ANTI-MESOTHELIN CHIMERIC ANTIGEN RECEPTOR SPECIFICALLY BINDING TO MESOTHELIN

Abstract

Provided is an anti-mesothelin chimeric antigen receptor specifically binding to mesothelin. The anti-mesothelin chimeric antigen receptor according to an aspect exhibits an ability to specifically bind to mesothelin, and thus may be usefully applied to preventing or treating mesothelin-overexpressing cancers.

Claims

1. An anti-mesothelin antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the following heavy chain CDRs and a light chain variable region comprising the following light chain CDRs: a heavy chain CDR1 comprising an amino acid sequence consisting of SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence consisting of SEQ ID NO: 2, a heavy chain CDR3 comprising an amino acid sequence consisting of SEQ ID NO: 3, and a light chain CDR1 comprising an amino acid sequence consisting of SEQ ID NO: 4, a light chain CDR2 comprising an amino acid sequence consisting of SEQ ID NO: 5, and a light chain CDR3 comprising an amino acid sequence consisting of SEQ ID NO: 6; or a heavy chain CDR1 comprising an amino acid sequence consisting of SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence consisting of SEQ ID NO: 14, a heavy chain CDR3 comprising an amino acid sequence consisting of SEQ ID NO: 15, and a light chain CDR1 comprising an amino acid sequence consisting of SEQ ID NO: 16, a light chain CDR2 comprising an amino acid sequence consisting of SEQ ID NO: 17, and a light chain CDR3 comprising an amino acid sequence consisting of SEQ ID NO: 18.

2. The anti-mesothelin antibody or antigen-binding fragment thereof according to claim 1, comprising: a heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 19 or 23.

3. The anti-mesothelin antibody or antigen-binding fragment thereof according to claim 1, comprising: a light chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 20 or 24.

4. The anti-mesothelin antibody or antigen-binding fragment thereof according to claim 1, comprising: a heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 19; and a light chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 20.

5. The anti-mesothelin antibody or antigen-binding fragment thereof according to claim 1, comprising: a heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 23; and a light chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 24.

6. An isolated nucleic acid encoding the anti-mesothelin antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. A vector comprising the isolated nucleic acid according to claim 6.

8. An isolated host cell transformed with the vector according to claim 7.

9. A method of preparing an anti-mesothelin antibody, the method comprising expressing the antibody by culturing the host cell according to claim 8.

10. A chimeric antigen receptor comprising an antigen-binding domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain, wherein the antigen-binding domain is an anti-mesothelin antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the following heavy chain CDRs and a light chain variable region comprising the following light chain CDRs: a heavy chain CDR1 comprising an amino acid sequence consisting of SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence consisting of SEQ ID NO: 2, a heavy chain CDR3 comprising an amino acid sequence consisting of SEQ ID NO: 3, and a light chain CDR1 comprising an amino acid sequence consisting of SEQ ID NO: 4, a light chain CDR2 comprising an amino acid sequence consisting of SEQ ID NO: 5, and a light chain CDR3 comprising an amino acid sequence consisting of SEQ ID NO: 6; or a heavy chain CDR1 comprising an amino acid sequence consisting of SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence consisting of SEQ ID NO: 14, a heavy chain CDR3 comprising an amino acid sequence consisting of SEQ ID NO: 15, and a light chain CDR1 comprising an amino acid sequence consisting of SEQ ID NO: 16, a light chain CDR2 comprising an amino acid sequence consisting of SEQ ID NO: 17, and a light chain CDR3 comprising an amino acid sequence consisting of SEQ ID NO: 18.

11. The chimeric antigen receptor according to claim 10, wherein the antigen-binding domain is an anti-mesothelin antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 19; and a light chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 20.

12. The chimeric antigen receptor according to claim 10, wherein the antigen-binding domain is an anti-mesothelin antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 23; and a light chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 24.

13. The chimeric antigen receptor according to claim 10, wherein the antigen-binding fragment is a single chain variable fragment (scFv).

14. A polynucleotide encoding the chimeric antigen receptor according to claim 10.

15. The polynucleotide according to claim 14, wherein the polynucleotide comprises a base sequence consisting of SEQ ID NO: 26 or 27.

16. A vector comprising the polynucleotide according to claim 14.

17. An isolated cell transformed with the vector according to claim 16.

18. The isolated cell according to claim 17, wherein the cell is a T cell, an NK cell, an NKT cell, or a gamma delta (γδ) T cell.

19. A pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition comprising the isolated cell according to claim 18.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0082] FIG. 1 shows an illustration showing a process of screening for antibodies through panning of phage display antibody libraries;

[0083] FIG. 2A shows a phage output titer and FIG. 2B shows an elution titer ratio, according to rounds of panning as a result of solid phase panning;

[0084] FIG. 3A shows a phage output titer and FIG. 3B shows an elution titer ratio, according to rounds of panning as a result of magnetic bead-mediated solution panning;

[0085] FIG. 4 shows results of comparative analysis of specific binding to antigen MSLN of clones obtained through phage ELISA;

[0086] FIG. 5 shows flow cytometry results of examining whether clones selected using a mesothelin-overexpressing cell line actually bind to mesothelin present on the cell membrane;

[0087] FIG. 6 shows relative peak shift values showing binding specificity to mesothelin of clones selected using a mesothelin-overexpressing cell line;

[0088] FIG. 7 shows SDS-PAGE results of analyzing purified anti-MSLN-scFv antibodies (2 μg of each protein loaded) (NR: Non-reducing condition, R: Reducing condition (100° C., 10 minutes));

[0089] FIG. 8 shows ELISA results of analyzing affinity of anti-MSLN-scFv antibody for antigen MSLNs (A: MSLN 34 clone, B: MSLN 37 Clone, C: MSLN 38 Clone);

[0090] FIG. 9 shows an illustration of an anti-MSLN-CAR expression system including an MSLN-specific antigen-binding domain according to an aspect;

[0091] FIG. 10 shows results of examining CAR expression in anti-MSLN-CAR-introduced T cells and measuring a percentage of CD4+ and CD8+ T cells in CD3-positive T cells, after a first round of transduction;

[0092] FIG. 11 shows results of examining CAR expression in anti-MSLN-CAR-introduced T cells and measuring a percentage of CD4+ and CD8+ T cells in CD3-positive T cells, after a second round of transduction;

[0093] FIG. 12 shows results of examining cell killing effects of anti-MSLN-CAR-introduced T cells using various cancer cell lines;

[0094] FIGS. 13 to 16 show results of examining cell killing effects of anti-MSLN34-CAR-T and anti-MSLN38-CAR-T;

[0095] FIG. 17 shows results of examining cancer cell-killing efficacy and body weight changes in mesothelioma animal models due to anti-MSLN34-CAR-T and anti-MSLN38-CAR-T (A: change in tumor volume, B: change in body weight, C: change in tumor weight); and

[0096] FIG. 18 shows results of examining cancer cell-killing efficacy and body weight changes in pancreatic cancer animal models due to anti-MSLN34-CAR-T and anti-MSLN38-CAR-T (A: change in tumor volume, B: change in body weight, C: change in tumor weight).

MODE OF DISCLOSURE

[0097] Hereinafter, an aspect will be described in more detail with reference to exemplary embodiments. However, these exemplary embodiments are only for illustrating an aspect, and the scope of an aspect is not limited to these exemplary embodiments, and exemplary embodiments of an aspect are provided to more completely explain an aspect to a person having ordinary knowledge in the art.

Example 1: Panning of Phage Display Antibody Libraries

[0098] To select antibodies binding to mesothelin (MSLN) which is a target antigen, four rounds of phage panning for MSLN (Acro Biosystems) were performed using KBIO human synthetic scFv phage display library KscFv-I according to a phage panning protocol established by New Drug Development Support Center, Osong Advanced Medical Industry Promotion Foundation. A schematic illustration of the panning process of phage display antibody libraries is shown in FIG. 1.

[0099] Panning was performed by two methods (solid, bead) according to antigen immobilization. For solid phase panning, 1 mL of a human mesothelin protein (in PBS, 1.sup.st: 10 μg/mL, 2.sup.nd: 5 μg/mL, 3.sup.rd; 2.5 μg/mL, 4.sup.th: 1.25 μg/mL) was fixed in an immunotube, and mixed with 1.3×10.sup.13 c.f.u. of phage library blocked with 5 mL of PBS (MPBS) containing 5% skim milk in the immunotube, and allowed to bind at 37° C. for 1.5 hours. Thereafter, the immunotube was washed with 5 mL of PBS-Tween20 (0.05%) (PBS-T) to remove unbound phages (1.sup.st: washed three times, 2.sup.nd to 4.sup.th: washed five times). 1 mL of 100 mM trimethylamine (TEA) was added to the tube, and allowed to react at room temperature for 10 minutes to elute bound phages, and the eluted phages were transferred to a 50 mL Falcon tube, and neutralized by mixing well with 0.5 mL of 1 M Tris-HCl (pH 7.4). The eluted phages were transfected to 8.5 mL of E. coli TG1 (OD600=0.5˜0.8) at a mid-log phase. Plasmid DNA was extracted from a portion of the transfected E. coli TG1 for sequencing, and a portion thereof was subjected to antibody screening through phage ELISA. In the magnetic bead-mediated solution panning, the same protocol as in the solid phase panning was performed, except that magnetic beads, instead of the immunotube, were treated with the mesothelin, which was then fixed. In common, during panning, panning of a PBS control to which MSLN protein was not fixed was also performed, and its output titer was compared at every round of panning, and the degree of phage enrichment was monitored through an elution titer ratio (a value obtained by dividing the output titer by the output titer of the control group). The results are shown in FIGS. 2 and 3.

[0100] In the solid phase panning, the enrichment started from the 3.sup.rd round, and the output titer for antigen MSLN showed a significant difference of about 53.4-fold (3.sup.rd round) and 1061.6-fold (4.sup.th round), as compared with the PBS control group (FIG. 2). In the panning using magnetic beads, the enrichment degrees of antigen MSLN at the 3.sup.rd round and the 4.sup.th round were about 2.0-fold (3.sup.rd) and 1.6-fold (4.sup.th), as compared with those of the PBS control group (FIG. 3), indicating no difference.

Example 2: Selection of Positive Clone by Phage-Specific ELISA

[0101] To select clones specifically binding to antigen MSLN from the phages obtained according to the phage panning of Example 1, 282 clones (94 colonies×3 plates) obtained from the 2.sup.nd round of panning using the immunotube were subjected to single-clone phage ELISA. In detail, 30 μL of 1 μg/mL human MSLN protein (antigen) was added to each well of a 96-half-well ELISA plate, and coated by incubation at 4° C. overnight. As a negative control, 30 μL of PBS was added to each well of another plate, followed by incubation at 4° C. overnight. Next day, contents in the plate were removed, and the plate was blocked with 150 μL of 5% MPBS at room temperature for 1 hour. Then, contents in the plate were removed, and 30 μL of the phage (˜10.sup.11 c.f.u.) was added, followed by incubation at room temperature for 1.5 hours. As a negative control, 30 μL of PBS, instead of the phage, was added. The plate was washed with a PBS-T (PBS-0.05% Tween 20) solution four times, and anti-M13-HRP (diluted 1:5,000 in PBS) was added and incubated at 37° C. for 1 hour. The plate was washed with the PBS-T solution four times, and 30 μL of TMB substrate reagent was added to each well, and incubated at room temperature for 8 minutes to induce color development. After stopping the color development by adding 30 μL of 2N H2504 per well, absorbance (O.D.) at 450 nm was measured.

[0102] As a result, when the absorbance cut-off for antigen MSLN was set at 0.4 or higher and the selection was performed, respectively, a total of 56 positive clones were obtained in the 2.sup.nd round. Additionally, the clones obtained in the 3.sup.rd and 4.sup.th rounds of panning using the immunotube were also subjected to single-clone phage ELISA in the same manner. 752 clones (94 colonies×8 plates) obtained in the 3.sup.rd round of panning were subjected to phage ELISA, and the absorbance cut-off was set at 0.7 or 0.4 or higher, and selection was performed. As a result, a total of 173 positive clones were obtained. Further, 188 clones (94 colonies×2 plates) obtained in the 4.sup.th round of panning were subjected to phage ELISA, and the absorbance cut-off was set at 0.4 or higher, and selection was performed. As a result, a total of 2 positive clones were obtained (Tables 1 to 4).

TABLE-US-00002 TABLE 1 2.sup.nd round of panning Absorbance Number of positive Number of unique (2 Round) (450 nm) clones clones 2R-1 >0.4 25 1 2R-2 >0.4 14 1 2R-3 >0.4 17 1 Sum 56 3

TABLE-US-00003 TABLE 2 3.sup.rd round of panning Absorbance Number of positive Number of unique (3 Round) (450 nm) clones clones 3R-6 >0.7 6 0 3R-7 >0.4 9 1 3R-8 >0.4 40 1 3R-9 >0.4 9 0 3R-10 >0.4 31 3 3R-11 >0.4 37 0 3R-12 >0.4 23 1 3R-13 >0.4 18 0 Sum 173 6

TABLE-US-00004 TABLE 3 4.sup.th round of panning Absorbance Number of positive Number of unique (4 Round) (450 nm) clones clones 4R-4 >0.4 1 0 4R-5 >0.4 1 0 Sum 2 0

TABLE-US-00005 TABLE 4 2.sup.nd + 3.sup.rd + 4.sup.th rounds of panning Sum Number of positive clones 231 Number of unique clones 23 Number of unique clones 9 (excluding overlapping clones)

[0103] Next, to further select clones specifically binding to antigen MSLN from the phages obtained according to the phage panning of Example 1, the clones obtained in the 3.sup.rd and 4.sup.th rounds of panning using the magnetic beads were also subjected to single-clone phage ELISA in the same manner. 188 clones (94 colonies×2 plates) obtained in the 3.sup.rd round of panning were subjected to phage ELISA, and the absorbance cut-off was set at 0.4 or higher, and selection was performed. As a result, a total of 4 positive clones were obtained. Further, 376 clones (94 colonies×4 plates) obtained in the 4.sup.th round of panning were subjected to phage ELISA, and the absorbance cut-off was set at 0.4 or higher, and selection was performed. As a result, a total of 7 positive clones were obtained (Tables 5 to 7).

TABLE-US-00006 TABLE 5 3.sup.rd round of panning Absorbance Number of positive Number of unique (3 Round) (450 nm) clones clones B-3R-1 >0.4 4 2 B-3R-2 >0.4 0 0 Sum 4 2

TABLE-US-00007 TABLE 6 4.sup.th round of panning Absorbance Number of positive Number of unique (4 Round) (450 nm) clones clones B-4R-1 >0.4 0 0 B-4R-2 >0.4 2 2 B-4R-3 >0.4 5 3 B-4R-4 >0.4 0 0 Sum 7 5

TABLE-US-00008 TABLE 7 3.sup.rd + 4.sup.th pounds of panning Sum Number of positive clones 11 Number of unique clones 7 Number of unique clones 7 (excluding overlapping clones)

Example 3: Sequencing and ELISA for Selecting Anti-MSLN Antibody Fragment Candidates

[0104] Phages were recovered from a total of 415 positive clones selected in Example 2, and then DNA sequencing was performed, and the sequences were aligned and grouped according to the Kabat numbering system. As a result, 16 kinds of unique clones for the antigen MSLN, the clones having different CDR sequences, were selected. In order to examine the specific binding of 16 kinds of the clones to antigen MSLN, each phage was purified and the phage titer was equally adjusted (1.2E+11 pfu/well), and then compared through ELISA. As a negative control, TLR4 antigen conjugated to a histidine tag as in MSLN was used, and as a positive control, clone MSLN3, of which excellent binding ability to mesothelin was confirmed in a previous study, was used (see Table 8 below). The results are shown in FIG. 4.

TABLE-US-00009 TABLE 8 SEQ Clone Region Amino acid sequence ID NO: MSLN3 HCDR1 DYAMS 32 HCDR2 AISSSGGTTYYADSVKG 33 HCDR3 EEEGEWREYFDV 34 LCDR1 RASQSISSYLN 35 LCDR2 ATSTLQS 36 LCDR3 QQSYTFPYT 37 VH EVQLVESGGGLVQPGGSL 38 RLSCAASGFTFSDYAMSWVR QAPGKGLEWVSAISSSGGTT YYADSVKGRFTISRDNSKNT LYLQMNSLRAEDTAVYYCAK EEEGEWREYFDVWGQGTLVT VSS VL DIQMTQSPSSLSASVGDR 39 VTITCRASQSISSYLNWYQQ KPGKAPKLLIYATSTLQSGV PSRFSGSGSGTDFTLTISSL QPEDFATYYCQQSYTFPYTF GQGTKVEIK

[0105] As shown in FIG. 4, it was confirmed that among 16 kinds of clones, 13 clones, except for MSLN26, MSLN30, and MSLN31, specifically bind to the antigen MSLN.

Example 4: Examination of Binding Ability Using Mesothelin-Overexpressing Cell Line

[0106] In order to determine whether 16 kinds of the phage clones selected in Example 3 actually bind to mesothelin present on the cell membrane, a pancreatic cancer cell line AsPC-1 which is a mesothelin-overexpressing cell line, and a human chronic myelogenous leukemia cell line K562 as a control were used to perform flow cytometry analysis.

[0107] In detail, K562 and AsPC-1 cells were prepared at a density of 10.sup.6 cells/well, and washed with 300 μL of PBS. Cells were blocked with 300 μL of 4% MPBS at 4° C. for 30 min. At the same time, phage clones (10′.sup.2/well) were blocked at room temperature for 1 hour in the same manner, and then the phage were incubated together with the cells at 4° C. for 2 hours. The cells were washed with PBS, and then treated with 1 μg/mL of anti-M13-FITC, followed by incubation at 4° C. for 1 hour. The cells were washed with PBS, and then resuspended in PBS, and the results were analyzed using a flow cytometer (BD biosciences). The results are shown in FIGS. 5 and 6.

[0108] As shown in FIG. 5, it was confirmed that MSLN34, MSLN37, and MSLN38 showed a relative peak shift value of 5.0% or more in the pancreatic cancer cell line AsPC-1. In the control K562 cell line, a significant level of peak shift was not observed. These results indicate that, among 16 kinds of clones, MSLN34, MSLN37, and MSLN38 actually exhibit high binding affinity for mesothelin present on the cell membrane.

[0109] In addition, as shown in FIG. 6, when the results of flow cytometry were quantified, it was confirmed that MSLN34, MSLN37, and MSLN38 showed relative peak shift values of 16.2%, 5.9%, and 30.8%, respectively, as compared to the control K562 cells. These results confirmed that all three clones specifically bind to the mesothelin-overexpressing cell line, and finally, they were selected as clones for the production of anti-MSLN antibody fragments.

Example 5: Production and Purification of Anti-MSLN Antibody Fragment

[0110] The three kinds of clones selected in Example 4 were used to transform Top10F′ competent E. coli which is an antibody fragment-expressing strain. Then, E. coli strains transformed with the three kinds of clones were cultured in 200 mL of TB medium, respectively, and protein expression was induced with IPTG (final concentration of 0.5 mM), followed by incubation at 30° C. overnight. Cells were obtained by centrifugation of the culture medium, and water-soluble proteins were obtained through periplasmic extraction, and then anti-MSLN-scFv antibody was purified through affinity chromatography using a protein L resin. The purified antibody protein was analyzed by SDS-PAGE, and the results are shown in FIG. 7.

[0111] Amino acid sequences of the three kinds of purified antibodies (MSLN34, MSLN37, and MSLN38) were examined and shown in Table 9, below. Specifically, heavy chain CDR1-3 amino acid sequences of MSLN34 are shown in SEQ ID NOS: 1 to 3, and light chain CDR1-3 amino acid sequences thereof are shown in SEQ ID NOS: 4 to 6, heavy chain CDR1-3 amino acid sequences of MSLN37 are shown in SEQ ID NOS: 7 to 9, and light chain CDR1-3 amino acid sequences thereof are shown in SEQ ID NOS: 10 to 12, and heavy chain CDR1-3 amino acid sequences of MSLN38 are shown in SEQ ID NOS: 13 to 15, and light chain CDR1-3 amino acid sequences thereof are shown in SEQ ID NOS: 16 to 18, respectively.

TABLE-US-00010 TABLE 9 SEQ Amino acid  ID Clone Region sequence NO: MSLN3 HCD DYGMH 1 4 R1 HCD SIYGSGGHTGYADSVKG 2 R2 HCD QHAYRYSYAFDV 3 R3 LCD RASQSISNWLN 4 R1 LCD ATSSLQS 5 R2 LCD QQSYSFPFT 6 R3 MSLN3 HCD SYAMH 7 7 R1 HCD GISGSGGTTYYADSVKG 8 R2 HCD EVEGQSQEYFDI 9 R3 LCD RASQSIANYLN 10 R1 LCD AASNLQS 11 R2 LCD QQSYSFPYT 12 R3 MSLN3 HCD SYAMS 13 8 R1 HCD GISGSGGSTGYADSVKG 14 R2 HCD HGQVGGISVFDI 15 R3 LCD RASQSISNWLN 16 R1 LCD ATSRLQS 17 R2 LCD QQSYSFPWT 18 R3 MSLN3 VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHW 19 4 VRQAPGKGLEWVSSIYGSGGHTGYADSVKGRFTISRD NSKNTLYLQMNSLRAEDTAVYYCAKQHAYRYSYAFDV WGQGTLVTVSS VL DIQMTQSPSSLSASVGDRVTITCRASQSISNWLNWYQQ 20 KPGKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQSYSFPFTFGQGTKVEIK MSLN3 VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWV 21 7 RQAPGKGLEWVSGISGSGGTTYYADSVKGRFTISRDN SKNTLYLQMNSLRAEDTAVYYCAKEVEGQSQEYFDIW GQGTLVTVSS VL DIQMTQSPSSLSASVGDRVTITCRASQSIANYLNWYQQ 22 KPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQSYSFPYTFGQGTKVEIK MSLN3 VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWV 23 8 RQAPGKGLEWVSGISGSGGSTGYADSVKGRFTISRDN SKNTLYLQMNSLRAEDTAVYYCAKHGQVGGISVFDIWG QGTLVTVSS VL DIQMTQSPSSLSASVGDRVTITCRASQSISNWLNWYQQ 24 KPGKAPKLLIYATSRLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQSYSFPWTFGQGTKVEIK

Example 6: Analysis of Affinity of Anti-MSLN Antibody for Antigen

[0112] The three kinds of anti-MSLN antibody proteins prepared in Example 5 were used to compare and analyze affinity thereof for antigen MSLN through ELISA. In detail, a MaxiSorb ELISA plate (Nunc) was coated with 30 μL of human mesothelin protein at a concentration of 1 μg/mL per well, and incubated at 4° C. overnight. The contents in the plate were removed, and the plate was blocked with 300 μL of 5% MPBS at room temperature for 1 hour. The purified antibody was serially diluted with PBS, 30 μL thereof was added to each well, and incubated at room temperature for 2 hours. As a negative control, 60 μL of PBS, instead of the purified antibody, was added and incubated at 37° C. for 2 hours.

[0113] The plate was washed with a PBS-T (PBS-0.05% Tween 20) solution four times, and 30 μL of anti-StrepMAB HRP (diluted 1:5,000 in PBS) was added and incubated at room temperature for 1 hour. The plate was washed with the PBS-T solution four times, and 30 μL of TMB substrate reagent was added to each well, and incubated at room temperature for 8 minutes to induce color development. After stopping the color development by adding 30 μL of 2N H2504 per well, absorbance (O.D.) at 450 nm was measured. The results are shown in FIG. 8.

[0114] As shown in FIG. 8, it was confirmed that MSLN34 showed an EC.sub.50 value of 83 nM, indicating the highest binding affinity among the three kinds of antibodies.

Example 7: Construction of Anti-MSLN Chimeric Antigen Receptor

[0115] Based on MSLN34 and MSLN38 showing high binding specificity to the mesothelin-overexpressing cell line, among the anti-MSLN antibody proteins prepared in Example 5, an anti-MSLN chimeric antigen receptor (anti-MSLN-CAR) was constructed.

[0116] 7-1: Anti-MSLN-CAR Lentiviral Vector Cloning

[0117] The Vector Belongs to the Second-Generation CAR Lentiviral Vector (pLV Lentiviral vector) system owned by the New Drug Development Support Center, in which the system includes pMDLg/pRRE (addgene) encoding gag/pol, and an envelope plasmid pRSV-Rev (addgene) encoding Rev protein, and an envelope plasmid pMD2.G (addgene) encoding VSV-G protein.

[0118] First, gene cloning was performed for the anti-MSLN scFv (antigen-binding domain) prepared in Example 5. Each anti-MSLN scFv of MSLN34 and MSLN38 and lentiviral vector were digested with XhoI (R0146S, NEB) and EcoRI (R0101, NEB) at 37° C. for 2 hours, followed by agarose gel electrophoresis. The identified products were purified using a FavorPrep Gel/PCR purification Mini kit (Favorgen). Each purified anti-MSLN scFv (100 ng) and vector (50 ng) were ligated by reacting at a ratio of 2:1 at 16° C. for 16 hours, and then transformed into Stbl3 competent cells to obtain colonies. The colonies were taken and grown in 5 mL of LB medium (ampicillin) to obtain plasmid DNA using a DNA plasmid mini-prep method. The plasmid DNA was digested with XhoI and EcoRI to confirm whether each inserted anti-MSLN scFv was well cloned into the vector. After sequencing, the DNA sequence was finally identified.

[0119] To the anti-MSLN scFv, CD8 hinge and CD8 TM (transmembrane) as a transmembrane domain, a cytoplasmic region of 4-1BB as a signaling domain, and an intracellular domain of CD3 zeta (CD3z) as a T cell activation domain were sequentially linked to construct anti-MSLN-CAR. Specifically, anti-MSLN-CAR consists of a CD8 signal sequence (Signal peptide, SP) (SEQ ID NO: 25), an anti-MSLN34 scFv (SEQ ID NO: 26) or an anti-MSLN38 scFv (SEQ ID NO: 27), a CD8 hinge domain (SEQ ID NO: 28), a CD8 transmembrane domain (SEQ ID NO: 29), a 4-1BB signaling domain (SEQ ID NO: 30), and a CD3 zeta signaling domain (SEQ ID NO: 31). Each domain was sequentially linked using each restriction enzyme, and specific nucleotide sequence information corresponding to each domain is summarized in Table 10 below.

TABLE-US-00011 TABLE 10 SEQ ID Name Nucleotide sequence (5′-3′) NO: CD8 ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTT 25 GCTGCTCCACGCCGCCAGGCCG MSLN GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACC 26 34 GGGTGGTTCACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTA scFv CTTTCTCTGATTATGGTATGCACTGGGTTCGTCAGGCGCCGG GCAAGGGTCTCGAATGGGTTTCATCTATCTACGGTTCTGGTG GTCACACTGGTTATGCCGATTCAGTGAAGGGTCGCTTTACCA TTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAA CTCGCTGCGTGCCGAAGACACGGCCGTCTATTATTGCGCCA AACAGCATGCATACCGTTACTCTTACGCATTCGATGTTTGGG GTCAGGGCACTTTAGTGACCGTCTCATCGGGTGGAGGCGGT TCAGGCGGAGGTGGATCCGGCGGTGGCGGATCGGACATTCA AATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCG ATCGTGTGACAATTACTTGTCGCGCTAGCCAGTCTATCTCTAA TTGGCTGAACTGGTATCAGCAGAAACCGGGCAAGGCGCCAA AATTGCTGATTTACGCAACTTCCTCTCTGCAGTCTGGTGTACC GTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCT GACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTAT TGTCAGCAATCTTACTCTTTTCCGTTTACGTTCGGGCAGGGA ACTAAAGTGGAAATTAAAGCCAGCACC MSLN GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACC 27 38 GGGTGGTTCACTGCGTCTGAGCTGCGCCGCCTCGGGTTlTA scFv CTTTCTCTTCTTATGCAATGTCTTGGGTTCGTCAGGCGCCGG GCAAGGGTCTCGAATGGGTTTCAGGTATCTCTGGTTCTGGTG GTTCTACTGGTTATGCCGATTCAGTGAAGGGTCGCTTTACCA TTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAA CTCGCTGCGTGCCGAAGACACGGCCGTCTATTATTGCGCCA AACATGGTCAGGTTGGTGGTATCTCTGTTTTCGATATCTGGG GTCAGGGCACTTTAGTGACCGTCTCATCGGGTGGAGGCGGT TCAGGCGGAGGTGGATCCGGCGGTGGCGGATCGGACATTCA AATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCG ATCGTGTGACAATTACTTGTCGCGCTAGCCAGTCTATCTCTAA TTGGCTGAACTGGTATCAGCAGAAACCGGGCAAGGCGCCAA AATTGCTGATTTACGCAACTTCCCGTCTGCAGTCTGGTGTAC CGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCC TGACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTA TTGTCAGCAATCTTACTCTllTCCGTGGACGTTCGGGCAGGG AACTAAAGTGGAAATTAAAGCCAGCACC CD8 ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCA 28 hinge CCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGC CGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTG GACTTCGCCTGTGAT CD8 ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCT 29 TM TCTCCTGTCACTGGTTATCACCCTTTACTGC 4-1BB AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCA 30 TTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGT AGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACT G CD3z AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAC 31 AGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGAC GAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGG GACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCA GGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGG AGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGG GGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGC CACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGC CCCCTCGC

[0120] 7-2: Production of Anti-MSLN-CAR-Loaded Lentivirus

[0121] Anti-MSLN-CAR lentivirus was produced by introducing the recombinant vector prepared in Example 7-1 into HEK293T cells. A schematic illustration of the anti-MSLN-CAR expression system according to an aspect, the system including the MSLN-specific antigen-binding domain, is shown in FIG. 9. First, the day before DNA transduction, HEK293T cells were seeded in a 100 mm tissue culture dish at a density of 6×10.sup.6 cells/dish. Next day, when the cell density reached 70% to 80%, transduction of MSLN-CAR-pLV, pMDLg/pRRE (addgene), pRSV-Rev (addgene), and pMD2.G (addgene) (5.5 μg: 3.5 μg:1.5 μg:2 μg) was performed using Lipofectamine 3000 (Thermofisher) according to the package insert. As a control, CD19 (FMC63) was used. 4 hours after transduction, DMEM medium containing 3% FBS (Gibco) was replaced, and after 48 hours, a virus culture medium was harvested. 10 mL of 20% sucrose solution was put in a centrifugation tube, 20 mL of the harvested virus culture medium was carefully placed thereon, and then mounted on a SW32T rotor, followed by ultra-high speed centrifugation at 25,000 rpm at 4° C. for 90 minutes. After centrifugation, the supernatant was discarded while being careful not to disturb the virus pellet at the bottom of the tube, and 400 μL of RPMI1640 medium (Gibco) was added and incubated in a refrigerator for 16 hours. Then, the pellet was resuspended and divided into 100 μL aliquots, which were then stored at −80° C.

[0122] 7-3: Lentivirus Titration

[0123] One day before lentivirus infection, HeLa cells were seeded in a 6-well plate at a density of 1.5×10.sup.5 cells/well. Next day, virus was diluted 1/100 and 1/1,000 with 500 μL of a virus infection medium, and added together with 8 μg/mL of polybrene to infect the cells. In one well, cells were treated with Trypsin-EDTA (0.05%) and harvested, followed by cell counting. After 4 hours, 1 mL of the cell culture medium was added, and after 48 hours, the virus titer was determined by FACS analysis. The virus titer was calculated by the following equation.

Virus titer (TU/mL)=Number of cells×Percentage (%) of FACS positive cells×Dilution factor×2  [Equation 1]

[0124] As a result, it was confirmed that the virus titer of MSLN34-CAR scFv was 9.6×10.sup.7 TU/mL, and the virus titer of MSLN38-CAR scFv was 1.6×10.sup.8 TU/mL.

Example 8: Preparation of Anti-MSLN-CAR-Introduced Cells

[0125] 8-1: Lentivirus Transduction

[0126] Transduction was performed a total of twice. Anti-CD3 (1 μg/mL) and anti-CD28 (3 μg/mL) antibodies were prepared at a predetermined concentration in 5 mL of DPBS, followed by vortexing. Then, each antibody was coated onto a 24-well plate at a density of 500 μl/well, and stored in a refrigerator at 4° C. overnight. Next day, PBMC (human primary PBMC) was dissolved in 9 mL of T cell culture medium (10% FBS+RPMI1640+200 IU IL-2), and centrifuged at 1,500 rpm for 5 minutes. Thereafter, the supernatant was removed, and the resultant was resuspended in 1 mL of a culture medium, followed by cell counting. After dilution to 1×10.sup.6 cells/mL, cells were seeded in the antibody-coated 24-well plate, and then incubated in a CO.sub.2 incubator at 37° C. After 3 days, all PBMC cells were harvested. For lentivirus infection, of 5 in 5×10.sup.5 of lentivirus was adjusted at multiple of infection (MOI) of 5, and 10 μg/mL of protamine sulfate was added to cells, which were then seeded in a new 24-well plate (a). The 24-well plate was centrifuged at 300 g, 32° C. for 90 minutes, and then incubated in a CO.sub.2 incubator at 37° C. (b). Next day, all T cells were harvested and the above (a) and (b) were performed once more. Then, all T cells were harvested and centrifuged at 1,500 rpm for 5 minutes to remove the supernatant, and the T cells were resuspended in the culture medium and cultured again.

[0127] 8-2: Examination of Anti-MSLN-CAR Expression

[0128] The presence or absence of CAR expression was examined in T cells into which the anti-MSLN-CAR prepared in Example 8-1 was introduced. Five days after the completion of lentivirus transduction of T cells, a portion of anti-MSLN-CAR-T cells were harvested, and biotin-MSLN (Acrobiosystems or Biolegend) was added thereto, followed by incubation on ice for 20 minutes. Then, cells were washed, and 1 μL of PE-anti-biotin was added, followed by incubation on ice for 20 minutes. After washing the cells, an expression rate of CAR was examined using FACS Canto II (BD). Further, the expression of finally differentiated T cells (CD3) was analyzed by FACS while incubating anti-MSLN-CAR-T for 14 days, and a percentage of CD4+ and CD8+ T cells in CD3-positive T cells was measured. The results are shown in Table 11 below and FIGS. 10 and 11.

TABLE-US-00012 TABLE 11 Round of CD3-positive T transduction Clone cells % CAR expression % 1.sup.st round CD19 (FMC63) 93.3% 29.2% 1.sup.st round MSLN34 CAR scFv 94.3% 22.4% 1.sup.st round MSLN38 CAR scFv 97.3% 8.99% 2.sup.nd round CD19(FMC63) 91.2% 30.9% 2.sup.nd round MSLN34 CAR scFv 92.9% 27.2% 2.sup.nd round MSLN38 CAR scFv 91.3% 27.9%

[0129] As shown in FIGS. 10 and 11, as a result of the 1.sup.st round of transduction, a percentage of CD4+:CD8+ was 20%:70% on average, and as a result of the 2.sup.nd round of transduction, a percentage of CD4+:CD8+ was 10%:80%.

Example 9: Examination of Cell-Killing Effects of Anti-MSLN-CAR-T Cells

[0130] Cell-killing effects on cancer cells were examined using the anti-MSLN-CAR-T cells prepared in Example 8 by a Calcein-AM assay.

[0131] First, to examine MSLN expression levels of various cancer cell lines (AsPC-1, MIA PaCa-2, NCI-H2052, and OVCAR-3), a portion of the cells was taken during culture, and bound with biotin-anti-MSLN antibodies, followed by FACS analysis. As a result, MSLN expression was observed all in AsPC-1 which is a pancreatic cancer cell line, OVCAR-3 which is an ovarian cancer cell line, and NCI-H2052 which is a malignant pleural mesothelioma cell line. However, MIA PaCa-2 which is a pancreatic cancer cell line showed significantly low MSLN expression, as compared with other cancer cell lines (FIG. 12).

[0132] The cancer cell lines (AsPC-1, MIA PaCa-2, NCI-H2052, and OVCAR-3) were resuspended in each culture medium at a density of 1×10.sup.6 cells/mL, 5 μL of calcein-AM (1 mg/mL) was added, and mixed well, followed by incubation for 1 hour in a 37° C. incubator. CD19-CAR-T cells and anti-MSLN-CAR-T cells which are effector cells were prepared by diluting at various E:T (effector cell:target cell) ratios while adding the cell culture medium. 1 hour after calcein-AM staining of the cancer cell lines, centrifugation was performed at 1,200 rpm for 5 minutes, followed by washing and resuspending by adding 10 mL of culture medium. Then, 100 μL (1×10.sup.4 cells/100 μL) of the stained cancer cell line was seeded in a 96-well round plate, and 100 μL of effector cells were seeded thereon. As a control group, a calcein-AM-stained cancer cell line treated with only 100 μL of culture medium (spontaneous value) or treated with 2% Triton X-100 (maximum value) was used. The 96-well round plate was centrifuged at 100 g for 1 minute, and then incubated for 4 hours in a 37° C. incubator. After 4 hours, the cells in the well were mixed five times with a pipette, centrifuged at 100 g for 5 minutes, and 100 μL of only the supernatant was taken and transferred to an assay 96-well plate. Calcein emission was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm with a fluorescent microplate reader using the 96-well plate containing the supernatant. The cell killing effect was calculated using the measured values according to the following equation. The results are shown in FIGS. 13 to 15.

Cell killing effect (%)=(Experimental release−Spontaneous release)/(Maximum release−Spontaneous release)×100  [Equation 2]

[0133] As shown in FIGS. 13 to 15, both anti-MSLN34-CAR-T and anti-MSLN38-CAR-T showed a significant antigen-specific cell-killing effect on the ovarian cancer cell line OVCAR-3 and the malignant pleural mesothelioma cell line NCI-H2052, in which high MSLN expression was confirmed, as compared with the negative control CD19(FMC63)-CAR-T. However, both anti-MSLN34-CAR-T and anti-MSLN38-CAR-T showed no specific cell-killing effect on MIA PaCa-2 which is a cancer cell line showing low MSLN expression, as compared with the negative control. These results confirmed that the cell-killing effect of the anti-MSLN-CAR-T cells according to one aspect is specific to mesothelin expressed in cancer cells.

[0134] The cell killing effects on cancer cells were also examined using the anti-MSLN-CAR-T cells prepared in Example 8 and a cancer cell line expressing green fluorescent protein (GFP). AsPC-1 which is a GFP-expressing pancreatic cancer cell line was resuspended in a culture medium at a density of 1×10.sup.6 cells/mL, and incubated. Then, the effector cells, CD19-CAR-T cells and anti-MSLN-CAR-T cells, were added to the cell culture medium, and co-cultured at an E:T ratio of 10:1. Results of measuring GFP up to 48 hours in real-time using an incucyte are shown in FIG. 16. As shown in FIG. 16, it was confirmed that the cell killing effect on the pancreatic cancer cell line AsPC-1 was observed according to the treatment with the anti-MSLN-CAR-T cells according to an aspect.

Example 10: Examination of Cancer Cell-Killing Effect of Anti-MSLN-CAR-T Cells, Based on Tumor Animal Model

[0135] Based on the cell-killing effect of anti-MSLN-CAR-T cells on cancer cells, as confirmed in Example 9, a tumor animal model was constructed and the tumor killing ability was examined.

[0136] In this experiment, 5-week-old male NOG (NOD/Shi-scid/IL-2Rγnull) mice were used. When the animals were supplied, the inspection and quarantine of the animals was conducted with reference to the health monitoring report of the test system provided by the supplier. After acclimatization for a week, the experiment was conducted. The breeding environment for this experiment was as follows: a temperature of 22° C.±2° C., relative humidity of 50%±10%, ventilation of 10 times to 20 times/hr, lighting time of 12 hours (light-up at 8 am˜light-out at 8 pm), and illuminance of 150 Lux to 300 Lux. After autoclaving chip-type bedding materials (121° C., sterilization time of 20 minutes, drying time of 5 minutes), an appropriate amount of the chip-type bedding materials was placed in a polycarbonate breeding box (W 278 (mm)×L 420 (mm)×H 230 (mm)) to breed the mice. A feed supplied during the experiment was a solid feed for laboratory animals, sterilized by irradiation (+40 RMM-SP-10, U8239G10R, SAFE-DIETS, France), and RO water in a water bottle was sterilized by autoclaving, and mice were allowed free access to the water.

[0137] Cells used in pancreatic cancer and mesothelioma animal models were tested for Mycoplasma pneumoniae, Murine coronavirus (Mouse hepatitis virus, MHV), and Murine respirovirus (Sendai virus, SeV), and the cells were confirmed to be negative before use. The compositions of transplanted cancer cells and CAR-T cells and test groups are shown in Table 12 below.

TABLE-US-00013 TABLE 12 N Cell line Administration Administration Dose Group (number) (cells/mouse) Material Route (CAR-Ts/mouse) Volume G1 6 AsPC-1 HBSS I.V — 200 uL G2 6 (5 × 10.sup.6) Mock — G3 6 CD19-CAR-T 5 × 10.sup.6 G4 6 Anti-MSLN34-CAR-T 5 × 10.sup.6 G5 6 Anti-MSLN38-CAR-T 5 × 10.sup.6 G6 6 NCI-H2052 HBSS — G7 6 (1 × 10.sup.7) Mock — G8 6 CD19-CAR-T 5 × 10.sup.6 G9 6 Anti-MSLN34-CAR-T 5 × 10.sup.6 G10 6 Anti-MSLN38-CAR-T 5 × 10.sup.6

[0138] Concentrations of the cells were adjusted using PBS, and each 200 uL thereof was subcutaneously transplanted into mice. Groups were divided according to tumor size by randomization. Individual identification was performed using an ear-punch method during the experiment period, and an identification card for each group was attached to the breeding box.

[0139] After dividing the experimental groups, anti-MSLN-CAR-T cells were administered once via the tail vein, and the body weight and tumor size of the experimental groups were measured twice a week from the beginning of administration. Based on the body weight on the beginning day of administration, changes in the body weight were observed until the end of the experiment. Body weight gain or loss (%) was calculated using the following equation.

Body weight gain or loss (%)=(Body weight/Body weight on day 0)×100  [Equation 3]

[0140] The tumor volume (mm.sup.3) was calculated using the following equation after measuring the short axis (A) and long axis (B) of the tumor using calipers.

Tumor volume (mm.sup.3)=½×[{A (mm)}.sup.2×B (mm)]  [Equation 4]

[0141] After the last measurement, the body weight and tumor volume were statistically analyzed using a post-hoc Dunnett's test of one-way ANOVA by comparing HBSS-administered groups and anti-MSLN-CAR-T-administered groups, each for pancreatic cancer and mesothelioma (*:p<0.05, **:p<0.01, ***:p<0.001).

[0142] As a result of validation of the tumor-killing ability of anti-MSLN-CAR-T cells in the mesothelioma (NCI-H2052) model, no tumors were observed in all animals of the anti-MSLN34-CAR-T-administered groups and the anti-MSLN38-CAR-T-administered groups, except for one individual (G4-4). As compared with the HBSS-administered group, the tumor size was significantly reduced in the anti-MSLN34-CAR-T-administered groups and the anti-MSLN38-CAR-T-administered groups from the 13.sup.th day of administration (p<0.05). As compared with the control group (G1, HBSS-administered group), the anti-MSLN38-CAR-T-administered group showed weight loss (p<0.05) and convulsions, and two mice died on the 20.sup.th day after administration. The results are shown in FIG. 17.

[0143] As a result of validation of the tumor-killing ability of anti-MSLN-CAR-T cells in the pancreatic cancer (AsPC-1) model, the tumor size was significantly reduced in the anti-MSLN34-CAR-T-administered groups and the anti-MSLN38-CAR-T-administered groups from the 13.sup.th day of administration (p<0.05), as compared with the HBSS-administered group. At autopsy, the tumor weight decreased in both groups at the same time (p<0.05). As compared with the control group (G6, HBSS-administered group), the anti-MSLN38-CAR-T-administered group showed weight loss (p<0.05) and convulsions, and one mouse died on the 34.sup.th day after administration. The results are shown in FIG. 18.

[0144] The above results taken together, it was confirmed that both anti-MSLN34-CAR-T and anti-MSLN38-CAR-T show the cancer cell-killing effects on cancer cells of both pancreatic cancer and mesothelioma as well as on animal models thereof.

[0145] The above description is for illustrating, and it will be understood by those skilled in the art that the present disclosure may be implemented in a different specific form without changing the technical spirit or essential characteristics thereof. Therefore, it should be understood that the embodiments described herein are not limitative, but illustrative in all aspects.