Chimeric antigen receptor comprising anti-mesothelin scFv, and use thereof

Abstract

Provided is an anti-mesothelin chimeric antigen receptor that specifically binds to mesothelin. The anti-mesothelin chimeric antigen receptor according to an aspect exhibits a specific binding ability for mesothelin, and thus, may be useful for preventing or treating cancer in which mesothelin is overexpressed.

Claims

1. An anti-mesothelin antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region comprising 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; and a heavy chain CDR3 comprising an amino acid sequence consisting of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence consisting of SEQ ID NO: 4; 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.

2. The anti-mesothelin antibody or an antigen-binding fragment thereof according to claim 1, wherein the anti-mesothelin antibody or an antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 7.

3. The anti-mesothelin antibody or an antigen-binding fragment thereof according to claim 1, wherein the anti-mesothelin antibody or an antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 8.

4. The anti-mesothelin antibody or an antigen-binding fragment thereof according to claim 1, wherein the anti-mesothelin antibody or an antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 8.

5. An isolated nucleic acid encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-4.

6. A vector comprising an isolated nucleic acid according to claim 5.

7. An isolated host cell transformed with a vector according to claim 6.

8. A method for preparing an anti-mesothelin antibody or an antigen binding fragment thereof, comprising culturing a host cell according to claim 7 to express an antibody or an antigen binding fragment thereof.

9. 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 an antigen-binding fragment thereof, comprising: a heavy chain variable region comprising 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; and a heavy chain CDR3 comprising an amino acid sequence consisting of SEQ ID NO: 3; and a light chain variable region comprising 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.

10. The chimeric antigen receptor according to claim 9, wherein the antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 8.

11. The chimeric antigen receptor according to claim 9, wherein the antigen-binding domain is a scFv (single chain variable fragment).

12. A nucleotide encoding a chimeric antigen receptor according to claim 9.

13. A vector comprising a nucleotide according to claim 12.

14. An isolated cell transformed with a vector according to claim 13.

15. The isolated cell according to claim 14, wherein the cell is a T cell, an NK cell, an NKT cell or a gamma delta () T cell.

16. A pharmaceutical composition for treating cancer, comprising an isolated cell according to claim 15.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a diagram schematically illustrating a process of antibody screening through phage display antibody library panning.

(2) FIG. 2 shows results of solid phase panning, in which FIG. 2A is a diagram showing phage output titers according to panning rounds, and FIG. 2B is a diagram showing elution titer ratios (B), according to panning rounds.

(3) FIG. 3 is a diagram showing results of a comparative analysis of specific binding for an MSLN antigen of clones obtained through phage ELISA.

(4) FIG. 4 is a diagram showing results of confirming through flow cytometry whether clones selected by using a mesothelin-overexpressing cell line bind to mesothelin present in actual cell membranes.

(5) FIG. 5 is a diagram showing binding specificity for mesothelin of clones selected by using a mesothelin-overexpressing cell line, with relative peak shift values.

(6) FIG. 6 is a diagram showing results of SDS-PAGE analyses of purified anti-MSLN-scFv antibodies (loading 2 g of each protein). NR: Non-reducing condition, R: Reducing condition (100 C., 10 minutes).

(7) FIG. 7 is a diagram showing results of analyzing the affinity of anti-MSLN-scFv antibodies for an MSLN antigen through ELISA.

(8) FIG. 8 is a schematic diagram of an anti-MSLN-CAR expression system according to an aspect, including an MSLN-specific antigen-binding domain.

(9) FIG. 9A is results of confirming expression of CAR in T cells into which anti-MSLN-CAR was introduced; and FIG. 9B is a diagram showing results of measuring ratios of CD4+ and CD8+ T cells among CD3 positive T cells.

(10) FIG. 10 is a diagram confirming changes by date in body weight of ovarian cancer (OVCAR-3) mouse models administered with HBSS (G1), Mock (G2), CD19-CAR-T (G3), and CAR-T cells (G4) into which anti-MSLN3-CAR was introduced.

(11) FIG. 11 is a diagram confirming changes in tumor volumes by date in ovarian cancer (OVCAR-3) mouse models administered with HBSS (G1), Mock (G2), CD19-CAR-T (G3), and CAR-T cells (G4) into which anti-MSLN3-CAR was introduced (*p<0.05 vs. the vehicle control treated group (G1))

(12) FIG. 12 is a diagram confirming tumor volumes on day 27 in ovarian cancer (OVCAR-3) mouse models administered with HBSS (G1), Mock (G2), CD19-CAR-T (G3), and CAR-T cells (G4) into which anti-MSLN3-CAR was introduced.

(13) FIG. 13 is a diagram confirming tumor weights on day 27 in ovarian cancer (OVCAR-3) mouse models administered with HBSS (G1), Mock (G2), CD19-CAR-T (G3), and CAR-T cells (G4) into which anti-MSLN3-CAR was introduced.

(14) FIG. 14 is a diagram showing results of confirming results of immunohistochemical staining of ovarian cancer (OVCAR-3) mouse models administered with HBSS (G1), Mock (G2), CD19-CAR-T (G3), and CAR-T cells (G4) into which anti-MSLN3-CAR as introduced, at a magnification of 5.

(15) FIG. 15 is a diagram showing results of confirming results of immunohistochemical staining of ovarian cancer (OVCAR-3) mouse models administered with HBSS (G1), Mock (G2), CD19-CAR-T (G3), and CAR-T cells (G4) into which anti-MSLN3-CAR was introduced, at a magnification of 20.

MODE FOR INVENTION

(16) Hereinafter, an aspect will be described in more detail through examples. However, these examples are intended to illustrate an aspect by way of example, and the scope of the aspect is not limited to these examples, and an example of an aspect provides a more complete description of the aspect to those skilled in the art.

Example 1: Phage Display Antibody Library Panning

(17) In order to select an antibody that binds to mesothelin (MSLN), which is a target antigen, phage panning for MSLN (Acro Biosystems) was performed up to 4 rounds by using the KBIO human synthetic scFv phage display library KscFv-I, according to the phage panning protocol established by the New Drug Development Support Center of the Osong Advanced Medical Industry Promotion Foundation. The phage display antibody library panning process is schematically shown in FIG. 1.

(18) Panning was performed in two ways (solid and bead) depending on a method of immobilizing the antigen. For solid phase panning, 1 mL of human mesothelin proteins (in PBS, 1st round: 10 g/mL, 2nd round: 5 g/mL, 3rd round: 2.5 g/mL, and 4th round: 1.25 g/mL) were immobilized on an immunotube, and 1.310.sup.13 c.f.u. of the phage library blocked with 5 mL of PBS (MPBS) including 5% skim milk was added to the immunotube, and then bound at 37 C. for 1.5 hours. Then, unbound phages were removed by washing with 5 mL of PBS-Tween20 (0.05%) (PBS-T) (1st round: washed 3 times, 2nd to 4th rounds: washed 5 times). Here, 1 mL of 100 mM trimethylamine (TEA) was added to the tube and reacted at room temperature for 10 minutes to elute the bound phages, and the eluted phages were moved to a 50 mL falcon tube, and mixed well with 0.5 mL of 1 M Tris-HCl (pH 7.4) to neutralize. The eluted phages were infected into 8.5 mL of mid-log phase E. coli TG1 (OD600=0.5 to 0.8). Some of the transfected E. coli TG1 were subjected to extraction of plasmid DNA for sequence confirmation, and some were subjected to antibody screening through phage ELISA. The results are shown in FIGS. 2A and 2B.

(19) As a result, as shown in FIG. 2, the samples started to concentrate from the third round of solid phase panning, and the yield titer for the MSLN antigen showed a large difference of about 53.4 times (third round) and 1061.6 times (fourth round) greater values compared to the PBS control group.

Example 2: Selection of Positive Clones Through Phage-Specific ELISA

(20) In order to select clones that specifically bind to the MSLN antigen among the phages obtained by the phage panning of Example 1, a monoclonal phage ELISA was performed for 470 (94 colonies5 lates) clones obtained from the third round of panning by using an immunotube. Specifically, 30 L per well of 1 g/mL human MSLN proteins (antigen) was added to a 96-half-well ELISA plate, followed by incubation at 4 C. overnight for coating. As a negative control group, 30 L per well of PBS was added to another plate and incubated overnight at 4 C. The next day, the contents of the plate were removed and the plate was blocked with 150 L of 5% MPBS for 1 hour at room temperature. Then, the contents of the plate were removed, and 30 L of phages (10 c.f.u.) was added and incubated for 1.5 hours at room temperature. For a negative control group, 30 L of PBS was added instead of the phages. The plate was washed 4 times with a PBS-T (PBS-0.05% Tween 20) solution, anti-M13-HRP (diluted to 1:5,000 in PBS) was added and incubated at 37 C. for 1 hour. The plate was washed 4 times with the PBS-T solution, and 30 L of TMB substrate reagent was added to each well and incubated for 8 minutes at room temperature to induce a color reaction. After stopping the color reaction by adding 30 L of 2 N H.sub.2SO.sub.4 per well, absorbance (O.D.) at 450 nm was measured.

(21) As a result, a total of 105 positive clones were secured in the third round when screening was performed by respectively setting the absorbance cut-off to 0.7 or higher, or 0.5 or higher, for the MSLN antigen. Additionally, monoclonal phage ELISA was performed in the same manner for the clones obtained in the fourth round of panning by using an immunotube. As a result of performing phage ELISA for 282 (94 colonies3 plates) clones obtained in the 4th round of panning, and screening by setting each of the absorbance cut-off to 0.7 or higher, a total of 15 positive clones were secured (Tables 1 to 3).

(22) TABLE-US-00002 TABLE 1 Panning at Number of Number of 3rd round Absorbance positive unique (Round 3) (450 nm) clones clones 3R-1 >0.7 35 6 3R-2 >0.7 4 1 3R-3 >0.7 8 2 3R-4 >0.5 34 9 3R-5 >0.5 24 2 Total sum 105 20

(23) TABLE-US-00003 TABLE 2 Panning at 4th Number of Number of round Absorbance positive unique (Round 4) (450 nm) clones clones 4R-1 >0.7 6 1 4R-2 >0.7 2 0 4R-3 >0.7 7 1 Total sum 15 2

(24) TABLE-US-00004 TABLE 3 Panning at 3rd and 4th rounds Total sum Number of positive clones 120 Number of unique clones 22

Example 3: Sequence Analysis and ELISA for Screening Anti-MSLN Antibody Fragment Candidates

(25) After recovering phages from a total of 120 positive clones selected in Example 2, a DNA sequence analysis was proceeded, and the sequences were aligned and grouped according to the Kabat numbering system. As a result, 22 unique clones for MSLN antigens with different CDR sequences were selected. In order to confirm specific binding of the 22 clones to the MSLN antigen, each phage was purified and the phage titer was equalized (3.3E+11 pfu/well), and then the phages were compared through ELISA. As a negative control, a TLR4 antigen conjugated to a histidine tag (His tag) was used in the same way as with MSLN. The results are shown in FIG. 3.

(26) As shown in FIG. 3, it was confirmed that 19 clones excluding MSLN1, MSLN18, and MSLN19 among the 22 clones specifically bind to the MSLN antigen.

Example 4: Confirmation of Binding Ability by Using Mesothelin-Overexpressing Cell Lines

(27) In order to know whether the 19 types of phage clones selected in Example 3 bind to mesothelin present in the actual cell membrane, a flow cytometry analysis was performed by using a pancreatic cancer cell line AsPC-1, which is a mesothelin-overexpressing cell line, and a human chronic myeloid leukemia cell line K562 as a control group.

(28) Specifically, K562 and AsPC-1 cells were prepared to be 10.sup.6 cells/well and washed with 300 L of PBS. The cells were blocked with 300 L of 4% MPBS at 4 C. for 30 minutes, and at the same time, the phage clones (10 2/well) were also blocked for 1 hour at room temperature, and then the phages and cells were incubated together at 4 C. for 2 hours. After washing the cells with PBS, the cells were treated with 1 g/mL of anti-M13-FITC and incubated at 4 C. for 1 hour. After washing the cells with PBS, the cells were resuspended in PBS and the results were analyzed by using a flow cytometer (BD biosciences). The results are shown in FIGS. 4 and 5.

(29) As shown in FIG. 4, it was confirmed that MSLN3, MSLN6, and MSLN10 exhibited a relative peak shift value of 3.0% or more in the pancreatic cancer cell line AsPC-1. In the K562 cell line, which is a control group, no significant peak shift was observed. From the above results, it was found that among the 19 clones, MSLN3, MSLN6 and MSLN10 showed high binding affinity to mesothelin present in the actual cell membrane.

(30) In addition, as shown in FIG. 5, as a result of quantifying flow cytometry results, it was confirmed that MSLN3, MSLN6, and MSLN10 showed relative peak shift values of 23.8%, 7.2%, and 4.8%, respectively, compared to the K562 cells, which is the control group. Through the above results, all three clones were confirmed to specifically bind to the cell line in which mesothelin is overexpressed, and finally selected as clones for producing anti-MSLN antibody fragments.

Example 5: Anti-MSLN Antibody Fragment Production and Purification

(31) By using the three clones selected in Example 4, Top10F competent E. coli, a strain expressing antibody fragments, was transformed. Then, each of the E. coli strains transformed with the three clones was cultured in 200 mL of TB medium, protein expression was induced with IPTG (final concentration: 0.5 mM), and the E. coli strains were cultured overnight at 30 C. The cells were obtained by centrifuging the culture medium, and after securing water-soluble proteins through periplasmic extraction, anti-MSLN-scFv antibodies were purified through affinity chromatography by using protein L resin. The purified antibody proteins were analyzed by SDS-PAGE, and the results are shown in FIG. 6.

Example 6: Analysis of Affinity of Anti-MSLN Antibodies for Antigens and Selection of Clones to be Used as Final Anti-MSLN Chimeric Antigen Receptors

(32) Affinity of each antibody to the MSLN antigen was compared and analyzed through ELISA by using the three types of anti-MSLN antibody proteins prepared in Example 5 above. Specifically, 30 L of human mesothelin proteins were coated on a MaxiSorb ELISA plate (Nunc) to a concentration of 1 g/mL per well, and incubated overnight at 4 C. The contents of the plate were removed and the plate was blocked with 300 L of 5% MPBS for 1 hour at room temperature. The purified antibodies were serially diluted in PBS, and added to each well by 30 L and incubated for 2 hours at room temperature. For a negative control group, 60 L of PBS was added instead of the purified antibodies and incubated at 37 C. for 2 hours.

(33) The plate was washed 4 times with a PBS-T (PBS-0.05% Tween 20) solution, 30 L of anti-StrepMAB HRP (diluted to 1:5,000 in PBS) was added and incubated at room temperature for 1 hour. The plate was washed 4 times with the PBS-T solution, and 30 L of TMB substrate reagent was added to each well and incubated for 8 minutes at room temperature to induce a color reaction. After stopping the color reaction by adding 30 L of 2 N H.sub.2SO.sub.4 per well, absorbance (O.D.) at 450 nm was measured. The results are shown in FIG. 7.

(34) As shown in FIG. 7, EC.sub.50 values of the three types of antibodies were 145 nM, 67 nM, and 91 nM for MSLN3, MSLN6, and MSLN10, respectively, confirming that MSLN3 showed the highest binding affinity among the three antibodies (see Table 4).

(35) TABLE-US-00005 TABLE 4 Ranking MSLN clone R.sup.2 EC.sub.50 (nM) 1 3 0.999 145 2 10 0.988 91 3 6 0.988 67

(36) Accordingly, the MSLN3 was finally selected as a clone for producing anti-MSLN antibody fragments, and the amino acid sequence of MSLN3 was confirmed and shown in Table 5 below. Specifically, the heavy chain CDR1-3 amino acid sequences of MSLN3 are respectively shown in SEQ ID NOS: 1 to 3, the light chain CDR1-3 amino acid sequences are respectively shown in SEQ ID NOS: 4 to 6, and the heavy chain amino acid sequence and light chain amino acid sequence are respectively shown in SEQ ID NOS: 7 and 8.

(37) TABLE-US-00006 TABLE5 HeavychainCDR1-3andlightchainCDR1-3amino acidsequencesofMSLN3ofMSLN3clones SEQID Region Aminoacidsequence NO: HCDR1 DYAMS 1 HCDR2 AISSSGGTTYYADSVKG 2 HCDR3 EEEGEWREYFDV 3 LCDR1 RASQSISSYLN 4 LCDR2 ATSTLQS 5 LCDR3 QQSYTFPYT 6 VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSW 7 VRQAPGKGLEWVSAISSSGGTTYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCAKEEEGEWREYF DVWGQGTLVTVSS VL DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWY 8 KQQKPGAPKLLIYATSTLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQSYTFPYTFGQGTKVEIK

Example 7: Construction of Anti-MSLN Chimeric Antigen Receptor

(38) An anti-MSLN chimeric antigen receptor was constructed (anti-MSLN-CAR) based on MSLN3, which showed high binding specificity with a cell line overexpressing mesothelin among the anti-MSLN antibody proteins prepared in Example 6.

(39) 7-1: Cloning of Anti-MSLN-CAR Lentiviral Vectors

(40) The vector belongs to the 2nd generation CAR lentiviral vector (pLV lentiviral vector) system retained by the New Drug Development Support Center, and the system includes all of pMDLg/pRRE (addgene) encoding gag/pol, an envelope plasmid pRSV-Rev (addgene) encoding Rev proteins, and an envelope plasmid pMD2.G (addgene) encoding VSV-G proteins.

(41) First, gene cloning was performed on the anti-MSLN scFv (antigen-binding domain) prepared in Example 6. Each anti-MSLN scFv of MSLN3 and lentiviral vectors were digested with Xhol (R0146S, NEB) and EcoRI (R0101, NEB) at 37 C. for 2 hours, subjected to agarose gel electrophoresis, and the identified products were purified by using the FavorPrep Gel/PCR purification Mini kit (Favorgen). Each purified anti-MSLN scFv (100 ng) and the vectors (50 ng) were reacted at a ratio of 2:1 at 16 C. for 16 hours for ligation, and then Stbl3 competent cells were transformed by the vectors, and then colonies were obtained. The colonies were taken and cultured in 5 mL of LB medium (ampicillin), and then plasmid DNA was obtained by using a DNA plasmid mini-prep method. The plasmid DNA was digested with Xhol and EcoRI to confirm whether each inserted anti-MSLN scFv was well cloned into the vector. Afterwards, sequencing was performed to finally confirm the DNA sequence.

(42) Anti-MSLN-CAR was constructed by sequentially connecting to the anti-MSLN scFv, a CD8 hinge and a CD8 transmembrane (TM) as transmembrane regions, a cytoplasmic region of 4-1BB as a signaling domain, and an intracellular domain of CD3 zeta (CD3z) as a T cell activation domain. Specifically, anti-MSLN-CAR consists of a CD8 signal peptide (SP) (SEQ ID NO: 10), anti-MSLN3 scFv (SEQ ID NO: 11), a CD8 hinge region (SEQ ID NO: 12), a CD8 transmembrane region (SEQ ID NO: 13), a 4-1BB signaling domain (SEQ ID NO: 14), and a CD3 zeta signaling domain (SEQ ID NO: 15). Each of the above domains was sequentially linked by using each restriction enzyme, and specific nucleotide sequence information corresponding to each domain is summarized in Table 6 below.

(43) TABLE-US-00007 TABLE6 SEQ ID Name Nucleotidesequence(5-3) NO: CD8 ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGC 10 CTTGCTGCTCCACGCCGCCAGGCCG MSLN3 GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCA 11 scFv ACCGGGTGGTTCACTGCGTCTGAGCTGCGCCGCCTCGG GTTTTACTTTCTCTGATTATGCAATGTCTTGGGTTCGT CAGGCGCCGGGCAAGGGTCTCGAATGGGTTTCAGCAAT CTCTTCTTCTGGTGGTACTACTTACTATGCCGATTCAG TGAAGGGTCGCTTTACCATTTCCCGTGACAACTCTAAG AATACTCTGTATCTGCAGATGAACTCGCTGCGTGCCGA AGACACGGCCGTCTATTATTGCGCCAAAGAAGAAGAAG CGTGAATGGCGTGAATACTTCGATGTTTGGGGTAGGGC ACTTTAGTGACCGTCTCATCGGGTGGAGGCGGTTCAGG CGGAGGTGGATCCGGCGGTGGCGGATCGGACATTCAAA TGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGC GATCGTGTGACAATTACTTGTCGCGCTAGCCAGTCTAT CTCTTCTTACCTGAACTGGTATCAGCAGAAACCGGGCA AGGCGCCAAAATTGCTGATTTACGCAACTTCCACTCTG CAGTCTGGTGTACCGTCCCGTTTCTCTGGCAGCGGTTC TGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGC CTGAAGATTTTGCCACCTATTATTGTCAGCAATCTTAC ACTTTTCCGTACACGTTCGGGCAGGGAACTAAAGTGGA AATTAAAGCCAGCACC CD8 ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCC 12 hinge CACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGG CCGTGCGGCCAGCGGGGGGGGCGCAGTGCACACGAGGG GGCTGGACTTCGCCTGTGAT CD8 ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGT 13 TM CCTTCTCCTGTCACTGGTTATCACCCTTTACTGC 4-1BB AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACA 14 TACCATTATGAGACCAGTACAAACTACTCAAGAGGAAG ATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGA GGATGTGAACTG CD3z AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTA 15 CACAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAAT CTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAG ACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAA GGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAG AAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGAT GGAAAGGCGAGCGCCGGAGGGCAAGGGGCACGATGGCC TTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTAC GACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

(44) 7-2: Production of Lentiviruses Loaded with Anti-MSLN-CAR

(45) Anti-MSLN-CAR lentiviruses were produced by introducing the recombinant vectors prepared in Example 7-1 into HEK293T cells. FIG. 8 shows a schematic diagram of an anti-MSLN-CAR expression system according to an aspect including an MSLN-specific antigen-binding domain. First, the day before DNA transfection, HEK293T cells were seeded in a 100 mm tissue culture dish at 610.sup.6 cells/dish. The next day, when cell density reached 70% to 80%, the cells were transformed with MSLN-CAR-pLV, pMDLg/pRRE (addgene), pRSV-Rev (addgene), pMD2.G (addgene) (5.5 g:3.5 g:1.5 g:2 g), by using lipofectamine 3000 (Thermofisher) according to instructions of the reagent. CD19 (FMC63) was used as a control group. 4 hours after the transformation, the medium was replaced with DMEM medium including 3% FBS (Gibco), and after 48 hours, the viral culture medium was harvested. 10 mL of a 20% sucrose solution was put into a centrifugal separation tube, and 20 mL of the harvested viral culture medium was carefully placed thereon, and the centrifugal separation tube was mounted on a SW32T rotor, and ultra-high-speed centrifugation was performed at 25,000 rpm at 4 C. for 90 minutes. After the centrifugation, the supernatant was carefully discarded not to drop the viral pellet at the bottom of the tube, 400 L of RPMI1640 medium (Gibco) was added, and the sample was incubated in the refrigerator for 16 hours, resuspend, and divided into 100 L each, and stored at 80 C.

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

(46) 8-1: Lentiviral Transduction

(47) Anti-CD3 (1 g/mL) and anti-CD28 (3 g/mL) antibodies were prepared in 5 mL of DPBS to the designated concentrations, vortexed, and then coated on a 24-well plate at 500 l/well and stored overnight in a refrigerator at 4 C. The next day, PBMC (primary human 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 remainder was resuspended in 1 mL of the culture medium, and the cells were counted, then the sample was diluted to 110.sup.6 cells/mL, seeded in an antibody-coated 24-well plate, and cultured in a CO.sub.2 incubator at 37 C. After 3 days, all the PBMC cells were harvested, and lentiviruses were adjusted to multiple of infection (MOI) of 5 in cells at a concentration of 510.sup.5 cells/500 L for lentiviral infection, and 10 g/mL of protamine sulfate was added, and the sample was seeded in a new 24-well plate (a). The 24-well plate was centrifuged at 300 g and 32 C. for 90 minutes, and then placed in a CO.sub.2 incubator at 37 C. and cultured (b). The next day, all T cells were harvested and processes (a) and (b) were performed once more. Then, all the T cells were harvested, 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.

(48) 8-2: Confirmation of Expression of Anti-MSLN-CAR

(49) Whether or not CAR was expressed was confirmed in the T cells into which the anti-MSLN-CAR prepared in Example 8-1 was introduced. Five days after the completion of lentiviral transduction of the T cells, some anti-MSLN-CAR-T cells were harvested, biotin-MSLN (Acrobiosystems or Biolegend) was added, and the cells were incubated on ice for 20 minutes, and then washed, and 1 L of PE-anti-biotin was added and incubated on ice for 20 minutes. After washing the cells, CAR expression rates were confirmed by using FACS Canto II (BD), and the results are shown in FIG. 9A and Table 7. In addition, while culturing anti-MSLN-CAR-T for 14 days, expression of ultimately differentiated T cells (CD3) was analyzed by FACS, and the ratio of CD4+ and CD8+ T cells among CD3 positive T cells was measured, and the results are shown in FIG. 9B.

(50) TABLE-US-00008 TABLE 7 CAR Clone expression % CD19 (FMC63) 50.8% MSLN3 CAR scFv 30.6%

(51) As confirmed in Table 7 and FIG. 9, as a result of confirming CAR expression rates of the prepared CAR-T, it was confirmed that the control group CD19 (FMC63)-CAR-T had a CAR expression rate of 50.8%, and MSLN3-CAR-T had an expression rate of 30.6%. As shown in FIG. 9, the ratio of CD4+:CD8+ as a result of the first round of transduction was measured to be 10%:80% on average.

Example 9: Confirmation of Cancer Cell Killing Effect of Anti-MSLN-CAR-T Cells Based on Tumor Animal Models

(52) 9-1: Administration of Anti-MSLN-CAR-T Cells to Tumor Animal Models

(53) An experiment was performed to confirm effectiveness of the MSLN3-CAR T cell therapeutic agent in ovarian cancer (OVCAR 3) animal models. Specifically, based on the cell killing effect of anti-MSLN3-CAR-T cells on cancer cells confirmed in Example 9, tumor ovarian cancer animal models were constructed to confirm the tumor killing ability.

(54) In this test, 6-week-old female NOG (NOD/Shi-scid/IL-2Rnull) specific-pathogen-free (SPF) mice were used, inspection and quarantine of the obtained animals were carried out by referring to the health monitoring report of the test system provided by the supplier, and the experiment was conducted after a week of acclimatization. The breeding environment for this test was at a temperature of 222 C., relative humidity of 5010%, ventilation frequency 10 times to 20 times/hr, lighting time 12 hours (lights on at 8:00 am and off at 8:00 pm), and illumination of 150 lux to 300 lux, after high-pressure steam sterilization (121 C., sterilization time 20 minutes, drying time 5 minutes) of chip-type litter, an appropriate amount of the litter was placed in a polycarbonate breeding box (W 278 (mm)L 420 (mm)H 230 (mm)) and the mice were bred. For the feed supplied for the experiment, solid feed (+40 RMM-SP-10, U8239G10R, SAFE-DIETS, France) for laboratory animals sterilized by irradiation was used, and reverse osmosis (RO) water was put in a water bottle and sterilized with a high-pressure sterilizer, and the mice were allowed to drink freely.

(55) The cells used in the ovarian cancer animal models were tested for Mycoplasma pneumoniae, Murine coronavirus (Mouse hepatitis virus, MHV), and Murine respirovirus (Sendai virus, SeV) and tested negative before use. The cells were cultured in a CO.sub.2 incubator at 37 C. and 5% CO.sub.2 by using a medium composed of RPMI 1640, 20% FBS, and 1% penicillin/streptomycin (P/S). RPMI 1640 (LM01 51, Welgene) medium was used. The composition of transplanted cancer cells and CAR-T cells and test groups are shown in Table 8 below.

(56) TABLE-US-00009 TABLE 8 Cell line Dosage (number of Administered Administration (CAR-T Group Number cells/animal) substance route cells/animal) Volume G1 6 OVCAR-3 HBSS I.V 200 L G2 6 (1 10.sup.7) Mock G3 6 CD19-CAR-T 5 10.sup.6 G4 6 antiMSLN3-CAR-T 5 10.sup.6

(57) For each cell, cell concentration was adjusted by using PBS, and 200 L of each cells were subcutaneously implanted into mice, and the groups were separated by a random distribution method based on tumor sizes, for individual identification, the ear-punch method was used during the test period, and identification cards were attached to breeding boxes for each group.

(58) 9-2: Confirmation of Anticancer Effect of Anti-MSLN-CAR-T Cells Based on Tumor Animal Models

(59) After isolating the experimental groups, anti-MSLN-CAR-T cells were administered once through a tail vein, and the body weight, tumor size, tumor volume, and tumor weight of the test groups were measured twice a week starting from the day of initial administration.

(60) Based on body weight on the day of initial administration, changes in body weight were observed until the day the test was ended. Body weight gain or loss (%) was calculated by using the equation below:
Body weight change(%)=(body weight/body weight at day 0)100(Equation 1)
Tumor volume (mm.sup.3) was calculated by measuring a short axis (A) and a long axis (B) of tumor by using calipers and using the following equation:
Tumor volume (mm.sup.3)=1/2[{A(mm)}.sup.2B(mm)](Equation 2)
For body weights and tumor volumes, after the last measurement, a statistical analysis was performed by using the post-hoc Dunnett's test of one-way ANOVA by comparing each HBSS-administered group with the anti-MSLN-CAR-T-administered group in ovarian cancer tumor models.

(61) Thereafter, results of confirming changes in body weight after the administration by dates are shown in FIG. 10, results of confirming changes in tumor volume after the administration by dates are shown in FIG. 11, results of confirming tumor volume on day 27 after a CAR-T administration for each group are shown in FIG. 12, and results of confirming tumor weight in the mouse models on day 27 after the CAR-T administration are shown in FIG. 13.

(62) As confirmed in FIG. 10, no statistically significant weight change was observed in the group administered with anti-MSLN3-CAR-T cells compared to the control group G1 (HBSS-administered) administered with excipients, in the ovarian cancer tumor models. However, in a subject G3-4, weight loss was observed on day 26 after the administration, and body weight of the subject become less than 80% of the initial weight, and liver fibrosis was confirmed in the subject at autopsy.

(63) As confirmed in FIG. 11, as a result of measuring tumor volumes, tumor volumes of the anti-MSLN3-CAR-T administered group, which is group G4, compared to that of groups G1, G2, and G3, hardly increased from the day of the administration, and the tumor was confirmed to be reduced from day 8 after the administration.

(64) In addition, FIG. 12 shows results of visually confirming tumor sizes of each test group and a control group on day 27 after the administration of CAR-T cells, and as confirmed in FIG. 12, it was confirmed that the tumor size of group G4 was significantly smaller than that of the the control group G1.

(65) As confirmed in FIG. 13, it was confirmed that tumor weight tended to significantly decrease in group G4 compared to group G1, which is the control group administered with excipients.

(66) Overall, as a result of evaluating the tumor killing efficacy of anti-MSLN3-CAR-T cells in ovarian cancer models, compared to the HBSS-administered group, G1, it was confirmed that the tumor weight did not increase after an CAR-T administration, and the tumor volume, tumor size, and tumor weight also significantly decreased, and thus, a cancer cell killing effect of anti-MSLN3-CAR-T was confirmed in ovarian cancer animal models expressing mesothelin.

(67) 9-3: Confirmation of Infiltration into Tumor Through Immunohistochemical Staining of Anti-MSLN-CAR-T Cells Based on Tumor Animal Models

(68) After isolating the experimental groups, anti-MSLN-CAR-T cells were administered once tail vein, and 50 mg/kg of Zoletil and 10 mg/kg of Xylazine were intraperitoneally injected to induce anesthesia, the abdominal cavity was opened, blood was collected from the abdominal vena cava, and euthanasia was performed by exsanguination. Then, 3 per group were randomly selected, and then slides were prepared after fixing parts of the isolated tumor in 10% neutral formalin, and immunohistochemical (IHC) staining was performed for hCD3c (cell signaling, Cat. No 58061). Subsequently, the slides were photographed by using PANNORAMIC SCAN II (3DHISTECH, Hungary) and analyzed by using a 3DHISTECH software, and results of confirming the immunostaining of each group at 5 magnification and 20 magnification, are shown in FIGS. 14 and 15, respectively.

(69) As confirmed in FIGS. 14 and 15, infiltration of T cells into tumor was confirmed in groups G2, G3, and G4, and thus, it was confirmed that the prepared MSLN-CAR-T cells effectively infiltrate T cells into tumor. In particular, a subject G4-4 was confirmed have tumor of a very small size at the time of autopsy, and complete response (CR) of the tumor was confirmed. In addition, in groups G1 and G3, necrosis was confirmed within the tumor. Therefore, overall, it was confirmed that T cell infiltration into tumors was observed to be relatively high in a large number of subjects in group G4, to which MSLN-CAR-T was administered.

(70) The above description is for illustrative purposes, and those skilled in the art to which the present disclosure belongs will be able to understand that the examples and embodiments can be easily modified without changing the technical idea or essential features of the disclosure. Therefore, it should be understood that the above examples are not limitative, but illustrative in all aspects.