Antibody specifically binding to c-terminus of mesothelin or antigen-binding fragment thereof, and uses thereof

Abstract

The present invention relates to an antibody specifically binding to C-terminus of mesothelin or an antigen-binding fragment thereof, and uses thereof. With ability to bind specifically to the C-terminus of mesothelin, an anti-mesothelin antibody or antigen-binding fragment thereof according to one aspect can recognize mesothelin present within the cell membrane even when mesothelin on the surface of cancer cells is cleaved due to shedding. Accordingly, the antibody or antigen-binding fragment thereof can be advantageously applied for prevention or treatment of cancer.

Description

Antibody or antigen-binding fragment thereof that specifically binds to the C-terminus of mesothelin and uses thereof

[0001] 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.

[0002] 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.

[0003] 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.

[0004] 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.

[0005] 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.

[0006] One aspect provides an anti-mesothelin antibody or an antigen-binding fragment thereof characterized by specifically binding to the C-terminus of mesothelin.

[0007] Another aspect provides an isolated nucleic acid encoding the above-mentioned anti-methothelin antibody or its antigen-binding fragment.

[0008] Another aspect provides a vector containing the isolated nucleic acid.

[0009] Another aspect provides isolated host cells transformed with the above vector.

[0010] Another aspect provides a method for producing the anti-mesothelin antibody or its antigen-binding fragment, comprising the step of culturing the isolated host cells to express the antibody.

[0011] Another aspect provides a chimeric antigen receptor comprising the above-mentioned anti-mesothelin antibody or an antigen-binding fragment thereof.

[0012] Another aspect provides a polynucleotide encoding the chimeric antigen receptor.

[0013] Another aspect provides a vector containing the above-mentioned polynucleotide.

[0014] Another aspect provides an isolated cell containing the above chimeric antigen receptor.

[0015] Another aspect provides a pharmaceutical composition for the prevention or treatment of cancer comprising the above-mentioned isolated cells.

[0016] Another aspect provides a method for preventing or treating cancer, comprising the step of administering the isolated cells to an individual.

[0017] Another aspect provides an antibody-drug conjugate comprising an antibody or its antigen-binding fragment; and a drug.

[0018] Another aspect provides a pharmaceutical composition for the prevention or treatment of cancer comprising the above antibody-drug conjugate.

[0019] Another aspect provides a mesothelin epitope comprising the amino acid sequence of SEQ ID NO. 41.

[0020] Another aspect provides a polynucleotide encoding the above epitope.

[0021]

[0022] Other objects and advantages of this application will become more apparent from the following detailed description, together with the appended claims and drawings. Anything not described in this specification is omitted, as it can be sufficiently recognized and inferred by those skilled in the art of this application or a similar art field.

[0023] Each description and embodiment disclosed in this application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application should not be considered limited by the specific descriptions provided below.

[0024]

[0025] One aspect provides an anti-mesothelin antibody or an antigen-binding fragment thereof characterized by specifically binding to the C-terminus of mesothelin.

[0026] The term "Mesothelin (MSLN)" in this specification refers to a cell surface glycoprotein (NCBI Gene ID: 10232) with a total amino acid length of 622 aa that is fixed to a cell membrane with a glycophosphatidylinositol (GPI) domain and is a protein that is restrictedly expressed in the mesothelium, which is a protective layer surrounding the cavities and internal organs of the human body. The Mesothelin is a precursor polypeptide of size 69–71 kDa (1–622 AA) that is cleaved by furin and divided into a membrane protein region containing GPI, which includes a C-terminus (296–622 AA) of size 40 kDa, and a megakaryocyte potentiating factor (MFP) containing an N-terminus of size 32 kDa. Mesothelin has three glycosylation sites, and it is expected to transmit signals into the cell through the presence of partner proteins that bind to it, but its function in the cell has not yet been clearly elucidated.

[0027] The above mesothelin is highly expressed in solid tumors and overexpression has been confirmed in esophageal cancer, breast cancer, triple-negative breast cancer (TNBC), gastric cancer, cholangiocarcinoma, pancreatic cancer, colon cancer, lung cancer, thymic carcinoma, mesothelioma, ovarian cancer, endometrial cancer, cervical cancer, uterine serous carcinoma (USC), and acute myeloid leukemia (AML), but shows low expression in normal mesothelial cells, so it can be used as a suitable target for the development of therapeutic agents targeting solid tumors.

[0028] 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.

[0029] The above anti-mesothelin antibody or its antigen-binding fragment may be characterized by specifically binding to the C-terminus of mesothelin, specifically, may specifically bind to the C-terminus of mesothelin containing the amino acid sequence of SEQ ID NO. 38, and more specifically, may specifically bind to the 581st to 600th amino acid sequence of SEQ ID NO. 38.

[0030] In one embodiment, the anti-mesothelin antibody or its antigen-binding fragment may not bind or bind weakly to a peptide consisting of the 269th to 580th amino acids of mesothelin [MSLN (269-580), SEQ ID NO. 39], while specifically binding to a peptide consisting of the 269th to 600th amino acids of mesothelin [MSLN (269-600), SEQ ID NO. 40].

[0031] The term "epitope" in this specification refers to a specific stereomolecular structure region that determines antigenic specificity. In this specification, the term "epitope" may be used interchangeably with "antigenic determinant." The epitope may include the amino acid sequence of SEQ ID NO. 41 below, and specifically may consist of the amino acid sequence of SEQ ID NO. 41.

[0032] In one embodiment, the anti-mesothelin antibody or the antigen-binding fragment thereof may specifically bind to an epitope of mesothelin comprising an amino acid sequence having 80% or more, 90% or more, 95% or more, 99% or more, or 100% sequence identity with the amino acid sequence of SEQ ID NO. 41, and specifically, may specifically bind to an epitope of mesothelin composed of an amino acid sequence having 80% or more, 90% or more, 95% or more, 99% or more, or 100% sequence identity with the amino acid sequence of SEQ ID NO. 41.

[0033] In this specification, the term "antibody" refers to a general term for proteins that act selectively on antigens and participate in biological immunity, and its types are not particularly limited. The heavy and light chains of antibodies have antigen-binding sites that recognize epitopes containing variable regions, and antigen specificity is exhibited according to sequence changes in the variable regions. The variable regions of the antigen-binding sites are divided into a framework region (FR) with low variability and a complementarity determining region (CDR) with high variability, and both the heavy and light chains have three CDR regions divided into CDR1, 2, and 3, and four FR regions. The CDRs of each chain are typically named sequentially as CDR1, CDR2, and CDR3 starting from the N-terminus, and are also identified by the chain in which a specific CDR is located.

[0034] In this specification, the term "complementary determining region" refers to a region within the variable region of an antibody that confers binding specificity with an antigen.

[0035] The above antibodies include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, and chimeric antibodies (e.g., humanized murine antibodies). Additionally, the above antibodies may include diabodies, triabadies, and tetrabodies.

[0036] In this specification, the term "antibody" includes an "antigen-binding fragment" or "antibody fragment" of an antibody possessing antigen-binding ability. The antigen-binding fragment may be selected from the group consisting of, for example, scFv, (scFv)2, scFv-Fc, Fab, Fab', and F(ab')2, which are antibody fragments comprising one or more complementarity determining regions. Among the antibody fragments, Fab has a structure having a variable region of the light and heavy chains, a constant region of the light chain, and a first constant region (CH1) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region comprising one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. The F(ab')2 antibody is generated when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds. Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region. In two-chain Fv, the variable regions of the heavy chain and the light chain are connected by non-covalent bonds. In single-chain Fv (scFv), the variable regions of the heavy chain and the light chain are generally connected by covalent bonds via peptide linkers or directly at the C-terminus, allowing them to form dimer-like structures similar to two-chain Fv.

[0037] In one embodiment, the anti-mesothelin antibody or its antigen-binding fragment may comprise 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):

[0038] 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

[0039] 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.

[0040] In one embodiment, the anti-mesothelin antibody or its antigen-binding fragment may comprise one or more selected from the antibodies or their antigen-binding fragments comprising the following heavy chain variable regions and light chain variable regions:

[0041] 1) an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO. 7 or a sequence having 80% or more sequence homology thereto, and a light chain variable region comprising an amino acid sequence of SEQ ID NO. 8 or a sequence having 80% or more sequence homology thereto, and

[0042] 2) An antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO. 16 or a sequence having 80% or more sequence homology thereto, and a light chain variable region comprising an amino acid sequence of SEQ ID NO. 17 or a sequence having 80% or more sequence homology thereto.

[0043] In one embodiment, the anti-mesothelin antibody or its antigen-binding fragment may comprise one or more selected from the antibodies or antigen-binding fragments comprising the following antigen-binding fragments:

[0044] 1) An antibody or an antigen-binding fragment comprising an amino acid sequence consisting of SEQ ID NO. 9 or a sequence having 80% or more sequence homology therewith, and

[0045] 2) An antibody or an antigen-binding fragment comprising an amino acid sequence consisting of SEQ ID NO. 18 or a sequence having 80% or more sequence homology therewith.

[0046] The antibody or its antigen-binding fragment according to the above-described aspect may include the sequence of the anti-mesothelin specific binding antibody described herein as well as biological equivalents thereof, within a range that can specifically recognize the C-terminus of mesothelin. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody.

[0047] For example, the amino acid sequence of the above antibody may be substituted through conservative substitution. Here, "conservative substitution" refers to a modification of a polypeptide comprising substituting one or more amino acids with amino acids having similar biochemical properties that do not cause a loss of the biological or biochemical function of the polypeptide. "Conservative amino acid substitution" is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain. A class of amino acid residues having a similar side chain is defined in the art. These classes include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). It can be expected that an antibody according to one aspect may still retain its original activity even if some of the amino acid sequences of the antibody are substituted through conservative amino acid substitution.

[0048] Considering the variant having the aforementioned biological equivalence activity, one aspect of the antibody (or antigen-binding fragment) or the nucleic acid molecule encoding it is interpreted to include a sequence that exhibits substantial identity with the sequence described in SEQ ID NO. The above substantial identity means a sequence that, when the said sequence is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art, exhibits at least 61% homology, more preferably 70% homology, more preferably 80% homology, more preferably 90% homology, more preferably 95% homology, and most preferably 98% homology. Alignment methods for sequence comparison are known in the art.

[0049]

[0050] Another aspect is to provide an isolated nucleic acid encoding the antibody or its antigen-binding fragment. The same parts described above apply equally to the nucleic acid.

[0051] In this specification, the term "nucleic acid" comprehensively includes DNA and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acids, include not only natural nucleotides but also analogues in which sugar or base sites are modified. The sequence of nucleic acid encoding a variable region of a heavy chain and light chain of one aspect may be modified. Such modification includes the addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0052] The above nucleic acid is interpreted to include nucleotide sequences that exhibit substantial identity with respect to the nucleotide sequence of the nucleic acid. Substantial identity means a nucleotide sequence that exhibits at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology when one aspect of the nucleotide sequence is aligned with any other sequence to correspond as much as possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0053] In one embodiment, the isolated nucleic acid encoding the anti-mesothelin antibody or its antigen-binding fragment may comprise one or more selected from nucleic acids comprising polynucleotide sequences including a polynucleotide sequence encoding a heavy chain variable region and a polynucleotide sequence encoding a light chain variable region:

[0054] 1) a polynucleotide sequence encoding a heavy chain variable region comprising an HCDR1 coding sequence comprising a polynucleotide sequence comprising SEQ ID NO. 19, an HCDR2 coding sequence comprising a polynucleotide sequence comprising SEQ ID NO. 20, and an HCDR3 coding sequence comprising a polynucleotide sequence comprising a polynucleotide sequence comprising SEQ ID NO. 21; and an isolated nucleic acid encoding an antibody or an antigen-binding fragment thereof comprising a polynucleotide sequence encoding a light chain variable region comprising an LCDR1 coding sequence comprising a polynucleotide sequence comprising SEQ ID NO. 22, an LCDR2 coding sequence comprising a polynucleotide sequence comprising SEQ ID NO. 23, and an LCDR3 coding sequence comprising a polynucleotide sequence comprising a polynucleotide sequence comprising SEQ ID NO. 24, and

[0055] 2) A polynucleotide sequence encoding a heavy chain variable region comprising an HCDR1 coding sequence comprising a polynucleotide sequence comprising SEQ ID NO. 28, an HCDR2 coding sequence comprising a polynucleotide sequence comprising SEQ ID NO. 29, and an HCDR3 coding sequence comprising a polynucleotide sequence comprising a polynucleotide sequence comprising SEQ ID NO. 30; and an isolated nucleic acid encoding an antibody or an antigen-binding fragment thereof comprising a polynucleotide sequence encoding a light chain variable region comprising an LCDR1 coding sequence comprising a polynucleotide sequence comprising SEQ ID NO. 31, an LCDR2 coding sequence comprising a polynucleotide sequence comprising SEQ ID NO. 32, and an LCDR3 coding sequence comprising a polynucleotide sequence comprising a polynucleotide sequence comprising SEQ ID NO. 33.

[0056] In one embodiment, the isolated nucleic acid encoding the anti-mesothelin antibody or its antigen-binding fragment may comprise one or more selected from nucleic acids comprising a polynucleotide sequence encoding a heavy chain variable region and a polynucleotide sequence encoding a light chain variable region:

[0057] 1) an isolated nucleic acid encoding an antibody or an antigen-binding fragment thereof, comprising a polynucleotide sequence consisting of SEQ ID NO. 25 or a sequence having 80% or more sequence homology thereto, encoding a heavy chain variable region, and a polynucleotide sequence consisting of SEQ ID NO. 26 or a sequence having 80% or more sequence homology thereto, encoding a light chain variable region, and

[0058] 2) A separated nucleic acid encoding an antibody or an antigen-binding fragment thereof, comprising a sequence encoding a heavy chain variable region including a polynucleotide sequence consisting of SEQ ID NO. 34 or a sequence having 80% or more sequence homology thereto, and a sequence encoding a light chain variable region including a polynucleotide sequence consisting of SEQ ID NO. 35 or a sequence having 80% or more sequence homology thereto.

[0059] In one embodiment, the isolated nucleic acid encoding the anti-mesothelin antibody or its antigen-binding fragment may comprise one or more selected from nucleic acids comprising a polynucleotide sequence encoding the following antigen-binding fragment:

[0060] 1) an antibody comprising a sequence encoding an antigen-binding fragment comprising a polynucleotide sequence consisting of SEQ ID NO. 27 or a sequence having 80% or more sequence homology therewith, or an isolated nucleic acid encoding an antigen-binding fragment thereof, and

[0061] 2) An antibody comprising a sequence encoding an antigen-binding fragment comprising a polynucleotide sequence consisting of SEQ ID NO. 36 or a sequence having 80% or more sequence homology therewith, or an isolated nucleic acid encoding the antigen-binding fragment thereof.

[0062]

[0063] Another aspect is to provide a vector containing the isolated nucleic acid. The same parts described above apply equally to the vector.

[0064] The above vector may be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma expressing a target antibody) for the expression of an antibody or its antibody fragment in a suitable host cell, with DNA encoding partial or full-length light and heavy chains, and may include essential regulatory elements operably linked to enable the expression of a DNA (gene) insert. "Operably linked" means that a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked to perform a general function, and that the gene is linked to the expression regulatory sequence so that the gene can be expressed.

[0065] The term "expression control sequence" above refers to a DNA sequence that regulates the expression of a linked DNA sequence operable in a specific host cell. Such a control sequence includes a promoter for carrying out transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, a sequence regulating the termination of transcription and translation, a start codon, a stop codon, a polyadenylation signal, and an enhancer. Those skilled in the art may recognize that the design of the expression vector may vary by selecting a different control sequence depending on factors such as the selection of the host cell to be transformed and the expression level of the protein.

[0066] The above vector is not particularly limited in type as long as it is a vector commonly used in the fields of cloning and antibody production; examples include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. The above plasmids include Escherichia coli-derived plasmids (pBR322, pBR325, pUC118, and pUC119, pET-21b(+)), Bacillus subtilis-derived plasmids (pUB110 and pTP5), and yeast-derived plasmids (YEp13, YEp24, and YCp50), and the above viruses may include animal viruses such as retroviruses, adenoviruses, or vaccinia viruses, and insect viruses such as baculoviruses. Vectores of the pComb3 family, commonly used for phage labeling, may be used, and vectors commonly used for expressing proteins in mammalian cells to express antibodies in mammalian cells, such as pcDNA or pVITRO, may be used.

[0067]

[0068] Another aspect is to provide isolated host cells transformed with the above-mentioned vector. The same parts described above apply equally to the said host cells.

[0069] In this specification, the term "transformation" refers to a molecular biological technique in which a fragment of DNA chain or a plasmid containing a type of foreign gene different from that of the original cell infiltrates between cells and binds to the DNA present in the original cell, thereby altering the genetic traits of the cell. The vector is transfected or transfected into a host cell. Various techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells for transfection or transfection may be used, such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection.

[0070] An antibody or its antigen-binding fragment according to one aspect may be expressed in eukaryotic cells, preferably mammalian host cells, considering the applicability to microorganisms such as bacteria (E. coli) or yeast, or to mammalian cells. The mammalian host cell may be, for example, any one selected from the group consisting of Chinese hamster ovary (CHO) cells, NSO myeloma cells, COS cells, SP2 cells, F2N cells, HEK293 cells, and antibody-producing hybridoma cells, but is not limited thereto.

[0071]

[0072] Another aspect provides a method for producing an anti-mesothelin antibody or an antigen-binding fragment thereof, comprising the step of culturing the isolated host cells to express antibodies. The same parts as described above apply equally to the method.

[0073] The above method may include the step of transforming a host cell for producing one type of antibody or its antigen-binding fragment into a vector operably linked to DNA encoding said antibody or its antigen-binding fragment. The type of selected host cell and recombinant expression vector is as described above, and this step may be carried out by selecting an appropriate transformation method. When the recombinant expression vector encoding said antibody gene is introduced into a mammalian host cell, the antibody may be produced by culturing the host cell for a period sufficient to cause the antibody to be expressed in the host cell, or more preferably, for a period sufficient to cause the antibody to be secreted into the culture medium in which the host cell is cultured.

[0074] In addition, the above method may further include the step of culturing the transformed isolated host cells to produce a polypeptide of an antibody or an antigen-binding fragment thereof according to one aspect from a recombinant expression vector introduced into the host cells. The composition of the culture medium, culture conditions, and culture time for culturing the selected host cells may be appropriately selected, and the antibody molecules produced in the host cells may be accumulated in the cytoplasm of the cells, secreted outside the cells or into the culture medium by an appropriate signal sequence, or targeted to periplasms, etc. In addition, the protein may be refolded and given a functional structure using methods known in the art so that the antibody according to one aspect maintains binding specificity to mesothelin. In addition, when producing an IgG-type antibody, the heavy chain and light chain may be expressed in separate cells and brought into contact at a separate stage to form a complete antibody, or the heavy chain and light chain may be expressed in the same cell to form a complete antibody inside the cell.

[0075] Additionally, the above method may further include a step of obtaining an antibody or its antigen-binding fragment produced in isolated host cells. The method of obtaining can be appropriately selected and controlled by considering the characteristics of the polypeptide of the antibody or its antigen-binding fragment produced in the host cells, the characteristics of the host cells, the mode of expression, or whether the polypeptide is targeted. For example, the antibody or its antigen-binding fragment secreted into the culture medium can be recovered by obtaining the medium in which the host cells were cultured and removing impurities through centrifugation, and if necessary, the cells may be lysed to a extent that does not affect the functional structure of the antibody or its antigen-binding fragment in order to release and recover the antibody present in specific organelles or cytoplasm within the cell to the outside.

[0076] The obtained antibody may undergo an additional process of further removing impurities and concentrating it through methods such as chromatography, filtration using a filter, or dialysis. The separation or purification of the obtained antibody may be performed by separation and purification methods commonly used for proteins, for example, by chromatography. The chromatography may include, for example, affinity chromatography including a protein A column, a protein G column, and a protein L column, ion exchange chromatography, or hydrophobic chromatography. In addition to the chromatography, the antibody may be separated and purified by combining filtration, ultrafiltration, salting out, dialysis, etc.

[0077]

[0078] Another aspect is a chimeric antigen receptor comprising an antigen-binding domain, wherein the antigen-binding domain provides a chimeric antigen receptor comprising the anti-mesothelin antibody or an antigen-binding fragment thereof. The same parts described above apply equally to the chimeric antigen receptor.

[0079] The chimeric antigen receptor is characterized by specifically binding to mesothelin, and therefore includes an antigen-binding domain that specifically binds to mesothelin.

[0080] The above antigen-binding domain may comprise one or more selected from the group consisting of an anti-mesothelin antibody or an antigen-binding fragment thereof comprising a heavy chain variable region including a heavy chain CDR and a light chain variable region including a light chain CDR:

[0081] 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

[0082] 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.

[0083] In this specification, the term "chimeric antigen receptor (CAR)" means that the structure of the chimeric antigen receptor includes an antigen binding (recognition) domain, a transmembrane domain, and an intracellular signaling domain.

[0084] In one embodiment, the antigen-binding fragment may be a scFv (single chain variable fragment).

[0085] In one embodiment, the antigen-binding domain may comprise one or more selected from an antibody comprising the following antigen-binding fragments or the antigen-binding fragments thereof:

[0086] 1) An antibody comprising an antigen-binding fragment comprising the amino acid sequence of SEQ ID NO. 9 or the antigen-binding fragment thereof, and

[0087] 2) An antibody or its antigen-binding fragment comprising an antigen-binding fragment comprising the amino acid sequence of SEQ ID NO. 18.

[0088] The chimeric antigen receptor may additionally include one or more selected from the group consisting of a hinge domain, a transmembrane domain, an intracellular signaling domain, and a costimulatory domain.

[0089] The hinge domain, transmembrane domain, intracellular signaling domain, and co-stimulation domain included in the above chimeric antigen receptor are well known in the art.

[0090] The above hinge domain is a domain that connects an anti-mesothelin antibody or its antigen-binding fragment (antigen-binding domain) with a transmembrane domain, is also named a spacer, and is intended to extend the antigen-binding domain from the T cell membrane. The above hinge domain may be a CD8 hinge domain, an IgG1 hinge domain, an IgG4 hinge domain, a CD28 extracellular domain, a KIR (killer immunoglobulin-like receptor) extracellular domain, and combinations thereof; specifically, a CD8 hinge domain (SEQ No. 43) may be used, but is not limited thereto, and any hinge domain commonly used in the art may be used.

[0091] The above-mentioned transmembrane domain can serve as a support for the chimeric antigen receptor molecule and simultaneously connect the hinge domain and the intracellular signaling domain. The transmembrane domain can penetrate the cell membrane so that the anti-mesothelin antibody of the chimeric antigen receptor or its antigen-binding fragment is located on the cell surface and the intracellular signaling domain is located inside the cell. The above-mentioned transmembrane domain may be a transmembrane region of CD3 zeta (CD3z), CD4, CD8, CD28, or KIR protein; specifically, the transmembrane domain of CD8 (SEQ No. 44) may be used, but any conventional transmembrane domain used in the manufacture of chimeric antigen receptors may be used without limitation.

[0092] The intracellular signaling domain described above receives a signal transmitted by an anti-mesothelin antibody or its antigen-binding fragment and serves to transmit it into a cell to which a chimeric antigen receptor is bound. The intracellular signaling domain is not particularly limited in type as long as it is a part that transmits a signal capable of inducing T cell activation when an antibody binds to an antigen-binding site located outside the cell, and various types of intracellular signaling domains may be used. The above intracellular signaling domain may be, for example, an immune receptor tyrosine-based activation motif or an ITAM, and the ITAM may include one derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, CD66d, or FcεRIγ, and specifically, the CD3 zeta signaling domain (SEQ No. 46) may be used, but is not limited thereto, and any intracellular signaling domain commonly used in the art may be used without limitation.

[0093] The above-mentioned co-stimulatory domain refers to the intracellular portion of a chimeric antigen receptor that includes the intracellular domain of a co-stimulatory molecule, which performs the role of transmitting a signal to T cells in addition to the signal by the intracellular signaling domain. The above-mentioned co-stimulatory molecule refers to a cell surface molecule that is necessary to bring about a sufficient response of lymphocytes to an antigen, and examples thereof may be CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1 (lymphocyte function-associated antigen-1), CD2, CD7, LIGHT, NKG2C, or B7-H3, and specifically, the 4-1BB signaling domain (SEQ ID No. 45) may be used, but is not limited thereto, and any co-stimulatory domain commonly used in the art may be used without limitation. The above-mentioned co-stimulatory domain may be the intracellular portion of a molecule selected from the group consisting of such co-stimulatory molecules and combinations thereof.

[0094] Each domain of the chimeric antigen receptor, including the above-mentioned transmembrane domain, intracellular signaling domain, and / or co-stimulation domain, may be optionally connected by a short oligopeptide or polypeptide linker. Any linker known in the art may be used, without being particularly limited in length, provided that the linker is capable of inducing T cell activation through the intracellular domain when the antigen binds to an antibody located outside the cell.

[0095] Additionally, the chimeric antigen receptor may include modified forms of the antibodies and domains as described above. In this case, the modification may be performed by substituting, deleting, or adding one or more amino acids to the amino acid sequence of the wild-type antibody and domain without altering the function of the antibodies and domains. Typically, the substitution may be alanine or a conservative amino acid substitution that does not affect the charge, polarity, or hydrophobicity of the entire protein.

[0096]

[0097] Another aspect provides a polynucleotide encoding the chimeric antigen receptor. The same parts described above apply equally to the polynucleotide.

[0098] The above polynucleotide may undergo various modifications to its coding region within a range that does not alter the amino acid sequence of the antigen receptor expressed from the coding region, due to codon degeneracy or in consideration of codons preferred by the organism intended to express the antigen receptor; and may also undergo various modifications or alterations to parts excluding the coding region within a range that does not affect gene expression, and those skilled in the art will understand that such modified genes are also included within the scope of the present invention. That is, the polynucleotide according to one aspect may be modified by substitution, deletion, insertion, or a combination thereof of one or more nucleic acid bases, as long as it codes for a protein having equivalent activity, and these are also included within the scope of the present invention.

[0099] In one embodiment, the polynucleotide encoding the chimeric antigen receptor may comprise one or more nucleotide sequences selected from the group consisting of SEQ ID NOs 27 and 36.

[0100]

[0101] Another aspect is to provide isolated cells containing the chimeric antigen receptor mentioned above. The same parts described above apply equally to said cells.

[0102] The isolated cell may contain the chimeric antigen receptor, and specifically may express the chimeric antigen receptor.

[0103] The isolated cells may be T cells, NK cells, NKT cells, gamma delta (γδ) T cells, CAR-T cells, and / or CAR-NK cells. Additionally, the isolated cells may be obtained from or produced from bone marrow, peripheral blood, peripheral blood mononuclear cells, or umbilical cord blood.

[0104] In one embodiment, the isolated cell may be a vector comprising a polynucleotide encoding a chimeric antigen receptor or transformed into said vector.

[0105] The above vector may utilize various vectors known in the art, and depending on the type of host cell intended to produce the antigen receptor, expression-regulating sequences such as promoters, terminators, and enhancers, as well as sequences for membrane targeting or secretion, may be appropriately selected and combined in various ways according to the purpose. The vector of the present invention includes, but is not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. Suitable vectors may include signal sequences or leader sequences for membrane targeting or secretion in addition to expression-regulating elements such as promoters, operators, start codons, stop codons, polyadenylation signals, and enhancers, and may be manufactured in various ways according to the purpose. Furthermore, the above vector may be introduced into cells to transform the cells.

[0106]

[0107] Another aspect is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising the isolated cells described above. The same parts as described above apply equally to the composition.

[0108] In this specification, the term "prevention" refers to any act of suppressing cancer (tumor) or delaying its onset by administering a pharmaceutical composition according to the present invention.

[0109] In this specification, the term "treatment" refers to any act in which symptoms of cancer (tumor) are improved or beneficially altered by the administration of a pharmaceutical composition according to the present invention.

[0110] In this specification, the term “cancer” collectively refers to a disease caused by cells having aggressive characteristics in which the cells divide and grow by disregarding normal growth limits, invasive characteristics in which they infiltrate surrounding tissues, and metastatic characteristics in which they spread to other parts of the body or outside. In this specification, the term cancer is used interchangeably with malignant tumor, and preferably may be a mesothelin-positive or mesothelin-overexpressing cancer.

[0111] The above cancer may preferably be a solid tumor, for example, more preferably a mesothelin-positive or mesothelin-overexpressing solid tumor. For example, the above solid tumor may be any one selected from the group consisting of esophageal cancer, breast cancer, triple-negative breast cancer (TNBC), gastric cancer, cholangiocarcinoma, pancreatic cancer, colon cancer, lung cancer, thymic carcinoma, mesothelioma, ovarian cancer, endometrial cancer, cervical cancer, uterine serous carcinoma (USC), non-small cell lung cancer, and acute myeloid leukemia (AML), but is not limited thereto.

[0112] In one embodiment, the anti-mesothelin antibody of the present invention or its antigen-binding fragment specifically binds to the C-terminus of mesothelin, so that the isolated cell or the pharmaceutical composition containing it can effectively target cancer cells expressing mesothelin in the presence of sMSLN (Soluble mesothelin) and induce apoptosis. In addition, even when mesothelin on the cell surface is shed due to protease, the isolated cell or the pharmaceutical composition containing it can specifically bind to mesothelin present on the cell membrane and induce apoptosis of the cancer cells.

[0113] The above pharmaceutical composition may contain 10 to 95 weight percent of a cell of one aspect that is an active ingredient based on the total weight of the pharmaceutical composition. In addition, the pharmaceutical composition of the present invention may additionally contain one or more active ingredients exhibiting the same or similar functions in addition to the above active ingredient.

[0114] The dosage of the above cells may be adjusted according to various factors, including the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the pharmaceutical composition, the type of formulation, and the patient's age, weight, general health condition, gender, diet, time of administration, route of administration, duration of treatment, and concurrently used drugs. However, for a desirable effect, the effective amount of cells included in the pharmaceutical composition according to the present invention is 1 x 10⁶ 5 Up to 1 x 10 11 It may be in cells / kg. In this case, administration may be performed once a day or divided into several doses. The effective amount of the cells or pharmaceutical composition presented herein can be determined empirically without excessive experimentation.

[0115] The above pharmaceutical composition may include a pharmaceutically acceptable carrier. The term "pharmaceuticalally acceptable carrier" may refer to a carrier or diluent that does not irritate living organisms and does not impair the biological activity and properties of the injected compound. Here, "pharmaceuticalally acceptable" means that the target of application (prescription) does not possess toxicity beyond an acceptable level without inhibiting the activity of the active ingredient. Any type of carrier that is commonly used in the relevant technical field and is pharmaceutically acceptable may be used in the above pharmaceutical composition. Non-limiting examples of the above carriers include lactose, dextrose, maltodextrin, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, glycerol, ethanol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. These may be used alone or in a mixture of two or more. The above pharmaceutical composition may be prepared into an oral or parenteral formulation according to the route of administration by conventional methods known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. The above pharmaceutical compositions may each be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, or sterile injectable solutions according to conventional methods.

[0116] When formulating the above pharmaceutical composition, it may be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, or surfactants, but is not limited thereto.

[0117] When the above pharmaceutical composition is prepared as an oral formulation, it may be prepared in the form of powder, granules, tablets, pills, coated tablets, capsules, liquids, gels, syrups, suspensions, wafers, etc., in accordance with methods known in the art together with a suitable carrier. Examples of pharmaceutically acceptable suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol; starches such as corn starch, potato starch, and wheat starch; celluloses such as cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; polyvinylpyrrolidone; water; methylhydroxybenzoate; propylhydroxybenzoate; magnesium stearate; mineral oil; malt; gelatin; talc; polyols; vegetable oils, etc. In the case of formulation, the formulation may include diluents and / or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants as needed.

[0118] When the above pharmaceutical composition is prepared as a parenteral formulation, it may be formulated in the form of an injectable, transdermal, nasal inhalant, and suppository according to methods known in the art with a suitable carrier. When formulated as an injectable, suitable carriers may include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof; preferably, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, isotonic solutions such as 5% dextrose, etc. When formulated as a transdermal formulation, it may be formulated in the form of an ointment, cream, lotion, gel, topical solution, paste, liniment, aerosol, etc. In the case of nasal inhalers, they can be formulated in the form of an aerosol spray using suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide, and when formulated as suppositories, the base may be Witepsol, Tween 61, polyethylene glycols, cocoa starch, laurin starch, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, and sorbitan fatty acid esters.

[0119] The above pharmaceutical composition may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to medical treatment or prevention, and the effective dose level may be determined based on factors including the severity of the disease, drug activity, patient's age, weight, health, gender, patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and elimination rate, the duration of treatment, drugs combined or used concurrently with the composition of the present invention used, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered alone or in combination with a component known to exhibit therapeutic effects for known diseases. It is important to administer an amount that obtains maximum effect with minimum amount without side effects, taking all of the above factors into consideration.

[0120] The dosage of the above pharmaceutical composition may be determined by a person skilled in the art by taking into consideration the purpose of use, the degree of toxicity of the disease, the patient's age, weight, gender, medical history, or the type of substance used as an active ingredient. For example, the pharmaceutical composition of the present invention may be administered at a dose of about 0.1 ng to about 1,000 mg / kg, preferably 1 ng to about 100 mg / kg per adult, and although the frequency of administration of the composition of the present invention is not particularly limited thereto, it may be administered once a day or administered several times by dividing the dose. The above dosage or frequency of administration does not limit the scope of the present invention in any way.

[0121]

[0122] Another aspect provides a method for treating or preventing cancer comprising the step of administering the isolated cells or a pharmaceutical composition containing the same to an individual. The same parts as described above apply equally to the method.

[0123] In this specification, the term "individual" refers to a subject requiring treatment for a disease, and more specifically, to mammals such as human or non-human primates, rodents (rats, mice, guinea pigs, etc.), mice, dogs, cats, horses, cattle, sheep, pigs, goats, camels, and antelopes.

[0124] The above pharmaceutical composition may be administered as a single or multiple doses in pharmaceutically effective amounts. In this case, the composition may be administered in the form of a liquid, powder, aerosol, injection, intravenous fluid (Ringer's gel), capsule, pill, tablet, suppository, or patch. The route of administration of the pharmaceutical composition for the prevention or treatment of the disease may be any general route as long as it can reach the target tissue.

[0125] The above pharmaceutical composition is not particularly limited thereto, but may be administered via routes such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration, depending on the purpose. However, when administered orally, it may be administered in an unformulated form, and since the active ingredient of the above pharmaceutical composition may be denatured or degraded by gastric acid, the oral composition may be administered into the mouth in a form coated with the active agent or formulated to protect it from degradation in the stomach, or in the form of an oral patch. Additionally, the above composition may be administered by any device capable of transporting the active substance to target cells.

[0126]

[0127] Another aspect provides an antibody-drug conjugate (ADC) comprising the above-mentioned anti-mesothelin antibody or an antigen-binding fragment thereof; and a drug. The same parts described above apply equally to the above-mentioned antibody-drug conjugate.

[0128] The term "antibody-drug conjugate (ADC)" as used herein refers to a conjugate in which an antibody (or its antigen-binding fragment) is linked to a drug, and may also be referred to as an immunoconjugate. The antibody-drug conjugate may be prepared using various methods known in the art. The anticancer drug must remain stably bound to the antibody until the anticancer drug is delivered to target cancer cells. Once delivered to the target, the drug must be released from the antibody to induce apoptosis in the target cells. To achieve this, the drug must be stably bound to the antibody while possessing sufficient cytotoxicity to induce apoptosis in the target cells upon release.

[0129] The antibody-drug conjugate may be one in which the drug and the antibody or its antigen-binding fragment are directly bound, or the drug may be indirectly linked to the antibody or its antigen-binding fragment by any means such as a linker. The linker may include a non-cleavable linker or a cleavable linker. Generally, the antibody-drug conjugate may be designed so that the drug is released when the linker is cleaved inside a targeted cell after being indirectly bound by means such as a linker.

[0130] The term "drug" in this specification may include, without limitation, a substance that can be used to treat a disease by binding to the antibody of the present invention to increase the efficiency of the therapeutic antibody itself, increasing the half-life of the antibody in the blood, or reaching a target site of the antibody to kill the cause of the disease, such as cancer, within said target. Examples include, but are not limited to, cytotoxic drugs, toxins, cytokines, chemokines, antibiotics, enzymes such as nucleases, radionuclides, photosensitizers, photothermal nanomaterials, nanoparticles, and micelles.

[0131] The above-mentioned cytotoxic drugs refer to drugs that can be used to treat diseases, and examples include drugs with anticancer activity (anticancer agents), such as microtubulin structure formation inhibitors, meiosis inhibitors, topoisomerase inhibitors, or DNA intercalators.The above cytotoxic drugs are camptothecin, maytansinoid, auristatin, dolastatin, trichothecene, CC-1065 (NSC 298223), calicheamicin, enediynes, taxane, anthracycline, methotrexate, adriamycin, vindesine, vinca alkaloid, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, daunomycin, etoposide, teniposide, It may include, but is not limited to, carminomycin, aminopterin, dactinomycin, bleomycin, esperamicin, 5-fluorouracil, melphalan, nitrogen mustard (mechlorethamine HCl), cis-platinum and its homologues, cisplatin, CPT-11, docetaxel, monomethyl auristatin E, monomethyl auristatin F, emtansine (DM1), or derivatives thereof.

[0132] The antibody-drug conjugate may have a molar drug-antibody / antibody fragment ratio (DAR) of 1 or more, that is, one or more drug molecules are attached to the antibody / antibody fragment. The antibody-drug conjugate may have a DAR of about 1 to 20.

[0133]

[0134] Another aspect provides a pharmaceutical composition for the prevention or treatment of cancer comprising the antibody-drug conjugate described above. The same parts as described above apply equally to the composition.

[0135]

[0136] Another aspect provides a method for treating or preventing cancer comprising the step of administering the antibody-drug conjugate or a pharmaceutical composition containing the same to an individual. The same parts as described above apply equally to the method.

[0137]

[0138] Another aspect is to provide an isolated cell containing the chimeric antigen receptor or an antibody-drug conjugate for the prevention or treatment of cancer. The same parts described above apply equally to the said use.

[0139]

[0140] Another aspect is to provide a mesothelin epitope comprising the amino acid sequence of SEQ ID NO. 41. The same parts as described above apply equally to the epitope.

[0141] In one embodiment, the epitope may include an amino acid sequence having 80% or more, 90% or more, 95% or more, 99% or more, or 100% sequence identity with the amino acid sequence of SEQ ID NO. 41, and specifically, may be composed of an amino acid sequence having 80% or more, 90% or more, 95% or more, 99% or more, or 100% sequence identity with the amino acid sequence of SEQ ID NO. 41.

[0142]

[0143] Another aspect is to provide a polynucleotide encoding the above epitope. The same parts as described above apply equally to the polynucleotide.

[0144] An anti-mesothelin antibody or its antigen-binding fragment according to one aspect 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, so it can be usefully used for the prevention or treatment of cancer.

[0145] Figure 1 is a schematic diagram of the biopanning process for discovering antibodies that specifically bind to the C-terminus of mesothelin.

[0146] Figures 2 and 3 are diagrams showing the results of biopanning to discover antibodies that specifically bind to the C-terminus of mesothelin.

[0147] Figure 4 is a diagram showing the screening results for selecting clones that bind to MSLN (269-600) but not to MSLN (269-580).

[0148] Figures 5 to 7 show the results of analyzing the mesothelin binding affinity of five types of phage clones selected through monoclonal phage ELISA.

[0149] Figure 8 is a diagram showing the results of antibody production from selected clones.

[0150] Figures 9 to 11 show the results of analyzing the mesothelin binding ability of antibodies produced using selected clones.

[0151] Figure 12 shows the FACS results of an anti-mesothelin antibody against the K562 cell line, which is a cell line that does not express mesothelin, and the AsPC-1 cell line, which is a cell line that expresses mesothelin.

[0152] Figures 13 and 14 show the results of confirming, via FACS, whether five types of antibodies or fragments thereof (scFvs / Fab) can bind to mesothelin-expressing cell lines.

[0153] Figure 15 is a diagram showing the results of confirming whether anti-MSLN-CAR specifically binds to the C-terminus of mesothelin.

[0154] Figure 16 is a diagram showing the results of confirming the apoptotic effect of anti-MSLN-CAR-T cells on ovarian cancer cell lines.

[0155] Figure 17 is a diagram showing the results of confirming the apoptotic effect of anti-MSLN-CAR-T cells on lung cancer cell lines.

[0156] Figure 18 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.

[0157] 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.

[0158]

[0159] Example 1: Discovery of an antibody that specifically binds to the C-terminus of mesothelin

[0160] 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.

[0161] 1.1: Biopanning using an immune tube

[0162] 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).

[0163] The conditions for performing panning are as follows:

[0164] - Magnetic beads: Dynabeads MyOne Streptavidin T1 (Invitrogen, Cat#65601)

[0165] - Phage library: Kab-1, Kab-II, Kab-III

[0166] - Immobilization: Biotinylated MSLN peptide (269-600)

[0167] - Blocking: MPBS (5% skim milk in PBST)

[0168] - Washing: PBST (0.05% Tween20 in PBS)

[0169] - Elution: 0.2 M Glycine-HCl pH2.2

[0170]

[0171] Meanwhile, the sequence information of the above-mentioned mesothelin, the peptide composed of the 269th to 580th amino acids of MSLN [MSLN (269-580)], the peptide composed of the 269th to 600th amino acids of MSLN [MSLN (269-600)], and the peptide composed of the 581st to 600th amino acids of MSLN [MSLN (581-600)] is listed in Table 1 below.

[0172]

[0173]

[0174] Next, in order to select clones that specifically bind to the C-terminus of mesothelin among the phages obtained through the above panning, monoclonal phage ELISA was performed on each of the clones obtained through 3 rounds of panning using an immunotube.

[0175] Through the above experiment, monoclonal phage ELISA was performed using 376 clones obtained from 3 rounds (188) and 4 rounds (188) after panning using the Fab library, and the sequences of 14 types of phages that bind to MSLN (269-600) but not to MSLN (269-580) were identified, and based on this, a total of 5 types of unique clones (3 types of Fab, 2 types of scFv) were obtained (Fig. 4).

[0176]

[0177] 1.2: Analysis of binding affinity between phages and the mesothelin C-terminus

[0178] In order to discover antibodies or fragments (scFv) that recognize the C-terminus of mesothelin, we used an ELISA method to determine whether the five types of phage clones selected through the monoclonal phage ELISA did not bind to MSLN (296-580) but only to MSLN (296-600).

[0179] Specifically, to perform a monoclonal phage ELISA, 30 μL of antigen protein (1 μg / mL in PBS) or control (PBS) was added to each well of a 96-well ELISA plate and incubated at 4°C for 16 hours. After removing the solution from the 96-well ELISA plate, 150 μL of MPBS was added to each well, and blocking was performed at room temperature for 1 hour. After removing the solution from the 96-well ELISA plate, 30 μL of phage was added to each well and incubated at 37°C for 1 hour. After the incubation, the 96-well ELISA plate was washed four times with PBST, and 30 μL of anti-M13-HRP (1:5,000 dilution) was added to each well and incubated at 37°C for 1 hour. After the reaction, the 96-well ELISA plate was washed four times with PBST, and 30 μL of TMB substrate was added to each well, followed by a color reaction at 25°C for 5 minutes. After stopping the reaction by adding 30 μL of 17% phosphoric acid to each well of the 96-well ELISA plate, the absorbance was measured at 450 nm using a microplate reader.

[0180] As a result, it was confirmed that all five types of phages selected in Example 1.1 did not bind to MSLN (296-580) but bound only to MSLN (296-600) (Figs. 5 to 7).

[0181]

[0182] 1.3: Production of Selected Candidate Antibodies

[0183] To produce candidate antibodies from the phages selected in Example 1.2 above, a gene fragment was first synthesized from the selected phages. The antibody light and heavy chain regions of the synthesized gene fragment were amplified via PCR and cleaved with an appropriate restriction enzyme to obtain the antibody gene fragment. Additionally, the IgG1 expression vector (light chain expression vector, heavy chain expression vector) was cleaved with an appropriate restriction enzyme to obtain the vector fragment. The antibody gene fragment and the IgG1 expression vector fragment were linked using T4 ligase to produce a recombinant vector, and the DH5a strain was transformed with the recombinant vector. The nucleotide sequences of the transformants were analyzed to confirm that the variable and constant region sequences of the light and heavy chains matched. Next, the recombinant vector produced above was purified to a high degree of purity, then transfected into Expi293 cells and cultured, and candidate antibodies were obtained from the cultured cells.

[0184] Five clones selected from the Fab library and the scFv library were transferred to a Fab or scFv expression vector, expressed and purified, then produced into antibodies and analyzed by SDS-PAGE to confirm expression, and it was confirmed that all five clones were expressed (Fig. 8).

[0185]

[0186] 1.4: Analysis of the binding affinity between the synthesized antibody and mesothelin

[0187] To confirm whether the antibodies (soluble Fab or scFv) produced from the above-mentioned five types of phages do not bind to MSLN (296-580) but bind only to MSLN (296-600), an ELISA was performed.

[0188] Specifically, 30 μL of antigen protein (2 μg / mL in PBS) was added to each well of a 96-well ELISA plate and incubated at 4°C for 16 hours. After the reaction, the solution on the 96-well ELISA plate was removed, and 150 μL of MPBS was added to each well to perform blocking at room temperature for 1 hour. The purified antibody was serially diluted (3-fold serial dilution) using PBS to a set concentration range. The solution on the 96-well ELISA plate was removed, and 30 μL of the serially diluted antibody was added to each well and incubated at 37°C for 1 hour. After the reaction, the 96-well ELISA plate was washed four times with PBST, and 30 μL of detection antibody (1:10,000 dilution) was added to each well and incubated at 37°C for 1 hour. Goat anti-Human Kappa Light Chain Antibody HRP Conjugated (Fab detection) or StrepMAB-Classic HRP (scFv detection) were used as the detection antibodies. The 96-well ELISA plates were washed four times with PBST, and 30 μL of TMB substrate was added to each well to induce a color reaction at 25 °C for 4 minutes. After stopping the reaction by adding 30 μL of 17% phosphate to each well of the 96-well ELISA plate, the absorbance was measured at 450 nm using a microplate reader. The EC50 values ​​were calculated by generating a graph from the derived absorbance data using 4-parameter logistic regression analysis.

[0189] If binding to the antigen protein was not confirmed by the above method, antigen binding analysis was performed by the following method. Specifically, biotin was conjugated to MSLN (296-580) using the EZ-Link Sulfo-NHS-Biotinylation Kit. Pierce Streptavidin Coated Plates were washed three times with wash buffer (25 mM Tris, 150 mM NaCl, 0.1% BSA, 0.05% Tween-20, pH 7.5), and 50 μL of biotinylated MSLN (296-580) and MSLN (296-600) was added to each well, followed by incubation at 25°C for 2 hours. The purified antibodies were serially diluted (3-fold serial dilution) to a set concentration range using PBS. The plates were washed three times with wash buffer, and 50 μL of the serially diluted antibodies was added to each well, followed by incubation at 25°C for 1 hour. The above plate was washed three times with wash buffer, and 50 μL of the above detection antibody (1:10,000 dilution) was added to each well, followed by a reaction at 25°C for 1 hour. The above plate was washed three times with wash buffer, and 50 μL of TMB substrate was added to each well to perform a colorimetric reaction at 25°C for 4 minutes. After terminating the reaction by adding 30 μL of 17% phosphoric acid to each well of the above plate, the absorbance was measured at 450 nm using a microplate reader. The EC50 value was calculated by generating a graph using 4-parameter logistic regression analysis of the calculated absorbance data.

[0190] As a result of the above experiment, it was confirmed that among the five types of antibodies, 1D8 and 2C6 bind to MSLN (269-580), and 1A4, 1G6 and 1E9 do not bind to MSLN (269-580) but bind only to MSLN (296-600) (Figs. 9 to 11).

[0191]

[0192] 1.5: Confirmation of the binding ability of the synthesized antibody to mesothelin-expressing cells

[0193] To determine whether antibodies 1A4, 1G6, and 1E9, which specifically bind to the C-terminus of mesothelin selected in Example 1.4 above, can also bind to cells expressing mesothelin, the following experiment was performed.

[0194] Specifically, 10 K562 cell lines or AsPC-1 cell lines per well in a 96-well plate 6 Cells were dispensed. Each purified antibody (100 μg / mL) was dispensed into each well containing cells and incubated at 4°C for 1 hour. After incubation, the cells were centrifuged at 1000 rpm for 5 minutes to remove the supernatant, then resuspended in DPBS and washed by centrifugation; this process was repeated twice. After washing and resuspending in DPBS, 10 μL of anti-kappa-FITC was added to the wells containing the Fab antibody, and 10 μL of anti-his-FITC was added to the wells containing the scFv antibody, and the cells were reacted at 4°C for 1 hour. Each reacted cell was centrifuged at 1000 rpm for 5 minutes to remove the supernatant, then resuspended in DPBS and centrifuged. After resuspending each cell in DPBS, fluorescence was measured using a flow cytometer.

[0195] First, K562 cell line, a cell line that does not express mesothelin, and AsPC-1 cell line, a cell line that expresses mesothelin, were analyzed by FACS using a known mesothelin antibody conjugated with FITC. As a result, it was confirmed that the mesothelin antibody bound only to the AsPC-1 cell line (Fig. 12).

[0196] Next, as a result of confirming by FACS using the same method as above whether the five types of scFvs / Fab (1G6, 1E9, 1A4, 1D8, and 2C6) obtained through screening in Example 1 above bind to mesothelin expressed on the cell membrane, it was confirmed that antibodies 1G6 and 1E9 bind to AsPC-1 cell lines expressing mesothelin (Figs. 13 and 14).

[0197]

[0198] 1.6: Derivation of Final Candidate Antibodies

[0199] Based on the above experiments, 1G6 and 1E9 were selected as antibodies that specifically recognize and bind to the C-terminus of mesothelin. The sequence information (light chain CDR sequence, heavy chain CDR sequence, light chain variable region, heavy chain variable region, and scFv) of the 1G6 and 1E9 antibodies is listed in Table 2 below.

[0200]

[0201] In addition, an example of a nucleotide sequence encoding the amino acid sequences of the above 1G6 and 1E9 antibodies is listed in Table 3 below.

[0202]

[0203]

[0204] Example 2: Construction of an Anti-MSLN Chimeric Antigen Receptor (CAR)

[0205] We intended to clone an anti-MSLN-CAR lentivirus vector to construct a chimeric antigen receptor containing the 1G6 and 1E9 antibodies identified in Example 1 above or their antigen-binding fragments.

[0206] First, vectors containing the scFv (antigen-binding domain) of 1G6 or 1E9 antibodies and lentivirus vectors 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 then purified using the FavorPrep Gel / PCR purification Mini kit (Favorgen). Each purified anti-MSLN scFv (100 ng) and vector (50 ng) were reacted in a 2:1 ratio at 16°C for 16 hours to perform ligation, after which they were transformed into Stbl3 competent cells to obtain colonies. These colonies were taken and cultured in 5 mL of LB medium (ampicillin), and plasmid DNA was obtained using the DNA plasmid mini-prep method. We confirmed whether each anti-MSLN scFv inserted by cleaving the above plasmid DNA with XhoI and EcoRI was successfully cloned into the vector. Subsequently, we performed sequencing to finally confirm the DNA sequence.

[0207] An anti-MSLN-CAR was constructed by sequentially linking the CD8 hinge and CD8™ (transmembrane) as the transmembrane region, the cytoplasmic region of 4-1BB as the signaling domain, and the intracellular domain of CD3 zeta (CD3z) as the T cell activation domain to the above anti-MSLN scFv. Specifically, it consists of a CD8 signal sequence (Signal peptide, SP), a 1G6 or 1E9 scFv, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB signaling domain, and a CD3 zeta signaling domain. Each of the above domains was sequentially linked using respective restriction enzymes. Specific nucleotide and amino acid sequence information corresponding to each domain is summarized in the table below.

[0208] Name Amino acid sequence SEQ ID number CD8MALPVTALLLPLALLLHAARP42CD8 hingeTTTPAPRPTPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD43CD8 TMIYIWAPLAGTCGVLLLSLVITLYC444-1BBKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL45CD3zRVKFSRSADAPAYQQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR46

[0209] Name Base Sequence (5'-3') Sequence Number CD8ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG47CD8 hingeACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT48CD8 TMATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC494-1BBAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGT ACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAAGGAGGATGTGAACTG50CD3zAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTA TAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGAT GGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC51

[0210]

[0211] Example 3: Preparation of anti-MSLN-CAR introduced cells

[0212]

[0213] 3-1. Production of Lentivirus Loaded with Anti-MSLN-CAR

[0214] The recombinant vector prepared in Example 2 above was introduced into HEK293T cells to produce an anti-MSLN-CAR lentivirus. First, 6 x 10 HEK293T cells were placed in a 100 mm tissue culture dish one day before DNA transformation. 6 Cells were seeded into a dish. The next day, when the cell density reached 70–80%, MSLN-CAR-pLV, pMDLg / pRRE (addgene), pRSV-Rev (addgene), and pMD2.G (addgene) (5.5 μg:3.5 μg:1.5 μg:2 μg) were transformed using Lipofectamine 3000 (Thermofisher) according to the reagent instructions. CD19 (FMC63) was used as a control. Four hours after transformation, the medium was replaced with DMEM containing 3% FBS (Gibco), and the virus cultures were harvested after 48 hours. 10 mL of 20% sucrose solution was placed in a centrifuge tube, and 20 mL of the harvested virus culture was carefully placed on top. The tube was then mounted on a SW32T rotor, and ultra-high-speed centrifugation was performed at 25,000 rpm at 4°C for 90 minutes. After centrifugation, the supernatant was carefully discarded to prevent the virus pellet at the bottom of the tube from falling off. 400 μL of RPMI1640 medium (Gibco) was added, and the mixture was incubated in a refrigerator for 16 hours. The mixture was then resuspended, dispensed in 100 μL aliquots, and stored at -80°C.

[0215]

[0216] 3-2. Lentivirus titration

[0217] 1.5 x 10⁶ HeLa cells in a 6-well plate the day before lentivirus infection 5Cells were seeded per well. The following day, the virus was diluted to 1 / 100 and 1 / 1,000 and added to 500 μL of virus infection medium, along with 8 μg / mL of polybrene, to infect the cells. In one well, cells were treated with Trypsin-EDTA (0.05%), harvested, and counted. After 4 hours, 1 mL of cell culture medium was added, and viral titers were measured via FACS analysis after 48 hours. Viral titers were calculated using the following formula.

[0218] Viral Titer (Tu / mL) = Number of Cells x Percentage of FACS Positive Cells (%) x Dilution Factor x 2

[0219] As a result, the viral titer was MSLN34 scFv 4.33 X 10 7 Tu / mL, 1G6 scFv 9.9 X 10 6 Tu / mL, 1E9 scFv 6.83 X 10 7 It was confirmed as Tu / mL.

[0220]

[0221] 3-3. Lentivirus transduction

[0222] Transduction was performed in a total of two cycles. Anti-CD3 (1 μg / mL) and anti-CD28 (3 μg / mL) antibodies were prepared to the specified concentrations in 5 mL of DPBS, respectively, and vortexed. The mixture was then coated onto 24-well plates at a rate of 500 μl / well and stored overnight in a 4°C refrigerator. The following day, PBMCs (human primary PBMCs) were 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. Subsequently, the supernatant was removed, and the cells were resuspended in 1 mL of culture medium for cell counting. The count was set to 1 x 10⁶ 6The cells were diluted to cells / mL, seeded into antibody-coated 24-well plates, and cultured in a CO2 incubator at 37°C. After 3 days, all PBMC cells were harvested, and 5 x 10⁶ cells were prepared for lentivirus infection. 5 Lentivirus was added to 500 μL of cells at an MOI (multiple of infection) of 5, and 10 μg / mL of protamine sulfate was added and seeded into a new 24-well plate. (a) The 24-well plate was centrifuged at 300 g and 32°C for 90 minutes, and (b) cultured in a CO2 incubator at 37°C. The next day, all T cells were harvested, and steps (a) and (b) were performed once more. Then, all 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.

[0223]

[0224] 3-4. Confirmation of Anti-MSLN-CAR Expression

[0225] The presence or absence of CAR expression in T cells introduced with the anti-MSLN-CAR prepared in Example 3-3 above was confirmed. Five days after the completion of lentiviral transduction of T cells, some anti-MSLN-CAR-T cells were harvested, biotin-MSLN [296-580] and biotin-MSLN [296-600] (Acrobiosystems) were added, and the cells were cultured on ice for 20 minutes. After washing the cells, 1 μL of PE-anti-biotin was added, and the cells were cultured on ice for 20 minutes. After washing the cells, CAR expression was confirmed using FACS Canto II (BD).

[0226]

[0227] Example 4: Confirmation of C-terminal specific binding of anti-MSLN-CAR to mesothelin

[0228] To confirm whether the anti-MSLN-CAR produced based on the antibody of the present invention specifically binds to the C-terminus of mesothelin, the following experiment was performed.

[0229] Specifically, a lentivirus expressing an anti-MSLN-CAR containing a fragment of 1E9 was transfected into HEK 293 cells, and then the expression level was confirmed by FACS after binding to fluorescently labeled MSLN (269-580) protein or MSLN (269-600) protein. Meanwhile, as a control, an anti-MSLN-CAR containing a fragment of MSLN34 (Korean Registered Patent No. 10-2309543) was used.

[0230] As a result of the above experiment, the control group bound to both MSLN (269-580) and MSLN (269-600) at a level of 99% or higher, but it was confirmed that the anti-MSLN-CAR containing 1E9 of the present invention binds to MSLN (269-600) but does not bind to MSLN (269-580) with a deleted C-terminus (Fig. 15).

[0231] Based on the above results, it can be seen that the CAR containing the antibody of the present invention can recognize and bind only to the C-terminal region of mesothelin.

[0232]

[0233] Example 5: Confirmation of the apoptotic effect of anti-MSLN-CAR-T cells on cancer cells

[0234] To confirm the apoptotic effect of the anti-MSLN-CAR-T cells produced in Example 3 above on cancer cells, the following experiment was performed.

[0235] Specifically, to confirm the apoptotic effect on cancer cells using the calcein-AM method, first, cancer cell lines (lung cancer cell line NCI-H2052, ovarian cancer cell line OVCAR-3) were placed in each culture medium at a rate of 1 x 10 6The cells were resuspended at cells / mL, 5 μL of Calcein-AM (1 mg / mL) was added and mixed well, and the cells were incubated in a 37°C incubator for 1 hour. Anti-MSLN-CAR-T effector cells were prepared by diluting the cells at various E:T (effector cell:target cell) ratios while adding cell culture medium. 1 hour after staining the cancer cell lines with Calcein-AM, the cells were washed by centrifugation at 1,200 rpm for 5 minutes, and 10 mL of culture medium was added to resuspend the cells. Then, 100 μL (1 x 10⁶) of the stained cancer cell lines were transferred to a 96-well round plate. 4 Cells were seeded with 100 μL of culture medium, and 100 μL of effector cells were seeded on top. As controls, a group of cancer cell lines stained with Calcein-AM treated with only 100 μL of culture medium (spontaneous value) and a group treated with 2% Triton X-100 (maximum value) were used. After centrifuging the 96-well round plates at 100g for 1 minute, they were incubated in a 37°C incubator for 4 hours. After 4 hours, the cells in the wells were mixed about 5 times with a pipette, and after centrifuging at 100g for 5 minutes, 100 μL of only the supernatant was taken and transferred to a test 96-well plate. Using the 96-well plate containing the supernatant, Calcein emission was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a fluorescence microplate reader. For the measured values, the apoptosis effect was calculated using the following formula.

[0236] Cell death effect (%)=[(experimental release -spontaneous release) / (maximum release - spontaneous release)]

[0237] Through the above experiment, the apoptotic effect on an ovarian cancer cell line (OVCAR-3), one of the cancers expressing mesothelin, using anti-MSLN-CAR-T cells containing a fragment of 1G6 or 1E9 as an anti-mesothelin antibody was confirmed, and it was confirmed that it has excellent killing ability (Fig. 16).

[0238] Next, to further confirm the apoptotic effect on other cancers expressing mesothelin, anti-MSLN-CAR-T cells containing a fragment of 1E9 were used to confirm the apoptotic effect on a lung cancer cell line (NCI-H322), one of the cancers expressing mesothelin, and it was confirmed that they had excellent killing ability (Fig. 17).

[0239] Based on the above results, it can be seen that anti-MSLN-CAR-T cells using the antibody of the present invention, which specifically binds to the C-terminus of mesothelin, have the ability to kill cancer cells.

[0240]

[0241] Example 6: Confirmation of the apoptotic effect of anti-MSLN-CAR-T cells on cancer cells in the presence of sMSLN

[0242] The antibody of the present invention, which specifically binds to the C-terminus of mesothelin, is characterized by not binding to sMSLN. Accordingly, to confirm whether the anti-MSLN-CAR-T cells produced in Example 3 above can effectively recognize cancer cells and induce apoptosis even in the presence of sMSLN, the following experiment was performed.

[0243] Specifically, anti-MSLN-CAR-T cells containing a fragment of 1E9 or anti-MSLN-CAR-T cells containing a fragment of MSLN34 (Korean Registered Patent No. 10-2309543) (control group) were incubated with sMSLN [MSLN(269-580)] for 1 hour. The incubated anti-MSLN-CAR-T cells were used to confirm the apoptotic effect on an ovarian cancer cell line (OVCAR-3).

[0244] To this end, 1 x 10⁶ ovarian cancer cell lines OVCAR-3 were placed in each culture medium. 6 The cells were resuspended at cells / mL, 5 μL of Calcein-AM (1 mg / mL) was added and mixed well, and incubated in a 37°C incubator for 1 hour. 10 μg of sMSLN [MSLN(269-580)] was added to anti-MSLN-CAR-T effector cells and reacted in a 37°C incubator for 1 hour; subsequently, the cells were diluted to various E:T (effector cell:target cell) ratios while adding cell culture medium. One hour after staining the cancer cell line with Calcein-AM, the cells were washed by centrifugation at 1,200 rpm for 5 minutes, and resuspended with 10 mL of culture medium. Then, 100 μL (1 x 10⁶) of the stained cancer cell line was transferred to a 96-well round plate. 4Cells were seeded with 100 μL of culture medium, and 100 μL of effector cells were seeded on top. As controls, a group of cancer cell lines stained with Calcein-AM treated with only 100 μL of culture medium (spontaneous value) and a group treated with 2% Triton X-100 (maximum value) were used. After centrifuging the 96-well round plates at 100g for 1 minute, they were incubated in a 37°C incubator for 4 hours. After 4 hours, the cells in the wells were mixed about 5 times with a pipette, and after centrifuging at 100g for 5 minutes, 100 μL of only the supernatant was taken and transferred to a test 96-well plate. Using the 96-well plate containing the supernatant, Calcein emission was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a fluorescence microplate reader. For the measured values, the cell death effect was calculated using the formula described in Example 4 above.

[0245] As a result of the above experiment, it was confirmed that anti-MSLN-CAR-T cells produced with MSLN34 capable of binding to sMSLN showed reduced killing ability against cancer cells in the presence of sMSLN, whereas anti-MSLN-CAR-T cells produced with 1E9 that does not bind to sMSLN maintained killing ability against cancer cells even in the presence of sMSLN (Fig. 18).

[0246] Based on the above results, it can be seen that the anti-MSLN-CAR-T cells produced using the antibody that specifically binds to the C-terminus of the present invention can effectively kill cancer cells even under conditions where the mesothelin present on the surface of the cancer cells is shed by protease.

[0247]

[0248] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

An anti-mesothelin antibody or an antigen-binding fragment thereof characterized by specifically binding to the C-terminus of mesothelin. 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. 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. 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. 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 comprising an antigen-binding fragment comprising the amino acid sequence of SEQ ID NO. 9 or the antigen-binding fragment thereof, and 2) An antibody or its antigen-binding fragment comprising an antigen-binding fragment comprising the amino acid sequence of SEQ ID NO.

18. 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. Isolated nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6. A vector comprising the isolated nucleic acid of claim 7. Isolated host cells transformed with the vector of claim 8. 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. 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. 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. A chimeric antigen receptor according to claim 11, wherein the antigen-binding fragment is a scFv (single chain variable fragment). Polynucleotide encoding the chimeric antigen receptor of claim 11. In claim 14, The above polynucleotide comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NOs 27 and 36. A vector comprising the polynucleotide of claim 14. Isolated cell comprising the chimeric antigen receptor of claim 11. In claim 17, the isolated cell is a T cell, NK cell, NKT cell, or gamma delta T cell (gamma delta (γδ) T cells). A pharmaceutical composition for the prevention or treatment of cancer comprising isolated cells of claim 17. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6; and Antibody-drug conjugate containing a drug. A pharmaceutical composition for the prevention or treatment of cancer comprising the antibody-drug conjugate of claim 20. An epitope of mesothelin comprising the amino acid sequence of SEQ ID NO.

41. Polynucleotide encoding the epitope of claim 22.

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