KR-20260065559-A - Antibody or antigen-binding fragment thereof specifically binding to the C-terminus of mesothelin and use thereof

Abstract

The present invention relates to an antibody or an antigen fragment thereof that specifically binds to the C-terminus of mesothelin and to the use thereof. According to one aspect, an anti-mesothelin antibody or an antigen-binding fragment thereof can specifically bind to the C-terminus of mesothelin, and thus can recognize mesothelin present inside the cell membrane even if the mesothelin on the surface of cancer cells is cleaved due to shedding, and thus can be usefully used for the prevention or treatment of cancer.

Inventors

• 안재형
• 최용준

Assignees

• 주식회사 셀렌진

Dates

Publication Date

20260508

Application Date

20251030

Priority Date

20241030

Claims (20)

1. An anti-mesothelin antibody or an antigen-binding fragment thereof characterized by specifically binding to the C-terminus of mesothelin.
2. The anti-mesothelin antibody or its antigen-binding fragment according to claim 1, wherein the antibody specifically binds to an epitope comprising the amino acid sequence of SEQ ID NO. 41.
3. The anti-mesothelin antibody or its antigen-binding fragment according to claim 1, wherein the anti-mesothelin antibody or its antigen-binding fragment comprises one or more selected from the group consisting of an antibody or its antigen-binding fragment comprising a heavy chain variable region including a heavy chain CDR (Heavy chain complementarity determining region) and a light chain variable region including a light chain CDR (Light chain complementarity determining region): 1) a heavy chain variable region comprising HCDR1 having an amino acid sequence of SEQ ID NO. 1, HCDR2 having an amino acid sequence of SEQ ID NO. 2, and HCDR3 having an amino acid sequence of SEQ ID NO. 3; and an antibody or an antigen-binding fragment thereof comprising a light chain variable region comprising LCDR1 having an amino acid sequence of SEQ ID NO. 4, LCDR2 having an amino acid sequence of SEQ ID NO. 5, and LCDR3 having an amino acid sequence of SEQ ID NO. 6, and 2) an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising an HCDR1 having an amino acid sequence of SEQ ID NO. 10, an HCDR2 having an amino acid sequence of SEQ ID NO. 11, and an HCDR3 having an amino acid sequence of SEQ ID NO. 12; and a light chain variable region comprising an LCDR1 having an amino acid sequence of SEQ ID NO. 13, an LCDR2 having an amino acid sequence of SEQ ID NO. 14, and an LCDR3 having an amino acid sequence of SEQ ID NO. 15.
4. The anti-mesothelin antibody or its antigen-binding fragment according to claim 1, wherein the anti-mesothelin antibody or its antigen-binding fragment comprises one or more selected from the group consisting of antibodies or their antigen-binding fragments comprising the following heavy chain variable regions and light chain variable regions: 1) an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 8, and 2) An antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 16 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 17.
5. The anti-mesothelin antibody or its antigen-binding fragment according to claim 1, wherein the anti-mesothelin antibody or its antigen-binding fragment comprises one or more selected from the group consisting of antibodies comprising the following antigen-binding fragments or antigen-binding fragments thereof: 1) An antibody or its antigen-binding fragment comprising an antigen-binding fragment comprising the amino acid sequence of SEQ ID NO. 9, and 2) An antibody or its antigen-binding fragment comprising an antigen-binding fragment comprising the amino acid sequence of SEQ ID NO. 18.
6. The anti-mesothelin antibody or its antigen-binding fragment according to claim 1, wherein the anti-mesothelin antibody or its antigen-binding fragment is unable to bind to a peptide containing the amino acid sequence of SEQ ID NO. 39, but can bind to a peptide containing the amino acid sequence of SEQ ID NO. 40.
7. Isolated nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6.
8. A vector comprising the isolated nucleic acid of claim 7.
9. Isolated host cells transformed with the vector of claim 8.
10. A method for producing an anti-mesothelin antibody or an antigen-binding fragment thereof, comprising the step of culturing the host cells of claim 9 to express an antibody.
11. A chimeric antigen receptor comprising an antigen-binding domain comprising an antibody according to any one of claims 1 to 6 or an antigen-binding fragment thereof.
12. A chimeric antigen receptor according to claim 10, wherein the chimeric antigen receptor further comprises one or more selected from the group consisting of a hinge domain, a transmembrane domain, an intracellular signaling domain, and a costimulatory domain.
13. A chimeric antigen receptor according to claim 11, wherein the antigen-binding fragment is a scFv (single chain variable fragment).
14. Polynucleotide encoding the chimeric antigen receptor of claim 11.
15. In claim 14, The above polynucleotide comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NOs 27 and 36.
16. A vector comprising the polynucleotide of claim 14.
17. Isolated cell comprising the chimeric antigen receptor of claim 11.
18. In claim 17, the isolated cell is a T cell, NK cell, NKT cell, or gamma delta T cell (gamma delta (γδ) T cells).
19. A pharmaceutical composition for the prevention or treatment of cancer comprising isolated cells of claim 17.
20. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6; and Antibody-drug conjugate containing a drug.

Description

Antibody or antigen-binding fragment thereof specifically binding to the C-terminus of mesothelin and use thereof The present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to the C-terminus of mesothelin and to the use thereof. Recently, while immunotherapies such as immune checkpoint inhibitors and CAR-T cell therapies have demonstrated efficacy in various cancers, it is reported that the therapeutic efficiency of these new forms of immunotherapies is not significantly high for solid tumors. This is presumed to be because the tumor microenvironment surrounding the tumor interferes with the immunotherapeutic response and makes drug delivery difficult. Therefore, there is a growing need for research on methods to effectively treat solid tumors by developing antibodies with high affinity that target proteins specifically overexpressed on the surface of solid tumor cells as cancer antigens, as a specific and more effective CAR-T cancer therapy method. Meanwhile, mesothelin is a glycoprotein anchored to the cell surface by a glycosylphosphatidylinositol (GPI) domain, and it is normally expressed at a limited low level in the mesothelium surrounding the cavities and internal organs of the human body, but it is known to be expressed abundantly in cancers such as pancreatic cancer, mesothelioma, ovarian cancer, and non-small cell lung cancer. Meanwhile, it is known that cancer cells can evade the activity of therapeutic agents, such as CAR-T cells that target mesothelin, by reducing mesothelin, a target protein present on the surface of cancer cells, through a shedding mechanism of mesothelin via proteases secreted by the cells. Accordingly, there is a need for technology to recognize shed mesothelin. Accordingly, the inventors of the present invention developed an antibody or an antigen-binding fragment thereof that specifically recognizes and binds to the C-terminus of mesoterin, thereby completing the present invention. Figure 1 is a schematic diagram of the biopanning process for discovering antibodies that specifically bind to the C-terminus of mesothelin. Figures 2 and 3 are diagrams showing the results of biopanning to discover antibodies that specifically bind to the C-terminus of mesothelin. Figure 4 is a diagram showing the screening results for selecting clones that bind to MSLN (269-600) but not to MSLN (269-580). Figure 5 is a diagram showing the results of analyzing the mesothelin binding affinity of five types of phage clones selected through monoclonal phage ELISA. Figure 6 is a diagram showing the results of antibody production from selected clones. Figure 7 is a diagram showing the results of analyzing the mesothelin binding affinity of antibodies produced using selected clones. Figure 8 shows the FACS results of an anti-mesothelin antibody against the K562 cell line, which does not express mesothelin, and the AsPC-1 cell line, which expresses mesothelin. Figures 9 and 10 show the results of confirming, via FACS, whether five types of antibodies or fragments thereof (scFvs/Fab) can bind to mesothelin-expressing cell lines. Figure 11 is a diagram showing the results of confirming whether anti-MSLN-CAR specifically binds to the C-terminus of mesothelin. Figure 12 is a diagram showing the results of confirming the apoptotic effect of anti-MSLN-CAR-T cells on ovarian cancer cell lines. Figure 13 is a diagram showing the results of confirming the apoptotic effect of anti-MSLN-CAR-T cells on lung cancer cell lines. Figure 14 is a diagram showing the results of confirming the apoptotic effect of anti-MSLN-CAR-T cells on ovarian cancer cell lines in the presence of sMSLN. One aspect is described in more detail below through examples. However, these examples are intended to illustrate one aspect and the scope of one aspect is not limited to these examples; the examples of one aspect are provided to more completely explain one aspect to those with average knowledge in the art. Example 1: Discovery of an antibody that specifically binds to the C-terminus of mesothelin To discover antibodies or fragments thereof (scFv, etc.) that do not bind to sMSLN (Soluble mesothelin) and specifically recognize only the C-terminal region of mesothelin, the following experiments were performed. 1.1: Biopanning using an immune tube First, using three types of Fab libraries, panning was performed by removing phages that bind to a peptide [MSLN (269-580), SEQ ID NO. 57] consisting of amino acids 269 to 580 of a biotin-attached MSLN (sequence No. 57), and then selecting phages that bind to a peptide [MSLN (269-600), SEQ ID NO. 58] consisting of amino acids 269 to 600 of a biotin-attached MSLN (Fig. 1), and phages that meet the above conditions were obtained (Figs. 2 and 3). The conditions for performing panning are as follows: - Magnetic beads: Dynabeads MyOne Streptavidin T1 (Invitrogen, Cat#65601) - Phage library: Kab-1, Kab-II, Kab-III - Immobilization: Biotinylated MSLN pep