the VL-FR3 of the light chain variable region comprises one or more mutations selected from the group consisting of mutations comprising a substitution of the amino acid at position 1 in the sequence of SEQ ID NO: 20 to threonine (T), serine (S) or tyrosine (Y), mutations comprising a substitution of the amino acid at position 3 in the sequence of SEQ ID NO: 20 to glutamine (Q), histidine (H) or glutamate (E), mutations comprising a substitution of the amino acid at position 14 in the sequence of SEQ ID NO: 20 to glycine (G), and mutations comprising a substitution of the amino acid at position 31 in the sequence of SEQ ID NO: 20 to valine (V).

22. The monoclonal antibody or antigen-binding fragment thereof according to claim 20, wherein the VL-FR3 of the light chain variable region comprises an amino acid sequence selected from the group consisting of those set forth in SEQ ID NOs: 20, 21, 22, and 23.

23. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region comprises a framework region 4 (VL-FR4) comprising the sequence set forth in SEQ ID NO: 24.

24. A nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof according to claim 1.

25. A vector comprising the nucleic acid molecule according to claim 24.

26. A host cell comprising the vector according to claim 25.

27. A method for preventing or treating cancer comprising administering a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof according to claim 1, a nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof or a vector comprising the nucleic acid molecule thereof to a subject.

28. A method for preventing or treating hypertension comprising administering a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof according to claim 1, a nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof or a vector comprising the nucleic acid molecule thereof to a subject.

29. A method for quantifying endothelin receptor type A in a sample, comprising treating the sample with the monoclonal antibody or antigen-binding fragment thereof according to claim 1.

30. A method for providing information necessary for the diagnosis of a disease caused by overexpression of endothelin receptor type A, comprising (a) separating a sample from a subject, (b) treating the sample with the monoclonal antibody or antigen-binding fragment thereof according to claim 1, and (c) determining whether the expression level of endothelin receptor type A in the sample from the subject is higher than that of endothelin receptor type A in a normal sample.

31. The method according to claim 30, wherein the disease caused by overexpression of endothelin receptor type A is cancer or hypertension.

32. A kit for quantifying endothelin receptor type A comprising the monoclonal antibody or antigen-binding fragment thereof according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0086] FIG. 1 shows constructed expression vectors for animal cells.

[0087] FIG. 2 shows the amino acid sequences of four antibodies with engineered framework regions.

[0088] FIG. 3 shows the results of analysis of four antibodies with engineered framework regions after purification in animal cells.

[0089] FIG. 4 shows the yield of MJ-F1, which selectively binds to endothelin receptor type A and whose framework regions were engineered to have trastuzumab framework sequences, in animal cells.

[0090] FIG. 5 shows the results of ELISA for four antibodies with engineered framework regions to human endothelin receptor type A.

[0091] FIG. 6 shows the production of five antibodies, which selectively bind to endothelin receptor type A and whose framework regions were engineered to have trastuzumab framework sequences, in animal cells.

[0092] FIG. 7 shows the results of ELISA for five antibodies, which have trastuzumab framework sequences and selectively bind to human endothelin receptor type A as an antigen.

[0093] FIGS. 8A and 8B show the results of ELISA for five antibodies, which have trastuzumab framework sequences and selectively bind to human endothelin receptor type A, and pH-dependent human FcRn pH 7.4 (FIG. 8A), pH 6.0 (FIG. 8B).

[0094] FIGS. 9A to 9D show the results of ELISA for five antibodies, which have trastuzumab framework sequences and selectively bind to human endothelin receptor type A, and human FcγRIIa variants FcγRIIa-131R (FIG. 9A), FcγRIIa-131H (FIG. 9B), FcγRIIa-158V (FIG. 9C), FcγRIIa-158F (FIG. 9D).

[0095] FIG. 10 shows the results of ELISA for five antibodies, which have trastuzumab framework sequences and selectively bind to human endothelin receptor type A, and human C1q.

[0096] FIGS. 11A and 11B show the inhibitory efficacies of four antibodies, all of which have trastuzumab framework sequences and selectively bind to human endothelin receptor type A, on the proliferation of colorectal cancer cell lines HT-29 (FIG. 11A) and HCT-116 (FIG. 11B).

[0097] FIG. 12A shows anticancer activities of four antibodies, all of which have trastuzumab framework sequences and selectively bind to human endothelin receptor type A, after administration to colorectal cancer xenograft models and FIG. 12B shows colorectal cancer tissues excised from the colorectal cancer xenograft models administered the antibodies.

[0098] FIGS. 13A and 13B show the inhibitory efficacies of four antibodies, all of which have trastuzumab framework sequences and selectively bind to human endothelin receptor type A, on the proliferation of pancreatic cancer cell lines AsPC-1 (FIG. 13A) and Panc-1 (FIG. 13B).

[0099] FIGS. 14A and 14B show the inhibitory efficacies of four antibodies, all of which have trastuzumab framework sequences and selectively bind to human endothelin receptor type A, on the proliferation of gastric cancer cell lines SNU-216 (FIG. 14A) and SNU-668 (FIG. 14B).

[0100] FIG. 15 shows the results of SEC-HPLC for four antibodies, all of which have trastuzumab framework sequences and selectively bind to human endothelin receptor type A.

[0101] FIG. 16 shows the results of glycan profiling for four antibodies, all of which have trastuzumab framework sequences and selectively bind to human endothelin receptor type A.

[0102] FIG. 17 shows the results of RP-HPLC for four antibodies, all of which have trastuzumab framework sequences and selectively binding to human endothelin receptor type A.

[0103] FIGS. 18A, 18B, 18C, and 18D show the thermal stability test results for MJ-F1-WT, MJ-F1-pFc29, GG12-pFc29, and FG12-pFc29, all of which have trastuzumab framework sequences and selectively bind to human endothelin receptor type A, respectively.

[0104] FIG. 19 shows the inhibitory activities of the antibodies (each 200 nM) described in Korean Patent Application No. 10-2017-0075129 on ET-1 (10 nM)-induced signaling in cells of the colorectal cancer cell line HT-29 overexpressing ET.sub.A.

MODE FOR CARRYING OUT THE INVENTION

[0105] The present invention will be more specifically explained with reference to the following examples. It will be evident to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

EXAMPLES

<Example 1> Production of Antibodies with Engineered Framework Regions Based on Sequences of Complementarity-Determining Regions (CDRs) of Antibodies Specifically Binding to Endothelin Receptor Type A

[0106] In this example, antibodies with improved stability and productivity were produced using optimal combinations of the framework region sequences of various antibody drugs and the CDR sequences of the ET.sub.A-specific antibodies (AG8, EF12, GG12, FG12, AB9) described in Korean Patent Application No. 10-2017-0075129 (FIG. 19) based on the sequences of the ET.sub.A-specific antibodies. To this end, the framework regions of four therapeutic antibodies (trastuzumab, bevacizumab, omalizumab, and adalimumab) were overlapped with the complementarity-determining regions (CDRs) of the heavy and light chain variable regions of the ET.sub.A-specific antibodies, which are known to play an important role in antigen-antibody interactions, by PCR. The four amplified heavy chain variable region genes and the four amplified light chain variable region genes were digested with the restriction enzymes XbaI/BssHII and inserted into expression vectors (pMAZ) for animal cells, which had been digested with the same restriction enzymes (FIG. 1). The fused antibody expression vectors were transformed into Escherichia coli Jude1 (F-mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ-rpsL nupG) and the DNA sequences of the constructed expression vectors were validated by Sanger sequencing. The sequences of the four produced antibodies (MJ-F1, MJ-F2, MJ-F3, and MJ-F4) are shown in FIG. 2.

<Example 2> Expression and Purification of the Four Antibodies with Engineered Framework Regions in Animal Cells

[0107] After transformation into E. coli, the amplified expression vectors were recovered and transiently transfected into Expi293 cells using a 150 mM NaCl solution containing polyethyleneimine (PEI, Polysciences, USA). Cells were cultured in Freestyle293 expression medium (Life Technologies, USA) at a temperature of 37° C. for 7 days. The expressed cell culture was centrifuged at 6,000 rpm for 20 min. The supernatant was collected and passed through a 0.22 μm filter. The filtrate was allowed to bind to 1 ml of Protein A resin (Amicogen, Korea) at 4° C. for 16 h. The bound resin was washed with 10 CV (column volume) of PBS solution, eluted with 100 mM glycine solution (pH 2.7), and neutralized with 1 M Tris-HCl (pH 8.0). After buffer change with PBS, the sizes and purities of the light and heavy chains of the four purified antibodies were analyzed by SDS-PAGE under reducing and non-reducing conditions (FIG. 3). The yields of AG8 and the four purified antibodies (MJ-F1, MJ-F2, MJ-F3, and MJ-F4) were compared and analyzed. The yield of MJ-F1 was 15 times higher than that of AG8 (FIG. 4).

<Example 3> Analysis of Binding Affinities Between Extracellularly Exposed Regions of Endothelin Receptor Type A as Antigen and the Produced Antibodies

[0108] In this example, the binding affinities of AG8 and the four prepared antibodies (MJ-F1, MJ-F2, MJ-F3, and MJ-F4) for endothelin receptor type A were analyzed by ELISA. To this end, human G.sub.αi3 was diluted with 0.05 M Na.sub.2CO.sub.3 (pH 9.6) as a buffer solution and allowed to bind to 96-well plates (Costar) at a concentration of 5 μg/well at 4° C. for 16 h. Each of the 96-well plates was added to a PBST solution (a PBS solution containing 0.05% Tween-20) containing 4% skim milk such that the concentration was 5% (w/v), followed by blocking at room temperature for 1 h. After the solution was discarded, the 96-well plate was washed 4 times with a PBS solution containing 0.05% Tween-20 (150 μl/well). Endothelin receptor type A (5 μg/ml) expressed and purified in E. coli and reconstituted with sarkosyl was immobilized onto the 96-well plate such that its extracellular regions were selectively exposed. AG8 and the four antibodies (MJ-F1, MJ-F2, MJ-F3, and MJ-F4) expressed and purified in animal cells were serially diluted four-fold and allowed to bind to 96-well plates (50 μl/well) at room temperature for 1 h. After each of the 96-well plates was washed 4 times with 0.05% PBST solution (150 μl/well), human IgG(H+L)-HRP conjugate was diluted 1:5,000 with PBS and allowed to bind to the 96-well plate at room temperature for 1 h. After the solution was discarded, the 96-well plate was washed 4 times with 0.05% PBST solution (150 μl/well). The reaction was performed with TMB (Thermo Scientific) (50 μl/well). 20 min later, the reaction was terminated with 4 N H.sub.2SO.sub.4 and the absorbance was measured at 450 nm. Analysis of the ELISA signals revealed that the binding affinities of the control antibody trastuzumab, MJ-F3, and MJ-F4 were too low to be measured whereas the equilibrium dissociation constant of MJ-F2 was 132.8 nM. Particularly, the visible equilibrium dissociation constant of MJ-F1 was 1.41 nM, which was improved by ˜19 times compared to that (26.1) of the antibody AG8 described in the prior art document (FIG. 5, Table 1).

TABLE-US-00001 TABLE 1 Calculated equilibrium dissociation constants of the four produced antibodies to human endothelin receptor type A as antigen by ELISA MJ-F1 MJ-F2 AG8 MJ-F3 MJ-F4 Herceptin K.sub.D (nM) 1.41 132.8 26.1 n.d. n.d. n.d.

<Example 4> Expression and Purification of Anti-ET.SUB.A .Antibodies Engineered to have Trastuzumab Framework Regions in Animal Cells

[0109] After transformation into E. coli, the amplified expression vectors were recovered and transiently transfected into Expi293 cells using a 150 mM NaCl solution containing polyethyleneimine (PEI, Polysciences, USA). Cells were cultured in Freestyle293 expression medium (Life Technologies, USA) at a temperature of 37° C. for 7 days. The expressed cell culture was centrifuged at 6,000 rpm for 20 min. The supernatant was collected and passed through a 0.22 μm filter. The filtrate was allowed to bind to 1 ml of Protein A resin (Amicogen, Korea) at 4° C. for 16 h. The bound resin was washed with 10 CV (column volume) of PBS solution, eluted with 100 mM glycine solution (pH 2.7), and neutralized with 1 M Tris-HCl (pH 8.0). After buffer change with PBS, the sizes and purities of the light and heavy chains of the purified anti-ET.sub.A antibodies (MJF1-WT, MJF1-pFc29, FG12-pFc29, and GG12-pFc29) (where the Fc region is wild type or PFc29 as an Fc variant having the sequence set forth in SEQ ID NO: 35) engineered to have trastuzumab framework regions were analyzed by SD S-PAGE under reducing and non-reducing conditions (FIG. 3). The anti-ET.sub.A antibodies engineered to have trastuzumab framework regions (MJF1-WT, MJF1-pFc29, FG12-pFc29, and GG12-pFc29) were identified by SDS-PAGE after purification (FIG. 6).

<Example 5> Analysis of Binding Affinities Between Regions of Endothelin Receptor Type A as Antigen and the Anti-ET.SUB.A .Antibodies Engineered to have Trastuzumab Framework Regions

[0110] In this example, the binding affinities of the five antibodies (MJF1-WT, MJF1-pFc29, FG12-pFc29, GG12-pFc29, and EF12-pFc29), which were engineered to have the framework regions of trastuzumab selected in Example 1, for endothelin receptor type A were analyzed by ELISA. To this end, human G.sub.αi3 was diluted with 0.05 M Na.sub.2CO.sub.3 (pH 9.6) as a buffer solution and allowed to bind to 96-well plates (Costar) at a concentration of 5 μg/well at 4° C. for 16 h. Each of the 96-well plates was added to a PBS solution containing 4% skim milk such that the concentration was 5% (w/v), followed by blocking at room temperature for 1 h. After the solution was discarded, the 96-well plate was washed 4 times with a PBST solution (a PBS solution containing 0.5% Tween-20) (150 μl/well). Endothelin receptor type A (5 μg/ml) expressed and purified in E. coli and reconstituted with sarkosyl was immobilized onto the 96-well plate such that its extracellular regions were selectively exposed. The five antibodies expressed and purified in animal cells were serially diluted four-fold and allowed to bind to 96-well plates (50 μl/well) at room temperature for 1 h. After each of the 96-well plates was washed 4 times with 0.5% PBST solution (150 μl/well), human IgG(H+L)-HRP conjugate was diluted 1:5,000 with PBS and allowed to bind to the 96-well plate at room temperature for 1 h. After the solution was discarded, the 96-well plate was washed 4 times with 0.5% PBST solution (150 μl/well). The reaction was performed with TMB (Thermo Scientific) (50 μl/well). 20 min later, the reaction was terminated with 4 N H.sub.2SO.sub.4 and the absorbance was measured at 450 nm. Analysis of the ELISA signals revealed that the binding affinity of the control antibody trastuzumab was too low to be measured whereas the equilibrium dissociation constant of MJF1-WT, MJF1-pFc29, AB9-pFc29, FG12-pFc29, EF12-pFc29, and GG12-pFc29 were 0.4873 nM, 0.3637 nM, 0.7706 nM, 0.4809 nM, 0.6044 nM, and 0.2875 nM, respectively (FIG. 7, Table 2).

TABLE-US-00002 TABLE 2 Calculated equilibrium dissociation constants of the six antibodies haying the framework sequences to endothelin receptor type as antigen A by ELISA MJF1-WT MJF1-pFc29 AB9-pFc29 FG12-pFc29 EF12-pFc29 GG12-pFc29 Trastuzumab K.sub.D (nM) 0.4873 0.3637 0.7706 0.4809 0.6044 0.2875 n.d.

<Example 6> Analysis of Binding Affinities Between the Anti-ET.SUB.A .Antibodies Engineered to have Trastuzumab Framework Regions and pH-Dependent FcRn

[0111] In this example, the binding affinities of the five antibodies (MJF1-WT, MJF1-pFc29, FG12-pFc29, GG12-pFc29, and EF12-pFc29), which were engineered to have the framework regions of trastuzumab selected in Example 1, for human FcRn were analyzed by ELISA. To this end, each of the five antibodies engineered to have trastuzumab framework regions was diluted with 0.05 M Na.sub.2CO.sub.3 (pH 9.6) as a buffer solution and allowed to bind to 96-well plates (Costar) at a concentration of 4 μg/well at 4° C. for 16 h. Each of the 96-well plates was added to a PBS solution containing 4% skim milk such that the concentration was 5% (w/v), followed by blocking at room temperature for 1 h. After the solution was discarded, the 96-well plate was washed 4 times with a PBST solution (a PBS solution containing 0.5% Tween-20) (150 μl/well). A fusion protein of human FcRn and glutathione S-transferase (GST) was serially diluted four-fold and allowed to bind to 96-well plates (50 μl/well) at pH 7.4 and pH 6.0 at room temperature for 1 h. After each of the 96-well plates was washed 4 times with 0.5% PBST solution (150 μl/well), anti-glutathione-HRP conjugate was diluted 1:5,000 with PBST and allowed to bind to the 96-well plate at room temperature for 1 h. After the solution was discarded, the 96-well plate was washed 4 times with 0.5% PBST solution (150 μl/well). The reaction was performed with TMB (Thermo Scientific) (50 μl/well). 20 min later, the reaction was terminated with 4 N H.sub.2SO.sub.4 and the absorbance was measured at 450 nm. The results are shown in FIGS. 8A and 8B. Analysis of the ELISA signals revealed that the binding affinities of the five antibodies expressed and purified in animal cells for pH-dependent human FcRn were similar to that of the control antibody trastuzumab, which has a long blood half-life (FIGS. 8A and 8B).

<Example 7> Analysis of Binding Affinities Between the Anti-ET.SUB.A .Antibodies Engineered to have Trastuzumab Framework Regions and Human FcγRlIa Variants

[0112] In this example, the binding affinities of the four antibodies (MJF1-WT, MJF1-pFc29, FG12-pFc29, and GG12-pFc29), which were engineered to have the framework regions of trastuzumab selected in Example 1, for human FcγRIIa variants (FcγRIIa-131R, FcγRIIa-131H, FcγRIIa-158V, and FcγRIIa-158F) were analyzed by ELISA. To this end, each of the four antibodies engineered to have trastuzumab framework regions was diluted with 0.05 M Na.sub.2CO.sub.3 (pH 9.6) as a buffer solution and allowed to bind to 96-well plates (Costar) at a concentration of 4 μg/well at 4° C. for 16 h. Each of the 96-well plates was added to a PBS solution containing 4% skim milk such that the concentration was 5% (w/v), followed by blocking at room temperature for 1 h. After the solution was discarded, the 96-well plate was washed 4 times with a PBST solution (a PBS solution containing 0.5% Tween-20) (150 μl/well). GST proteins fused with human FcγRIIa variants were serially diluted four-fold and allowed to bind to 96-well plates (50 μl/well) at room temperature for 1 h. After each of the 96-well plates was washed 4 times with 0.5% PBST solution (150 μl/well), anti-GST-HRP conjugate was diluted 1:5,000 with PBST and allowed to bind to the 96-well plate at room temperature for 1 h. After the solution was discarded, the 96-well plate was washed 4 times with 0.5% PBST solution (150 μl/well). The reaction was performed with TMB (Thermo Scientific) (50 μl/well). 20 min later, the reaction was terminated with 4 N H.sub.2SO.sub.4 and the absorbance was measured at 450 nm. The results are shown in FIGS. 9A to 9D. Analysis of the ELISA signals revealed that the binding affinities of the four antibodies expressed and purified in animal cells for the human FcγRIIa variants were similar to those of the control antibody trastuzumab (FIGS. 9A to 9D).

<Example 8> Analysis of Binding Affinities Between the Anti-ET.SUB.A .Antibodies Engineered to have Trastuzumab Framework Regions and Human C1q

[0113] In this example, the binding affinities of the four antibodies (MJF1-WT, MJF1-pFc29, FG12-pFc29, and GG12-pFc29), which were engineered to have the framework regions of trastuzumab selected in Example 1, for human C1q were analyzed by ELISA. To this end, each of the four antibodies engineered to have trastuzumab framework regions was diluted with 0.05 M Na.sub.2CO.sub.3 (pH 9.6) as a buffer solution and allowed to bind to 96-well plates (Costar) at a concentration of 4 μg/well at 4° C. for 16 h. Each of the 96-well plates was added to a PBS solution containing 4% skim milk such that the concentration was 5% (w/v), followed by blocking at room temperature for 1 h. After the solution was discarded, the 96-well plate was washed 4 times with a PBST solution (a PBS solution containing 0.5% Tween-20) (150 μl/well). Human C1q was serially diluted four-fold and allowed to bind to 96-well plates (50 μl/well) at room temperature for 1 h. After each of the 96-well plates was washed 4 times with 0.5% PBST solution (150 μl/well), anti-human C1q-HRP conjugate was diluted 1:400 with PBST and allowed to bind to the 96-well plate at room temperature for 1 h. After the solution was discarded, the 96-well plate was washed 4 times with 0.5% PBST solution (150 μl/well). The reaction was performed with TMB (Thermo Scientific) (50 μl/well). 20 min later, the reaction was terminated with 4 N H.sub.2SO.sub.4 and the absorbance was measured at 450 nm. The results are shown in FIG. 10. Analysis of the ELISA signals revealed that the binding affinities of the four antibodies expressed and purified in animal cells for human C1q were similar to that of the control antibody trastuzumab (FIG. 10).

<Example 9> Determination of Anti-Proliferative Efficacies of the Anti-ET.SUB.A .Antibodies Engineered to have Trastuzumab Framework Regions Against Colorectal Cancer Cells

[0114] In this example, a determination was made as to whether the anti-ET.sub.A antibodies with engineered framework regions had anti-proliferative efficacies against cells of the colorectal cancer cell lines HT-29 and HCT-116, which are widely used for studies on colorectal cancer cells. The degrees of cell proliferation were confirmed by measuring the amount of DNA in cells using a fluorescent dye (CyQUANT NF, Invitrogen, USA).

[0115] Each of the cell lines HT-29 and HCT-116 was treated with the four anti-ET.sub.A antibodies (MJF1-WT, MJF1-pFc29, FG12-pFc29, and GG12-pFc29) engineered to have trastuzumab framework regions and cultured in a cell incubator at 37° C. for 1 h. 72 h after inoculation, cells were treated with a fluorescent dye (CyQUANT NF). The amounts of fluorescence emitted by 485 nm excitation depending on the level of DNA present in cells were measured using a fluorescence spectrophotometer (TECAN, Switzerland) to determine the ability of the anti-ET.sub.A antibodies with engineered framework regions to inhibit the proliferation of colorectal cancer cells (FIGS. 11A and 11B). As shown in FIGS. 11A and 11B, the inventive antibodies effectively inhibited the proliferation of HT-29 and HCT-116 colorectal cancer cell lines.

<Example 10> Validation of Anticancer Activities of the Anti-ET.SUB.A .Antibodies Engineered to have Trastuzumab Framework Regions in Colorectal Cancer Animal Models

[0116] Colorectal cancer cell line HT-29 was injected subcutaneously into the flank region of BALB/c nude mice to establish xenograft models. The cell number was adjusted to 2×10.sup.6 cells/mouse depending on the amount of PBS (100 μl). From 5 days after injection, each of the inventive antibodies was directly injected into the colorectal cancer tissue at 2-day intervals (200 μg at a time) to validate its anticancer activity. 21 days after injection, the colorectal cancer tissue was excised. The results are shown in FIGS. 12A and 12B. In FIGS. 12A and 12B, “Vehicle” is a control group injected with PBS alone in the same amount as the antibody.

[0117] As shown in FIGS. 12A and 12B, the four inventive anti-ET.sub.A antibodies (MJF1-WT, MJF1-pFc29, FG12-pFc29, and GG12-pFc29) engineered to have trastuzumab framework regions showed inhibitory effects on the growth of colorectal cancer in the xenograft models of the colorectal cancer cell line HT-29. These results demonstrate that the four inventive anti-ET.sub.A antibodies engineered to have trastuzumab framework regions have inhibitory activities on the proliferation of colorectal cancer cells even in animal models, thus being suitable for use in therapeutic agents.

<Example 11> Determination of Anti-Proliferative Efficacies of the Anti-ET.SUB.A .Antibodies Engineered to have Trastuzumab Framework Regions in Pancreatic Cancer Cells

[0118] In this example, a determination was made as to whether the anti-ET.sub.A antibodies (MJF1-WT, MJF1-pFc29, FG12-pFc29, and GG12-pFc29) with engineered framework regions had anti-proliferative efficacies against cells of the cell lines AsPC-1 and Panc-1, which are widely used for studies on pancreatic cancer cells. To this end, the same procedure as in Example 9 was carried out. The results are shown in FIGS. 13A and 13B.

[0119] As shown in FIGS. 13A and 13B, the anti-ET.sub.A antibodies (MJF1-WT, MJF1-pFc29, FG12-pFc29, and GG12-pFc29) with engineered framework regions effectively inhibited the proliferation of cells of the pancreatic cancer cell lines. These results demonstrate that the inventive anti-ET.sub.A antibodies (MJF1-WT, MJF1-pFc29, FG12-pFc29, and GG12-pFc29) with engineered framework regions have inhibitory activities on the proliferation of pancreatic cancer cells.

<Example 12> Determination of Anti-Proliferative Efficacies of the Anti-ET.SUB.A .Antibodies Engineered to have Trastuzumab Framework Regions in Gastric Cancer Cells

[0120] In this example, a determination was made as to whether the anti-ET.sub.A antibodies (MJF1-WT, MJF1-pFc29, FG12-pFc29, and GG12-pFc29) with engineered framework regions had anti-proliferative efficacies against cells of the cell lines SNU-216 and SNU-668, which are widely used for studies on gastric cancer cells. To this end, the same procedure as in Example 9 was carried out. The results are shown in FIGS. 14A and 14B.

[0121] As shown in FIGS. 14A and 14B, the anti-ET.sub.A antibodies (MJF1-WT, MJF1-pFc29, FG12-pFc29, and GG12-pFc29) with engineered framework regions effectively inhibited the proliferation of cells of the pancreatic cancer cell lines. These results demonstrate that the inventive anti-ET.sub.A antibodies (MJF1-WT, MJF1-pFc29, FG12-pFc29, and GG12-pFc29) with engineered framework regions have inhibitory activities on the proliferation of gastric cancer cells.

<Example 13> Determination of Physical Properties and Characteristics of the Anti-ET.SUB.A .Antibodies Engineered to have Trastuzumab Framework Regions

[0122] The anti-ET.sub.A antibodies (MJF1-WT, MJF1-pFc29, GG12-pFc29, and FG12-pFc29) engineered to have trastuzumab framework regions and the control antibody trastuzumab were separated by SEC-HPLC. The results are shown in FIG. 15. As shown in FIG. 15, the degrees of aggregation were not more than 10%. The glycan profiles of the antibodies were analyzed. The results are shown in FIG. 16. As shown in FIG. 16, the glycan profiles of the developed antibodies did not show significant differences from that of the human IgG standard. The contents of impurities in the antibodies were confirmed by RP-HPLC. The results are shown in FIG. 17. The contents of impurities in all antibodies were found to be <8%. The thermal stabilities of the discovered antibodies were tested (nanoDSC). As a result, the Tm values of all antibodies were >70° C. (FIGS. 18A to 18D).

[0123] Although the particulars of the present invention have been described in detail, it will be obvious to those skilled in the art that such particulars are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.