Chimeric antigen receptor with increased affinity for mesothelin and use thereof

Abstract

The present invention relates to an anti-mesothelin chimeric antigen receptor that specifically binds to mesothelin with increased affinity for mesothelin. According to one embodiment, the anti-mesothelin chimeric antigen receptor exhibits specific binding ability to mesothelin with increased affinity for mesothelin and can be useful for preventing or treating cancers in which mesothelin is overexpressed. One embodiment provides an anti-mesothelin antibody, or an antigen-binding fragment thereof, with increased affinity for mesothelin. Another embodiment provides an isolated nucleic acid encoding the anti-mesothelin antibody, or an antigen-binding fragment thereof, with increased affinity for mesothelin.

Description

[Technical Field]

[0001] The present invention relates to an anti-mesothelin chimeric antigen receptor that specifically binds to mesothelin and has increased affinity for mesothelin, and uses thereof. [Background technology]

[0002] Recently, immune-mediated anti-cancer drugs such as immune checkpoint inhibitors and CAR-T cell therapy have proven effective against a variety of cancers. However, solid tumors have not shown significant therapeutic efficacy, even with these new forms of immune-mediated anti-cancer drugs. This is presumably because the fibrous tissue surrounding the tumor interferes with the immune-therapeutic response, making drug delivery difficult. Therefore, there is a growing need for research into specific and more effective CAR-T cancer treatment methods, such as the development of antibodies with high affinity that target proteins specifically overexpressed on the surface of solid tumor cells as cancer antigens, and the use of these antibodies to effectively treat solid tumors.

[0003] Mesothelin is a glycoprotein anchored to the cell surface by a glycosylphosphatidylinositol (GPI) domain. It is normally expressed at low levels in the mesothelium, which surrounds cavities and internal organs in the human body. However, it is known to be abundantly expressed in cancers such as pancreatic cancer, mesothelioma, ovarian cancer, and non-small cell lung cancer. Summary of the Invention

[0004] One aspect provides an anti-mesothelin antibody, or antigen-binding fragment thereof, that has increased affinity for mesothelin.

[0005] Another aspect provides an isolated nucleic acid encoding the anti-mesothelin antibody, or antigen-binding fragment thereof, that has increased affinity for mesothelin.

[0006] Yet another aspect provides a vector comprising the isolated nucleic acid.

[0007] A further aspect provides an isolated host cell transformed with the vector.

[0008] Yet another aspect provides a method for producing an anti-mesothelin antibody with increased affinity for mesothelin, comprising culturing the isolated host cell to express the antibody.

[0009] Yet another embodiment provides a chimeric antigen receptor with increased affinity for mesothelin, which comprises an antigen-binding domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain.

[0010] Yet another aspect provides a polynucleotide encoding the chimeric antigen receptor.

[0011] Yet another aspect provides a vector comprising the polynucleotide.

[0012] Yet another aspect provides an isolated cell transformed with the vector.

[0013] Yet other aspects provide pharmaceutical compositions comprising the isolated cells; pharmaceutical uses of the cells; and methods for preventing or treating cancer, comprising administering a therapeutically effective amount of the cells to an individual.

[0014] Other objects and advantages of the present application will become more apparent from the following detailed description taken in conjunction with the appended claims and drawings. Contents not described in this specification can be fully understood and inferred by those skilled in the art of the present application or a similar art, and therefore, the description thereof will be omitted. [Means for solving the problem]

[0015] Each description and embodiment disclosed in this application may be applied to each other description and embodiment. In other words, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of this application is not limited by the specific descriptions described below.

[0016] One aspect provides an anti-mesothelin (MSLN) antibody, or an antigen-binding fragment thereof, comprising: a heavy chain variable region comprising heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 19, heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence of SEQ ID NO: 20, and heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence of SEQ ID NO: 21; and a light chain variable region comprising light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 22, light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 23, and light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence of SEQ ID NO: 24; wherein the heavy chain variable region and the light chain variable region contain one or more amino acid substitutions.

[0017] Mesothelin (MSLN) is a cell surface glycoprotein (NCBI Gene ID: 10232) with a total amino acid length of 622 aa that is selectively expressed in some cells, particularly certain tumor cells. The amino acid sequence of the mesothelin protein is shown below. (SEQ ID NO: 60)

[0018] Mesothelin is expressed at low levels in normal mesothelial cells, but is highly expressed in solid tumors, including 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 childhood acute myeloid leukemia (AML) (Cancer Discov. 2016 Feb; 6(2): 133-46.). J Reprod Immunol. 2020; 139: 103115; Gynecol Oncol. 2007; 105(3): 563-570.; Eur J Haematol. 2007; 79(4): 281-286).

[0019] As used herein, the term "antibody" refers collectively to proteins that selectively act on antigens and are involved in biological immunity, and the type of antibody is not particularly limited. The heavy and light chains of the antibody have antigen-binding sites that recognize epitopes containing variable regions, and antigen specificity is determined by sequence variations in the variable regions. The variable regions of the antigen-binding sites are divided into framework regions (FRs) with low variability and complementarity-determining regions (CDRs) with high variability. Both the heavy and light chains have three CDR regions, each divided into CDR1, CDR2, and CDR3, and four FR regions. The CDRs of each chain are typically designated CDR1, CDR2, and CDR3, sequentially, starting from the N-terminus, and are identified by the chain in which a particular CDR is located.

[0020] As used herein, the term "complementarity determining region" refers to a site in an antibody variable region that confers antigen-binding specificity.

[0021] As used herein, the term "epitope" refers to a specific three-dimensional molecular structure within an antigen molecule to which an antibody can specifically bind.

[0022] The antibodies include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, and chimeric antibodies (e.g., humanized murine antibodies). The antibodies also include diabodies, triabodies, and tetrabodies.

[0023] As used herein, the term "antibody" includes "antigen-binding fragments" or "antibody fragments" of antibodies that retain antigen-binding ability. The antigen-binding fragments also include antibody fragments containing one or more complementarity-determining regions, such as those selected from the group consisting of scFv (single-chain variable fragment), (scFv)2, scFv-Fc, Fab, Fab', and F(ab')2. Among these antibody fragments, Fab has a structure comprising light-chain and heavy-chain variable regions, a light-chain constant region, and a heavy-chain first constant region (CH1), and possesses one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy-chain CH1 domain. F(ab')2 antibodies are produced by disulfide bonding between the cysteine ​​residues in the hinge region of Fab'. Fv is the smallest antibody fragment containing only the heavy-chain variable region and the light-chain variable region. In a two-chain Fv, the heavy-chain variable region and the light-chain variable region are linked by a non-covalent bond. In a single-chain Fv (scFv: single-chain Fv), the heavy-chain variable region and the light-chain variable region are generally linked by a covalent bond via a peptide linker or directly at the C-terminus, and can form a dimer-like structure like a two-chain Fv.

[0024] In one embodiment, the anti-mesothelin antibody, or antigen-binding fragment thereof, comprises one or more amino acid substitutions, and the one or more amino acid substitutions may comprise about 1 to 5, about 1 to 4, about 1 to 3, or about 1 to 2 amino acid substitutions within the amino acids of SEQ ID NO:1.

[0025] The one or more amino acid substitutions may also be at one or more positions selected from the group consisting of the first position of SEQ ID NO: 19 (the first amino acid of HCDR1), the 7th position of SEQ ID NO: 23 (the 7th amino acid of LCDR2), and the 4th position of SEQ ID NO: 24 (the 4th amino acid of LCDR3).

[0026] In one embodiment, the one or more amino acid substitutions also include one or more selected from the group consisting of the following groups 1) to 3): 1) the first amino acid of SEQ ID NO: 19 is substituted from D to K, W, L, or R; 2) The 7th amino acid of SEQ ID NO: 23 is substituted from S to F or R, and 3) The fourth amino acid of SEQ ID NO: 24 is substituted from Y to R.

[0027] In this specification, the substitution of the first amino acid of SEQ ID NO: 19 from D to K, W, L or R may be expressed as D31L, D31K, D31W or D31R, respectively; the substitution of the seventh amino acid of SEQ ID NO: 23 from S to F or R may be expressed as S192F or S192R, respectively; and the substitution of the fourth amino acid of SEQ ID NO: 24 from Y to R may be expressed as Y228R.

[0028] In one embodiment, the one or more amino acid substitutions are one or more selected from the group consisting of the following groups 1) to 3): 1) the first amino acid of SEQ ID NO: 19 is substituted from D to L; 2) The seventh amino acid of SEQ ID NO: 23 is substituted from S to R, and 3) The first amino acid of SEQ ID NO: 19 is substituted from D to L, and the seventh amino acid of SEQ ID NO: 23 is substituted from S to R.

[0029] In one embodiment, the anti-mesothelin antibody, or antigen-binding fragment thereof, is also selected from antibodies, or antigen-binding fragments thereof, comprising a heavy chain variable region comprising the following heavy chain CDRs and a light chain variable region comprising the following light chain CDRs: 1) an antibody, or an antigen-binding fragment thereof, comprising: a heavy chain variable region comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 27, HCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 29; and a light chain variable region comprising LCDR1 comprising the amino acid sequence of SEQ ID NO: 30, LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 32; 2) an antibody, or an antigen-binding fragment thereof, comprising: a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 35, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 36, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 38, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 39, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 40; and 3) An antibody, or an antigen-binding fragment thereof, comprising: a heavy chain variable region comprising HCDR1 having an amino acid sequence consisting of SEQ ID NO: 43, HCDR2 having an amino acid sequence consisting of SEQ ID NO: 44, and HCDR3 having an amino acid sequence consisting of SEQ ID NO: 45; and a light chain variable region comprising LCDR1 having an amino acid sequence consisting of SEQ ID NO: 46, LCDR2 having an amino acid sequence consisting of SEQ ID NO: 47, and LCDR3 having an amino acid sequence consisting of SEQ ID NO: 48.

[0030] In one embodiment, the anti-mesothelin antibody, or antigen-binding fragment thereof, is selected from the group consisting of antibodies, or antigen-binding fragments thereof, comprising the heavy chain variable region and light chain variable region: 1) an antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 34; 2) an antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 41 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 42; and 3) An antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 50.

[0031] In one embodiment, the anti-mesothelin antibody, or antigen-binding fragment thereof, is or comprises a single chain variable fragment (scFv), and the anti-mesothelin scFv comprising one or more amino acid substitutions comprises a substitution selected from the group consisting of the following amino acid substitutions: 1) the 31st amino acid of SEQ ID NO: 1 is substituted from D to L (D31L); 2) The 192nd amino acid of SEQ ID NO: 1 is substituted from S to R (S192R), and 3) The 31st amino acid of SEQ ID NO: 1 is substituted from D to L, and the 192nd amino acid of SEQ ID NO: 1 is substituted from S to R (D31L / S192R).

[0032] In one embodiment, the anti-mesothelin antibody, or antigen-binding fragment thereof, is selected from the group consisting of antibodies, or antigen-binding fragments thereof, including the following antigen-binding fragments: 1) an antibody comprising an antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof; 2) an antibody comprising an antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 3, or an antigen-binding fragment thereof; and 3) An antibody comprising an antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 4, or an antigen-binding fragment thereof. [Table 1]

[0033] The anti-mesothelin antibody or antigen-binding fragment thereof is characterized by comprising a heavy chain variable region having an amino acid sequence having 80% or more sequence homology, preferably 90% or more sequence homology, more preferably 95% or more sequence homology, and even more preferably 100% sequence homology, with the amino acid sequence of SEQ ID NO: 33, 41, or 49.

[0034] The anti-mesothelin antibody or antigen-binding fragment thereof is characterized by comprising a light chain variable region containing an amino acid sequence having 80% or more sequence identity, preferably 90% or more sequence identity, more preferably 95% or more sequence identity, and even more preferably 100% identity, to the amino acid sequence of SEQ ID NO: 34, 42, or 50.

[0035] Considering variations with biologically equivalent activity, an antibody of one embodiment, or a nucleic acid molecule encoding the antibody, may also be construed as including sequences that exhibit substantial identity to the sequence set forth in SEQ ID NO: The term "substantial identity" as used herein refers to a sequence that exhibits at least 61% homology, preferably 70% homology, more preferably 80% homology, even more preferably 90% homology, even more preferably 95% homology, even more preferably 98% homology, and most preferably 99% homology when the sequence is aligned with any other sequence for maximum correspondence and the aligned sequences are analyzed using algorithms commonly used in the art. Alignment methods for sequence comparison are known in the art.

[0036] When one or more amino acid substitutions are included, the affinity for mesothelin may be increased, thereby exhibiting improved mesothelin affinity. When one or more amino acid substitutions are included, the binding ability to the target mesothelin may be increased compared to the wild-type antibody lacking such substitutions.

[0037] Another embodiment provides an anti-mesothelin antibody, or antigen-binding fragment thereof, containing one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1. The same applies to the antibody or antigen-binding fragment thereof as described above.

[0038] In one embodiment, the anti-mesothelin antibody, or antigen-binding fragment thereof, is an anti-mesothelin scFv comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1. The one or more amino acid substitutions may include substitutions of about 1 to 5, about 1 to 4, about 1 to 3, or about 1 to 2 amino acids within the amino acid sequence of SEQ ID NO: 1.

[0039] The one or more amino acid substitutions may be at one or more positions selected from the group consisting of positions 31, 192, and 228 of SEQ ID NO: 1. For example, the one or more amino acid substitutions may be at one or more positions selected from the group consisting of the following groups (a)-c): a) D31K, D31W, D31L and D31R, b) S192F and S192R, and c) Y228R

[0040] When one or more amino acid substitutions are included, the affinity for mesothelin may be increased, thereby exhibiting improved mesothelin affinity. When one or more amino acid substitutions are included, the binding ability to the target mesothelin may be increased compared to a wild-type antibody lacking such substitutions.

[0041] The anti-mesothelin scFv containing one or more amino acid substitutions may be, for example, one or more selected from the group consisting of SEQ ID NOs: 2, 3, and 4. Yet another aspect provides an isolated nucleic acid encoding the antibody, or antigen-binding fragment thereof. The same applies as above.

[0042] As used herein, the term "nucleic acid" is intended to encompass both DNA molecules and RNA molecules. Nucleotides, the basic building blocks of nucleic acids, include not only natural nucleotides but also analogues in which the sugar moiety or base moiety is modified. In one embodiment, the nucleic acid sequences encoding the heavy chain variable region and the light chain variable region may be modified. Such modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides. The nucleic acid is also understood to include a nucleotide sequence that is substantially identical to the nucleotide sequence of the nucleic acid. The term "substantial identity" refers to a nucleotide sequence that is at least 80% identical, more preferably at least 90% identical, and most preferably at least 95% identical, when the nucleotide sequence of one embodiment is aligned with any other sequence to maximize correspondence and the aligned sequences are analyzed using algorithms commonly used in the art.

[0043] A further aspect provides a vector comprising the isolated nucleic acid. The same applies to the vector as described above.

[0044] The vector contains DNA encoding partial or full-length light and heavy chains for expression of an antibody or antibody fragment thereof in a suitable host cell, obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma expressing the antibody of interest), and also contains the necessary regulatory elements operably linked to enable expression of the DNA (gene) insert. "Operably linked" means that the nucleic acid expression control sequence and the nucleic acid sequence encoding the protein or RNA of interest are functionally linked to perform their general function, and is linked so that the gene can be expressed by the expression control sequence.

[0045] The term "expression control sequence" refers to a DNA sequence that regulates the expression of an operably linked DNA sequence in a specific host cell. Such regulatory sequences include a promoter for transcription, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, sequences regulating the termination of transcription and translation, an initiation codon, a termination codon, a polyadenylation signal, and an enhancer. Those skilled in the art will recognize that different regulatory sequences may be selected and expression vectors may be designed differently depending on factors such as the choice of host cell to be transformed and the expression level of the protein. The vector is not particularly limited in type, as long as it is a vector commonly used in the fields of cloning and antibody production, including, but not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. Examples of such vectors include E. coli-derived plasmids (pBR322, pBR325, pUC118, pUC119, and pET-21b(+)), Bacillus subtilis-derived plasmids (pUB110 and pTP5), and yeast-derived plasmids (YEp13, YEp24, and YCp50). Examples of such viruses include animal viruses such as retroviruses, adenoviruses, and vaccinia viruses, and insect viruses such as baculoviruses. pComb3-based vectors commonly used for phage display can also be used. To express antibodies in mammalian cells, vectors commonly used for expressing proteins in mammalian cells, such as pcDNA and pVITRO, can be used.

[0046] A further aspect provides an isolated host cell transformed with said vector, and the same applies as above to said host cell.

[0047] As used herein, the term "transformation" refers to a molecular biological technique in which a DNA fragment or plasmid carrying a foreign gene different from that contained in the original cell is introduced into the cell and combines with the DNA present in the original cell, thereby changing the genetic traits of the cell. The vector is then transfected into the host cell. For transfection or transfection, various techniques commonly used to introduce exogenous nucleic acid (DNA or RNA) into prokaryotic or eukaryotic host cells can be used, such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection.

[0048] In one embodiment, the antibody or antigen-binding fragment thereof may be expressed in a microorganism, such as bacteria (E. coli) or yeast, or in a eukaryotic cell, preferably a mammalian host cell, for potential application in mammalian cells, such as, but not limited to, Chinese hamster ovary (CHO) cells, NSO myeloma cells, COS cells, SP2 cells, F2N cells, HEK293 cells, and antibody-producing hybridoma cells. Yet another aspect provides a method for producing an anti-mesothelin antibody with increased affinity for mesothelin, comprising culturing the isolated host cell to express the antibody. The same provisions as above also apply to this method.

[0049] The method also includes a step of transforming a host cell for producing an antibody or antigen-binding fragment thereof of one embodiment with a vector to which DNA encoding the antibody or antigen-binding fragment thereof is operably linked. The host cell and the type of recombinant expression vector selected are as described above, and an appropriate transformation method can be selected to carry out this step. When the recombinant expression vector encoding the antibody gene is introduced into a mammalian host cell, the antibody can be produced by culturing the host cell for a period sufficient to express the antibody in the host cell, or more preferably, for a period sufficient to secrete the antibody into the culture medium in which the host cell is cultured.

[0050] The method may further include culturing the transformed, isolated host cells to produce a polypeptide of the antibody or antigen-binding fragment thereof according to one embodiment from the recombinant expression vector introduced into the host cells. By appropriately selecting the medium composition, culture conditions, and culture time for culturing the selected host cells, the antibody molecules produced in the host cells are accumulated in the cytoplasm of the cells, or are secreted to the outside of the cells or into the culture medium via an appropriate signal sequence, or are targeted to the periplasm, etc. Furthermore, to maintain the binding specificity of the antibody according to one embodiment to mesothelin, the protein is refolded to a functional structure using methods known in the art. When producing an IgG antibody, the heavy and light chains can be expressed in separate cells and contacted in a separate step to form a complete antibody, or the heavy and light chains can be expressed in the same cell to form a complete antibody within the cell.

[0051] The method may further include obtaining the antibody or antigen-binding fragment thereof produced in the isolated host cells. The method for obtaining the antibody or antigen-binding fragment thereof may be appropriately selected and adjusted taking into consideration the characteristics of the polypeptide of the antibody or antigen-binding fragment thereof produced in the host cells, the characteristics of the host cells, the expression method, and the targeting of the polypeptide. For example, an antibody or antigen-binding fragment thereof secreted into the culture medium can be recovered by collecting the medium from which the host cells were cultured and centrifuging it to remove impurities. If necessary, the cells can be lysed to an extent that does not affect the functional structure of the antibody or antigen-binding fragment thereof in order to release and recover the antibody present in specific intracellular organelles or the cytoplasm to the outside of the cells.

[0052] The obtained antibody may be further concentrated by removing impurities through methods such as chromatography, filtration, and dialysis. Separation or purification of the obtained antibody may be performed using a separation or purification method commonly used for proteins, such as chromatography. Chromatography may include affinity chromatography, including protein A columns, protein G columns, and protein L columns, ion exchange chromatography, or hydrophobic chromatography. In addition to the above chromatography, antibodies may be separated and purified by combining filtration, ultrafiltration, salting out, dialysis, and the like.

[0053] Yet another embodiment provides a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain, wherein the antigen-binding domain comprises the anti-mesothelin antibody or an antigen-binding fragment thereof. The same applies to the chimeric antigen receptor as described above. The chimeric antigen receptor is characterized by its specific binding to mesothelin and therefore comprises an antigen-binding domain that specifically binds to mesothelin.

[0054] The antigen-binding domain includes a heavy chain variable region comprising heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 19, heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence of SEQ ID NO: 20, and heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence of SEQ ID NO: 21; and a light chain variable region comprising light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 22, light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 23, and light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence of SEQ ID NO: 24, and also includes an anti-mesothelin antibody or antigen-binding fragment thereof, wherein the heavy chain variable region and the light chain variable region contain one or more amino acid substitutions.

[0055] In one embodiment, the one or more amino acid substitutions also include one or more selected from the group consisting of the following groups 1) to 3): 1) the first amino acid of SEQ ID NO: 19 is substituted from D to K, W, L, or R; 2) The 7th amino acid of SEQ ID NO: 23 is substituted from S to F or R, and 3) The fourth amino acid of SEQ ID NO: 24 is substituted from Y to R.

[0056] In this specification, the substitution of the first amino acid of SEQ ID NO: 19 from D to K, W, L or R may be expressed as D31L, D31K, D31W or D31R, respectively; the substitution of the seventh amino acid of SEQ ID NO: 23 from S to F or R may be expressed as S192F or S192R, respectively; and the substitution of the fourth amino acid of SEQ ID NO: 24 from Y to R may be expressed as Y228R.

[0057] In one embodiment, the one or more amino acid substitutions are one or more selected from the group consisting of the following groups 1) to 3): 1) The first amino acid of SEQ ID NO: 19 is substituted from D to L, 2) The seventh amino acid of SEQ ID NO: 23 is substituted from S to R, and 3) The first amino acid of SEQ ID NO: 19 is substituted from D to L, and the seventh amino acid of SEQ ID NO: 23 is substituted from S to R.

[0058] When one or more amino acid substitutions are included, the affinity for mesothelin may be increased, thereby exhibiting improved mesothelin affinity. When one or more amino acid substitutions are included, the binding ability to the target mesothelin may be increased compared to a wild-type antibody lacking such substitutions.

[0059] When one or more amino acid substitutions are included, the affinity for mesothelin may be increased, thereby exhibiting improved mesothelin affinity. When one or more amino acid substitutions are included, the binding ability to the target mesothelin may be increased compared to a wild-type antibody lacking such substitutions. Furthermore, when one or more amino acid substitutions are included, the compound may be shown to have excellent killing ability against cancer cells that express mesothelin, such as mesothelioma, ovarian cancer, and pancreatic cancer.

[0060] As used herein, the term "chimeric antigen receptor (CAR)" refers to a chimeric antigen receptor structure that includes an antigen binding (recognition) domain, a transmembrane domain, and an intracellular signaling domain.

[0061] In one embodiment, the antigen-binding fragment is also an scFv.

[0062] In one embodiment, the antigen-binding domain is selected from an antibody or antigen-binding fragment thereof, including the following antigen-binding fragments: 1) an antibody comprising an antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof; 2) an antibody comprising an antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 3, or an antigen-binding fragment thereof; and 3) An antibody comprising an antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 4, or an antigen-binding fragment thereof.

[0063] The hinge domain, transmembrane domain, and intracellular signaling domain contained in the chimeric antigen receptor are well known in the art.

[0064] The hinge domain, also known as a spacer, connects the anti-mesothelin antibody or its antigen-binding fragment to the transmembrane domain and serves to extend the antigen-binding domain from the T cell membrane. The hinge domain may be, but is not limited to, a CD8 hinge domain, an IgG1 hinge domain, an IgG4 hinge domain, a CD28 ectodomain, a KIR (killer immunoglobulin-like receptor) ectodomain, or a combination thereof. Any hinge domain commonly used in the art may be used.

[0065] The transmembrane domain may serve as a support for the chimeric antigen receptor molecule and connect the hinge domain and the intracellular signaling domain. The transmembrane domain may penetrate the cell membrane so that the anti-mesothelin antibody or its antigen-binding fragment of the chimeric antigen receptor is located on the cell surface and the intracellular signaling domain is located intracellularly. The transmembrane domain may be the transmembrane region of CD3 zeta (CD3z), CD4, CD8, CD28, or KIR protein. Preferably, the transmembrane domain of CD8 or CD28 may be used. However, any common transmembrane domain used in the construction of chimeric antigen receptors may be used without limitation.

[0066] The intracellular signaling domain receives a signal transmitted by an anti-mesothelin antibody or its antigen-binding fragment and transmits it into the cell where the chimeric antigen receptor binds. The intracellular signaling domain is not limited to any particular type, and various types of intracellular signaling domains can be used, as long as they transmit a signal capable of inducing T cell activation upon antibody binding to the extracellular antigen-binding site. The intracellular signaling domain can be, for example, an immunoreceptor tyrosine-based activation motif (ITAM), including, but not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, CD66d, or FcεRIγ.

[0067] Furthermore, according to one embodiment, the chimeric antigen receptor further comprises a costimulatory domain in addition to the intracellular signaling domain.

[0068] The costimulatory domain is a part that plays a role in transmitting a signal to T cells in addition to the signal from the intracellular signaling domain, and refers to the intracellular part of a chimeric antigen receptor that includes the intracellular domain of a costimulatory molecule.

[0069] The costimulatory molecule refers to a cell surface molecule required for sufficient lymphocyte response to an antigen, including, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1 (lymphocyte function-associated antigen-1), CD2, CD7, LIGHT, NKG2C, and B7-H3. The costimulatory domain refers to the intracellular portion of a molecule selected from the group consisting of such costimulatory molecules and combinations thereof.

[0070] Each domain of the chimeric antigen receptor, including the transmembrane domain and the intracellular signaling domain, can be optionally linked by a short oligopeptide or polypeptide linker. The linker is not particularly limited in length, and any linker known in the art can be used, as long as it can induce T cell activation via the intracellular domain when an antigen binds to an extracellularly located antibody.

[0071] The chimeric antigen receptor also includes modified forms of the antibodies and domains described above. The modifications can be achieved by substituting, deleting, or adding one or more amino acids in the amino acid sequence of the wild-type antibody or domain without altering the function of the antibody or domain. Typically, the substitutions are alanine or conservative amino acid substitutions that do not affect the charge, polarity, or hydrophobicity of the overall protein.

[0072] Yet another aspect provides a polynucleotide encoding the chimeric antigen receptor. The same applies to the polynucleotide as described above.

[0073] Those skilled in the art will readily understand that the polynucleotide may be modified in various ways in the coding region, taking into account codon degeneracy or preferred codons in the organism in which the antigen receptor is to be expressed, without changing the amino acid sequence of the antigen receptor expressed from the coding region, and that the portion excluding the coding region may also be modified in various ways without affecting gene expression, and that such modified genes are also within the scope of the present invention. That is, a polynucleotide according to one embodiment may be modified by substitution, deletion, insertion, or a combination of one or more nucleic acid bases, as long as it encodes a protein having equivalent activity, and these modifications are also within the scope of the present invention. Further aspects provide vectors containing the polynucleotides, and isolated cells transformed with the vectors. The same applies to the cells as described above.

[0074] The vector can be a variety of vectors known in the art, and expression regulatory sequences such as promoters, terminators, and enhancers, and sequences for membrane targeting or secretion can be appropriately selected and combined according to the type of host cell in which the antigen receptor is to be produced, and can be variously combined according to the purpose. Vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and virus vectors. Suitable vectors include not only expression regulatory elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals, and enhancers, but also signal sequences or leader sequences for membrane targeting or secretion, and can be prepared in a variety of ways according to the purpose.

[0075] The vector can also be introduced into cells to transform the cells, and the isolated cells can be, but are not limited to, T cells, NK cells, NKT cells, or gamma delta T cells. The isolated cells can be obtained or produced from bone marrow, peripheral blood, peripheral blood mononuclear cells, or umbilical cord blood.

[0076] Further aspects provide pharmaceutical compositions comprising the isolated cells, pharmaceutical uses of the isolated cells, and methods for preventing or treating cancer, comprising administering a therapeutically effective amount of the isolated cells to an individual. The same applies to the compositions and methods as described above.

[0077] Since the pharmaceutical composition utilizes the isolated cells described above, the commonality between the two will be omitted to avoid overcomplicating this specification.

[0078] Said pharmaceutical composition or said medical use may also be for the prevention or treatment of cancer.

[0079] As used herein, the term "prevention" refers to any action of suppressing cancer (tumor) or delaying the onset of the disease by administering the pharmaceutical composition of the present invention.

[0080] As used herein, the term "treatment" refers to any action that improves or favorably alters the symptoms of cancer (tumor) by administering the pharmaceutical composition according to the present invention.

[0081] As used herein, the term "individual" refers to a subject in need of treatment for a disease, and more specifically refers to a mammal such as a human or non-human primate, rodent (e.g., rat, mouse, guinea pig), mouse, dog, cat, horse, cow, sheep, pig, goat, camel, or antelope.

[0082] As used herein, the term "cancer" refers to a cellular disease characterized by aggressive cell division and growth, ignoring normal growth limits, invasive cell invasion into surrounding tissue, and metastatic cell spread to other sites both inside and outside the body. As used herein, "cancer" is used interchangeably with "malignant tumor," and preferably refers to mesothelin-positive cancer or cancer that overexpresses mesothelin.

[0083] The cancer is preferably a solid cancer, for example, more preferably a mesothelin-positive solid cancer or a mesothelin-overexpressing solid cancer. For example, the solid cancer 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), and childhood acute myeloid leukemia (AML), non-small cell lung cancer, and childhood acute myeloid leukemia (AML), but is not limited thereto.

[0084] The pharmaceutical composition may contain 10 to 95% by weight of the cells of one embodiment as an active ingredient based on the total weight of the pharmaceutical composition. The pharmaceutical composition of the present invention may also further contain, in addition to the active ingredient, one or more active ingredients exhibiting the same or similar functions.

[0085] The dosage of the cells can be adjusted depending on 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 dosage form, the age, weight, general health condition, sex and diet of the patient, the time of administration, the route of administration, the treatment period, and concurrently used drugs. However, for the desired effect, the effective amount of cells contained in the pharmaceutical composition of the present invention is 1x10 5 or 1x10 11The effective amount of the cells or pharmaceutical compositions provided herein can be determined empirically without undue experimentation.

[0086] The pharmaceutical composition may also be a formulation having a dosage form suitable for the purpose, prepared by methods commonly used in the pharmaceutical field. The composition may also be formulated into a unit-dose formulation suitable for administration to a patient's body, and administered by methods commonly used in the pharmaceutical field. In addition to the active ingredient, the pharmaceutical formulation may also contain one or more pharmaceutically acceptable inert carriers, such as preservatives, soothing agents, solubilizers, or stabilizers in the case of injections, or bases, excipients, lubricants, or preservatives in the case of topical administration formulations. Furthermore, the cells or pharmaceutical compositions containing the cells may be administered to an individual by various methods known in the art, including, but not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration. [Effects of the Invention]

[0087] According to one embodiment, the anti-mesothelin chimeric antigen receptor exhibits specific binding ability to mesothelin with increased affinity for mesothelin, and can be useful for preventing or treating cancers in which mesothelin is overexpressed. [Brief explanation of the drawings]

[0088] [Figure 1] FIG. 1 shows the PCR conditions used to clone affinity-improved antibody candidates. [Figure 2] This figure confirms the tertiary structure of the antibody MSLN34, calculated using Discovery studio 2021. [Figure 3]This figure shows representative docking models for each cluster among the various docking models confirmed through Discovery studio. [Figure 4] This figure shows the structure of the docking model involving the finally selected MSLN antibody and MSLN34 antibody. [Figure 5] This figure shows the SDS-PAGE results of purified seven mutant antibody candidate substances (D31K, D31R, D31W, D31L, S192F, S192R, Y228R), including the wild-type (WT). [Figure 6] FIG. 1 shows the results of ELISA-based measurements of the affinity of wild-type (WT) and mutant antibody D31W for mesothelin. [Figure 7] FIG. 1 shows the results of ELISA-based measurement of the affinity of mutant antibodies D31L and S192F for mesothelin. [Figure 8] FIG. 1 shows the results of ELISA-based measurement of the affinity of mutant antibodies S192R and Y228R for mesothelin. [Figure 9] FIG. 1 shows the affinity of MSLN34 wild-type (WT), a single D31L mutant, and mutants containing both D31L and other mutations for mesothelin. [Figure 10] FIG. 1 shows the affinity for mesothelin of a single S192R mutant and a mutant containing both the D31L and S192R mutations. [Figure 11] This figure shows the amino acid sequences of the scFvs of anti-MSLN chimeric antigen receptor vectors with increased and improved affinity for the wild-type (WT) MSLN34, the single mutants D31L and S192R, and the mutants containing both the D31L and S192R mutations. [Figure 12] This figure shows the characteristics of the mutant MSLNCAR-T with increased affinity, confirmed through Batch #1 experiments. [Figure 13]This figure shows the characteristics of the mutant MSLNCAR-T with increased affinity, confirmed through Batch #1 experiments. [Figure 14] This figure shows the characteristics of the mutant MSLNCAR-T with increased affinity confirmed through Batch #2 experiments. [Figure 15] This figure shows the characteristics of the mutant MSLNCAR-T with increased affinity confirmed through Batch #2 experiments. [Figure 16] This is a diagram showing the cell killing effect of the affinity-enhanced mutant MSLNCAR-T on mesothelioma cells and ovarian cancer cells, confirmed through Batch #1 experiments using calcein-AM (calcein release assay). [Figure 17] This is a diagram showing the cell killing effect of the affinity-enhanced mutant MSLNCAR-T on mesothelioma cells and ovarian cancer cells confirmed through Batch #2 experiments using calcein-AM (calcein release assay). [Figure 18] This figure shows the in vitro pancreatic cancer cell-killing effect of anti-MSLN-CAR-T cells with increased and improved affinity confirmed via an Incucyte-based real-time cytotoxicity assay. [Figure 19] FIG. 1 shows the weight change in a pancreatic cancer animal model after CAR-T cell treatment. [Figure 20] FIG. 1 shows tumor volume changes in a pancreatic cancer animal model after CAR-T cell treatment. [Figure 21] This is a photograph of a pancreatic cancer animal model observed with the naked eye on day 49 after CAR-T cell treatment. [Figure 22] This is a photograph of a tumor isolated from a pancreatic cancer animal model 49 days after CAR-T cell treatment and observed visually. [Figure 23] FIG. 1 shows tumor weight in a pancreatic cancer animal model after CAR-T cell treatment. [Figure 24]This figure shows the immunohistochemical staining results of tumor sections from a pancreatic cancer animal model after CAR-T cell treatment (magnification: 5x) (stained with human CD3ε antibody). [Figure 25] This figure shows the immunohistochemical staining results of tumor sections from a pancreatic cancer animal model after CAR-T cell treatment (magnification: 20x) (stained with human CD3ε antibody). DETAILED DESCRIPTION OF THE INVENTION

[0089] Hereinafter, one embodiment will be described in more detail through examples. However, these examples are provided to exemplify one embodiment, and the scope of one embodiment is not limited to these examples. The examples of one embodiment are provided to more completely explain one embodiment to those having average knowledge in the art.

[0090] Materials and Methods 1. Materials, equipment, and experimental methods used in experiments to generate high-affinity antibodies to mesothelin 1.1. Materials and Equipment In experiments to construct an anti-MSLN chimeric antigen receptor with improved affinity for mesothelin, the anti-mesothelin antibody MSLN34 was used as a control.

[0091] The reagents and equipment used in the specific experiments are listed in Tables 2 and 3 below. [Table 2] [Table 3]

[0092] 1.2. Design of antibodies with improved affinity to mesothelin Discovery studio 2021 was utilized to develop antibodies with improved affinity for mesothelin.

[0093] Specifically, the tertiary structure of the anti-mesothelin antibody MSLN34 was calculated using the Model Antibodies function in Discovery Studio 2021. The Docking function (ZDOCK) in Discovery Studio 2021 was used to calculate an antigen-antibody docking model, and the best docking model was selected from the calculated docking models, taking into account factors such as binding strength. The selected docking model was then used to design an antibody with improved affinity using the Mutation (binding energy) function in Discovery Studio 2021.

[0094] 1.3. Selection of antibodies with improved affinity to MSLN 1.3.1. Cloning of candidate antibodies with improved affinity Primers were designed to clone the gene sequence that can code for the designed amino acid based on the mutation information calculated in Discovery Studio 2021. The primer sequences used for mutation cloning are shown in Table 4 below, and the PCR conditions used are shown in Figure 1. [Table 4] The PCR products of each mutation were cloned using the Gibson assembly method and transformed into E. coli DH5α to obtain single clones, and the amino acid sequences of the final candidate group were confirmed through base sequence analysis by sequencing.

[0095] 1.3.2. Generation of Mutant Antibody Candidates The cloned gene was transformed into E. coli TOP10F', a protein expression strain, and plated onto LB solid medium containing the antibiotic ampicillin. Transformants were obtained by culturing the transformant colonies in 10 mL of LB medium containing ampicillin at 37°C for 16 hours with shaking. Five mL of the cultured cells were then inoculated into 500 mL of LB medium containing ampicillin. After culturing at 37°C, 0.5 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) was added when the cell density (OD600) reached 0.6 or higher. Expression was continued at 30°C, and the cells were harvested after 16 hours. The harvested E. coli was incubated in 1x TES buffer (50 mM Tris-HCl, 1 mM EDTA, 20% sucrose, pH 8.0) for 1 hour, followed by an additional 1 hour in 0.2x TES buffer to completely lyse. The lysed E. coli was centrifuged (15,000 rpm, 40 minutes, 4°C), and the supernatant was collected and loaded onto a column equilibrated with 1x PBS to bind to the resin inside. After washing the resin with 1x PBS, the antibody was recovered with 1x BXT buffer. The recovered protein was concentrated using a centrifugal filter (3 kDa).

[0096] 1.3.3. ELISA-based affinity measurements 30 μL of 1 μg / mL MSLN antigen was dispensed into each well of a polystyrene-coated 96-well ELISA plate and incubated at 4°C for 16 hours. After incubation, the MSLN antigen was removed and the plate was treated with a blocking solution of 5% MPBS (5% w / v skim milk powder in PBS). Then, 1 mg / mL antibody was diluted to 1 / 3, 1 / 9, 1 / 27, 1 / 81, or 1 / 243 and incubated at room temperature for 1 hour. After washing four times with PBST buffer, the plate was incubated with HRP-anti-strep for 1 hour at 37°C. After washing four times with PBST buffer, the plate was incubated with TMB substrate for 8 minutes at room temperature and then stopped with 2N H2SO4. Absorbance readings were measured at OD 450 nm using a microplate reader.

[0097] 2. Materials and equipment used in the experiment to confirm the anti-cancer cell killing ability of CAR-T containing the newly discovered MSLN34 mutant scFv with high affinity 2.1 Materials, cell lines, reagents, and equipment used Experiments were conducted to evaluate the killing ability of CAR-T equipped with MSLN34 scFv variants (MSLN34-D31L, MSLN34-S192R, and MSLN34-D31L / S192R), obtained through in silico affinity maturation of the scFv of the anti-mesothelin antibody MSLN34, against pancreatic cancer cell lines, mesothelioma cell lines, and ovarian cancer cell lines. The materials used in these experiments are as follows:

[0098] A lentiviral vector (pLV) was used as the vector expressing the anti-MSLN CAR, and the cell lines used are shown in Tables 5 to 7 below. [Table 5] [Table 6] [Table 7] [Table 8] Information on the lentiviral production vectors used is shown in Table 9 below. [Table 9] Other reagents and materials are shown in Table 10 below. [Table 10] The equipment used is shown in Table 11. [Table 11]

[0099] 2.2. Generation of pLV lentiviral expression vector expressing anti-MSLN CAR Three affinity-matured scFv sequences, MSLN34-D31L, MSLN34-S192R, and MSLN34-D31L / S192R, were obtained from the New Drug Development Support Center's Artificial Intelligence Structural Design Team. Vectors were constructed using the following method: Using the KOD plus mutagenesis kit, affinity-matured anti-MSLN scFv-containing CAR vectors (MSLN34-D31LCAR, MSLN34-S192RCAR, and MSLN34-D31L / S192RCAR) were constructed based on the MSLN34 CAR vector (REP-RD21-011) constructed in a second-generation KBIO CAR vector. The construction method was described in accordance with the KOD plus mutagenesis kit manual. Gene sequence analysis of the constructed vector confirmed that there were no abnormalities in the entire sequence of the anti-MSLN scFv gene.

[0100] 2.3 Extraction of plasmid DNA for lentivirus production Lentiviral packaging plasmids (pMDLg / pRRE, pRSV-Rev, pMD2.G) and pLV MSLN DNA vectors were introduced into E. coli (DH5α) bacteria using heat shock transformation. Plasmid DNA was extracted using the NucleoBond® Xtra® Maxi EF kits and the NucleoBond® Xtra Midi plasmid DNA purification manual. The concentration and purity of the extracted plasmid DNA were measured using a NanoDrop™ 2000 spectrophotometer.

[0101] 2.4 Lentivirus production and concentration / purification 293T cells were cultured in a 100 mm cell culture dish at 6.0 x 10 6 The cells were seeded at a concentration of 1000 cells / dish and cultured for 1 day. The third-generation lentiviral packaging plasmids pMDLg / pRRE, pRSV-Rev, and pMD2.G DNA, and the MSLN CAR vector DNA were each diluted in Opti-MEM at a specified ratio and transformed using Lipofectamine 3000. The transformation was performed according to the Lipofectamine 3000 user manual. The lentiviral culture medium after production was purified and concentrated using a 20% sucrose gradient purification method and finally stored at -80°C.

[0102] 2.5. Measuring the Infectious Titer of Lentiviruses Using Fluorescence-Activated Cell Sorting (FACS) Analysis HeLa cells, 1.5x10, were placed in a 6-well plate. 5 The cells were seeded at a concentration of 1000 cells / well and cultured for 1 day. The next day, the lentivirus to be titered and polybrene were added to each well at a final concentration of 8 μg / mL. After 48 hours of transduction, the cells were harvested for fluorescence-activated cell sorting (FACS) analysis.

[0103] Using recombinant MSLN protein as an antigen, the number of cells bound to the anti-MSLN scFv antibody site was measured by FACS analysis. The formula for converting the functional titer (infectious titer) is as follows: TU(Transducing Unit)= [(Seeded cells #) x (Frequency PE+ cells) x 1,000] / (μL of lenti virus vector)

[0104] 2.6.MSLN CAR-T cell culture 2.6.1. T cell activation Human PBMCs were thawed and diluted with 9 mL of T cell culture medium (RPMI-1640 medium + 10% FBS + 1% penicillin-streptomycin + IL-2 200 U / mL) and centrifuged at room temperature at 300 g for 7 minutes. The supernatant was then removed and resuspended in 10 mL of fresh T cell culture medium. The differentiation procedure from human PBMCs to T cells was performed according to the manual provided by the TransAct bead reagent manufacturer.

[0105] 2.6.2. MSLN CAR transduction One day after the start of T cell activation, all activated T cells in the culture were harvested and centrifuged at 300 g for 7 minutes at room temperature. Activated T cells were then plated in a new 24-well plate at a density of 5.0 x 10 5 The cells were cultured in a final volume of 0.5 mL, with lentivirus added at an MOI of 5 based on the infectious titer for each experimental group. Protamine sulfate was added to a final concentration of 1 μg / mL and seeded into a new 24-well plate. The 24-well plate was spin-infected at 300 g for 90 minutes at 32°C, followed by incubation in a 37°C, 5% CO2 incubator. The next day, T cells were harvested and centrifuged at 300 g for 7 minutes. The supernatant was removed and fresh culture medium was added for incubation.

[0106] Analysis of MSLN CAR-T characteristics The cultured MSLN CAR-T cells were counted at 1x10 using a cell counter. 6The cells were washed with wash buffer (PBS + 2% FBS), then added with biotin-MSLN antigen and stored in a refrigerator for 20 minutes. The cells were washed again with wash buffer, then added with PE-biotin antibody and APC-CD3 antibody and stored in a refrigerator in the dark for 20 minutes. Finally, the cells were washed with wash buffer and resuspended in 100 μL of wash buffer. Finally, FACS analysis of the differentiated CAR-T was performed.

[0107] 2.7. Evaluation of MSLN CAR-T in vitro efficacy: calcein release assay 2.7.1. Preparation of target cells stained with calcein-AM The required amount of cells was placed in a 1.5 mL tube, and calcein-AM was added to a final concentration of 10 μg / mL. The cells were stained at 37°C for 1 hour. The tube was centrifuged at 1,200 rpm for 5 minutes at room temperature, and the cells were washed three times with 1 mL of culture medium. 1.0 x 10 cells were placed in a 96-well plate (R type). 4 Calcein-AM stained cells were seeded at a cell / well concentration.

[0108] 2.7.2. Preparation of effector cells (MSLN CAR-T) The ratio of effector cells reacting with target cells was calculated by performing a 2-fold serial dilution starting from E:T = 10:1 to create a total of four E:T ratios. The CAR expression ratios for each were corrected based on the number of CAR-expressing T cells. However, in the case of mock T cells, the number of cells was calculated as the same as the highest number of cells corrected for CAR expression. Spontaneous release of calcein-AM was achieved by treatment with RPMI-1640 medium. Maximum release of calcein-AM was achieved by treatment with 2% Triton X-100.

[0109] 2.7.3. Performing the Calcein Release Assay Target and effector cells were mixed and co-cultured for 4 hours at 37°C and 5% CO2. Centrifugation was performed at room temperature for 5 minutes at 100 g. To measure the amount of calcein-AM released from the cells due to cell death, 100 μL of the co-culture medium was transferred to a black optical plate (F type). A fluorescence microplate reader was used to measure values ​​in the excitation wavelength range of 485 nm and emission wavelength range of 530 nm. The formula for calculating the cytotoxic effect was as follows: % of Specific Lysis = [(Test release - Spontaneous release) / (Maximum release - Spontaneous release)] x 100%

[0110] 2.8. Evaluation of MSLN CAR-T in vitro efficacy: Incucyte-based real-time cytotoxicity assay 2.8.1. Target Cell Preparation AsPC-1 / GFP cells, 1x10 4 100 μL of cells were seeded into a 96-well plate so that each well had 100 cells.

[0111] 2.8.2. Preparation of effector cells This study was conducted using Mock T (non-transducing T) cells and MSLN CAR-T (MSLN34, MSLN34-D31L, MSLN34-S192R, MSLN34-D31L / S192R) cells. The ratio of effector cells reacting with target cells was set at E:T = 0.5:1, and calculations were made by correcting the number of CAR-expressing T cells based on the expression rate of each CAR. However, in the case of mock T cells, calculations were made using the same number of cells as the highest when corrected for CAR expression rate.

[0112] 2.8.3. Real-time Cytotoxicity Analysis Target and effector cells were co-cultured in a 96-well plate. GFP was measured every 3 hours for 72 hours in an Incucyte™ system, and the co-cultured plate was then loaded. GFP measurements were analyzed after each Incucyte™ system run.

[0113] 2.9. Experimental Data and Statistical Analysis For the Incucyte assay and calcein release assay, four independent E+T co-culture wells per experimental group (tetra-plicated assay) were used to obtain experimental data. All experimental data were graphed using GraphPad Prism 9.0 software. Statistical significance was determined using the statistical analysis tool Two-Way ANOVA (Full model, Tukey, 95% confidence interval) included in GraphPad Prism 9.0 software (ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

[0114] 3. Materials, equipment, and experimental methods used in the experiment to confirm the anti-cancer effect of the newly discovered, high-affinity MSLN34 scFv-containing CAR-T on an animal model of pancreatic cancer 3.1. Creation of an animal model of pancreatic cancer (AsPC-1) 3.1.1. Mouse rearing To generate an animal model of pancreatic cancer, we used NOG (NOD / Shi-scid / IL-2Rγ) nullMale, 6-week-old (15.0-25.0 g) specific pathogen-free (SPF) mice of the Osong Advanced Medical Industry Foundation were used. The mouse rearing and related experiments were carried out in the animal room of the Experimental Animal Center of the Osong Advanced Medical Industry Foundation, with a temperature of 22±2°C, a relative humidity of 50±10%, a ventilation rate of 10-20 times / hr, a 12-hour lighting period (lights on at 8:00 AM - lights off at 8:00 PM), and an illumination level of 150-300 Lux. The mice had free access to food and water. The mouse-related experiments were conducted in accordance with the regulations of the Experimental Animal Management Committee of the Osong Advanced Medical Industry Foundation.

[0115] 3.1.2. Cell culture and transplantation The AsPC-1 cell line was used to generate an animal model of pancreatic cancer. It was tested for Mycoplasma pneumoniae, Murine coronavirus (Mouse hepatitis virus, MHV), and Murine respirovirus (Sendai virus, SeV) and tested negative before use. The cell line was cultured in RPMI-1640 medium containing 10% FBS and 1% P / S (Penicillin / Streptomycin) at 37°C and 5% CO2 in a CO2 incubator. The AsPC-1 cells were adjusted to a cell concentration using PBS and then subcutaneously implanted in 200 μL aliquots into mice.

[0116] 3.2. Composition of pancreatic cancer animal model test groups The pancreatic cancer animal models prepared above were divided into groups by random allocation according to tumor size, and the test groups were constructed as shown in Table 12 below. [Table 12]

[0117] The test groups were individually identified using ear punching during the test period, and cards were attached to the rearing cages to identify each group. After separating the groups, the test substance was administered once intravenously (IV) to the tail vein.

[0118] 3.4. Measurement of body weight and tumor size The body weight and tumor size of the test group were measured twice a week from the start of treatment. The body weight on the start of treatment (day 0) was used as the baseline, and weight changes were observed until the end of the test. Body weight (%) was calculated using the following formula: Body weight (%) = (Body weight / Body weight at Day 0) x 100

[0119] Tumor size (mm 3 ) was calculated using the following formula by measuring the short axis (A) and long axis (B) of the tumor using calipers. Tumor volume (mm 3 ) = ([A(mm)] 2 x B(mm)) / 2

[0120] Autopsy Zoletil TM Anesthesia was induced by intraperitoneal injection of 50 mg / kg of riboflavin and 10 mg / kg of Rompun. The abdominal cavity was opened, blood was collected from the abdominal vein, and the animals were then exsanguinated and euthanized. Serum was separated from the blood and stored frozen (below -80°C). A portion of the isolated tumor was stored frozen (below -80°C), and another portion was fixed in fixative (10% neutral formalin) for histopathological examination. Slides were prepared and stained with hematoxylin and eosin (H&E). The stained slides were photographed using a PANNORAMIC SCAN II (3DHISTECH, Hungary) and analyzed using 3DHISTECH software.

[0121] 3.6.Data Analysis Statistical comparisons of data were performed using SPSS 10.1. Data are presented as mean ± standard deviation (SD). Analysis was performed by one-way ANOVA followed by Tukey's post-hoc analysis for multiple comparisons (*: p<0.05, **: p<0.01, ***: p<0.001 vs. vehicle control-treated group (G1); #: p<0.05, ##: p<0.01, ###: p<0.001 vs. mock cell-treated group (G2)).

[0122] result Experimental example 1: Calculation of docking model of antigen (MSLN) and antibody (MSLN34) First, we calculated the tertiary structure of MSLN34, an antibody that specifically binds to the antigen mesothelin. MSLN34 was calculated using the Homology Model function in Discovery Studio 2021, and the calculated structure is shown in Figure 2. As can be seen in Figure 2, the CDR regions of the antibody are represented in pink (light chain) and blue (heavy chain), respectively.

[0123] Next, we used the ZDOCK program in Discovery Studio 2021 to calculate antigen-antibody docking models for mesothelin and MSLN34. As a result, as seen in Figure 3, we were able to observe various docking models that bind to the curved inner surface of MSLN, from the N-terminus to the C-terminus, and identify various binding positions. Through the docking program in Discovery Studio 2021, approximately 2,000 docking models were calculated. We then checked the structures of approximately 200 models in order of highest predicted binding strength, and ultimately selected the docking model shown in Figure 4, taking into account factors such as the specific binding between mesothelin and MSLN34.

[0124] As shown in Figure 4 above, the selected docking model bound to amino acids 385-569 of mesothelin, and this binding position was confirmed to be the position for which the most docking models were calculated in the docking results of Discovery Studio 2021. It was also confirmed that MSLN34 uses all six variant regions of the amino acids involved in binding to its target, mesothelin, resulting in a structure that forms a stable bond.

[0125] Experimental Example 2: Selection of MSLN34 antibodies with increased affinity 2.1. Design of affinity-enhanced mutation candidates based on in silico model structures An experiment was designed to identify antibodies that can increase binding strength based on the docking model selected through Experimental Example 1. Calculations were performed using the Mutagenesis (binding) function in Discovery Studio 2021, and the calculation results are shown in Table 13 below. [Table 13]

[0126] As shown in Table 13, the mutation candidates were arranged in order of decreasing mutation energy, and the mutation candidates that most significantly increased affinity in the docking model were selected.

[0127] Specifically, mutations at positions D31, S192, and Y228 were determined to maximize the affinity between the target mesothelin and MSLN34 in a docking model. Therefore, mutations were introduced into an anti-mesothelin antibody or its antigen-binding fragment. In the scFv of the anti-mesothelin antibody (MSLN34) (SEQ ID NO: 1), mutations were designed in which the aspartic acid (D) at amino acid position 31 (the first amino acid of HCDR1) was replaced with lysine (K), tryptophan (W), leucine (L), and arginine (R), the serine (S) at amino acid position 192 (the seventh amino acid of LCDR2) was replaced with phenylalanine (F) and arginine (R), and the tyrosine (Y) at amino acid position 228 (the fourth amino acid of LCDR3) was replaced with arginine (R). However, although the mutation energy is low, the Y102 and Y104 positions were excluded because they are amino acids directly involved in binding in the docking model. A group of candidate mutation sequences for the anti-mesothelin antibody or antigen-binding fragment thereof designed in this way with increased affinity was analyzed. The mutant sequences were those of scFvs in which the amino acids at each position in the MSLN34 scFv of SEQ ID NO: 1 were substituted with specific amino acids.

[0128] 2.2. Confirmation of affinity of single mutation-containing antibody candidates with increased and improved affinity to mesothelin An experiment was conducted to confirm whether the antibody candidate substances in Experimental Example 2.1 could actually be confirmed as anti-mesothelin antibodies or antigen-binding fragments thereof with increased and improved affinity. To produce the anti-mesothelin antibodies or antigen-binding fragments confirmed in Experimental Example 2.1, DNA sequences encoding the MSLN34 scFv mutant amino acid sequences were expressed in E. coli strain Top10F' and purified by affinity chromatography using a strep tag. The purity and production of a total of eight mutant antibody candidates, including the wild-type (WT), were confirmed by SDS-PAGE gel analysis. The results are shown in Figure 5. As can be seen in Figure 5, D31K and D31R were not expressed or purified, while the wild-type (WT), D31W, D31L, S192F, S192R, and Y228R were purified to purities of 95% or higher.

[0129] Next, the ELISA-based affinity of the six antibodies, including the purified wild-type (WT), was measured. Mesothelin (MSLN) was added to a 96-well plate, and the purified antibodies were serially diluted 1 / 3 and reacted. The affinity graphs are shown in Figures 6 to 8. As can be seen in Figures 6 to 8, the D31L, S192F, and S192R candidate substances showed significantly higher EC than the wild-type (WT). 50 The values ​​were even lower and the affinity increased.

[0130] 2.3 Confirmation of affinity of double-mutation-containing antibody candidates with increased and improved affinity to mesothelin In the single mutant antibodies identified through the above experimental examples, experiments were conducted to determine whether double mutations, involving the D31L mutation candidate group and the S192R mutation candidate group, which had increased affinity, would further increase affinity for mesothelin.

[0131] The specific experimental method was the same as that used to measure the affinity of the single mutant antibody in Experimental Example 2.2. The affinity of the wild-type (WT) MSLN34 with no mutations, the single mutants D31L and S192R, and the mutant containing both the D31L and S192R mutations was measured and the results are shown in Figures 9 and 10. As can be seen in Figures 9 and 10, the ELISA-based affinity measurements confirmed that the double mutant antibody had significantly higher affinity than the wild-type (WT).

[0132] The amino acid sequences of single mutants of MSLN34 scFv, D31L (the first amino acid of HCDR1 is replaced from D to L) or S192R (the seventh amino acid of LCDR2 is replaced from S to R), which were previously shown to have improved affinity, as well as a mutant containing both the D31L and S192R mutations (D31L / S192R), were confirmed and are shown in Figure 11.

[0133] The light chain CDR sequences, heavy chain CDR sequences, and amino acid sequences related to the light chain variable region and heavy chain variable region of the aforementioned MSLN34 and its improved variants are shown in Table 14. The substituted portions in the MSLN34 variants are shown in bold and underlined. [Table 14-1] [Table 14-2]

[0134] Experimental Example 3: Construction of anti-MSLN chimeric antigen receptor with increased and improved affinity 3.1. Cloning of anti-MSLN-CAR lentiviral vector To construct a chimeric antigen receptor containing the improved anti-mesothelin antibody or its antigen-binding fragment confirmed in Experimental Example 2, an anti-MSLN-CAR lentiviral vector was cloned.

[0135] The vector belongs to the second-generation CAR lentiviral vector (pLV lentiviral vector) system held by the New Drug Development Support Center, and includes pMDLg / pRRE (addgene) encoding gag / pol, envelope plasmid pRSV-Rev (addgene) encoding the Rev protein, and envelope plasmid pMD2.G (addgene) encoding the VSV-G protein. First, we performed gene cloning of the MSLN34 scFv (antigen-binding domain) and its mutants, which demonstrated excellent efficacy in Experimental Example 2. Each anti-MSLN scFv and lentiviral vector were digested with XhoI (R0146S (NEB)) and EcoRI (R0101 (NEB)) at 37°C for 2 hours. After agarose gel electrophoresis, the identified products were purified using the FavorPrep Gel / PCR Purification Mini Kit (Favorgen). Each purified anti-MSLN scFv (100 ng) and vector (50 ng) were reacted at a 2:1 ratio for 16 hours at 16°C. Ligation was then carried out, and the resulting products were transformed into Stbl3 competent cells to obtain colonies. The colonies were picked and grown in 5 mL of LB medium (ampicillin), and plasmid DNA was obtained using the DNA plasmid mini-prep method. The plasmid DNA was digested with XhoI and EcoRI to confirm whether each anti-MSLN scFv inserted into the vector was successfully cloned, and then sequencing was performed to finally confirm the DNA sequence.

[0136] The anti-MSLN-CAR was constructed by sequentially linking the CD8 hinge and CD8TM (transmembrane) to the transmembrane domain, the 4-1BB cytoplasmic domain to the signaling domain, and the CD3 zeta (CD3z) intracellular domain to the T cell activation domain to the anti-MSLN scFv. Specifically, the anti-MSLN-CAR was composed of the CD8 signal peptide (SP) (SEQ ID NO: 51), MSLN34 scFv and its mutants (SEQ ID NOs: 52-55), the CD8 hinge region (SEQ ID NO: 56), the CD8 transmembrane region (SEQ ID NO: 57), the 4-1BB signaling domain (SEQ ID NO: 58), and the CD3 zeta signaling domain (SEQ ID NO: 59). Each domain was sequentially linked using the respective restriction enzymes. Specific nucleotide sequence information corresponding to each domain is summarized in Table 15 below. [Table 15-1] [Table 15-2]

[0137] Cloning was completed, and the amino acid sequences of the scFvs of the anti-MSLN chimeric antigen receptor vectors with increased and improved affinity were confirmed for the wild-type (WT) MSLN34, the single mutants D31L and S192R, and the mutants containing both the D31L and S192R mutations.

[0138] 3.2. Production of anti-MSLN-CAR-loaded lentivirus and measurement of functional titers The results of confirming the functional titer of the CAR-carrying lentivirus in HeLa cells are shown in Table 16 [functional titer (infectious titer) measurement results (FACS analysis)]. [Table 16]

[0139] 3.3. Preparation of anti-MSLN-CAR-transduced cells Experiments were conducted to produce MSLN CAR-T cells transfected with the vector of Experimental Example 3.1 (Batch #1 and Batch #2), and their characteristics were analyzed by FACS analysis. The results of the characterization of the MSLN CAR-T produced in Batch #1 are shown in Figures 12 and 13, and the results of the characterization of the MSLN CAR-T produced in Batch #1 are shown in Figures 14 and 15. The results of the characterization of the MSLN CAR-T produced in Batch #1 and #2 are summarized in Table 17.

[0140] As shown in Figures 12 and 13, in MSLN CAR-TBatch #1, CAR-expressing cells were confirmed to be 46.1%, 39.3%, 54.6%, and 43.3% for MSLN34, MSLN34-D31L, MSLN34-S192R, and MSLN34-D31L / S192R, respectively. Most of the CAR-expressing cells were confirmed to be CD3+ T cells (≧99.3%).

[0141] 14 and 15, in MSLN CAR-T and MSLN CAR-TBatch #2, the CAR-expressing cells were MSLN34, MSLN34-D31L, MSLN34-S192R, and MSLN34-D31L / S192R, respectively, at 43.9%, 46.9%, 37.9%, and 52.9%. Most of the CAR-expressing cells were confirmed to be CD3+ T cells (≧99.0%). [Table 17]

[0142] Experimental Example 4: Confirmation of the cell killing effect of anti-MSLN-CAR-T cells with increased and improved affinity against mesothelioma and ovarian cancer Using the anti-MSLN-CAR-T cells prepared in Experimental Example 3, the cell killing effect on mesothelioma cells and ovarian cancer cells was confirmed using calcein-AM (calcein release assay).

[0143] First, the in vitro efficacy of CAR-T in the malignant pleural mesothelioma cell line NCI-H2052 and the ovarian cancer cell line OVCAR-3 was evaluated using calcein-AM to assess the cytotoxic effect on target cells in Batch #1 and Batch #2 experiments. Statistical significance was determined using two-way ANOVA (full model, Tukey's, 95% confidence interval) (ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (vs. mock)). Experimental results regarding the cytotoxic effect of anti-MSLN-CAR-T cells on target cells using calcein-AM are shown in Table 18. [Table 18]

[0144] Primary calcein-AM results using MSLN CAR-T Batch #1 are shown in Figure 16. As seen in Figure 11, the cytotoxic effect against the NCI-H2052 target cell line was confirmed to be 24.7% and 6.0% higher for MSLN34-D31L CAR-T and MSLN34-D13L / S192R CAR-T, respectively, compared to MSLN34 CAR-T cells, based on an E:T = 10:1 experimental group ratio, and 10.7% lower for MSLN34-S192R CAR-T. The cytotoxic effect against the OVCAR-3 target cell line was confirmed to be 19.3% and 9.3% higher for MSLN34-D31L CAR-T and MSLN34-D13L / S192R CAR-T, respectively, compared to MSLN34 CAR-T cells, based on an E:T = 10:1 experimental group standard, while the MSLN34-S192R CAR-T was confirmed to be 10.5% lower.

[0145] Secondary Calcein-AM results using MSLN CAR-T Batch #2 are shown in Figure 17. As seen in Figure 17, the cytotoxic effect on the NIC-H2052 target cell line was confirmed to be 16.7% and 7.5% higher for MSLN34-D31L CAR-T and MSLN34-D31L / S192R CAR-T, respectively, compared to MSLN34 CAR-T cells, based on an E:T = 10:1 experimental group ratio, and 4.4% lower for MSLN34-S192R CAR-T. The cytotoxic effect against the OVCAR-3 target cell line was confirmed to be 25.3% and 19.5% higher for MSLN34-D31L CAR-T and MSLN34-D13L / S192R CAR-T, respectively, compared to MSLN34 CAR-T cells, based on an E:T = 10:1 experimental group standard, while MSLN34-S192R CAR-T was confirmed to be 1.0% lower.

[0146] Therefore, it was confirmed that MSLN34-D31L and MSLN34-D31L / S192R exhibited even higher cancer cell killing ability compared to MSLN34 CAR-T in mesothelioma and ovarian cancer cell lines.

[0147] Experimental Example 5: Confirmation of the cell killing effect of anti-MSLN-CAR-T cells with increased and improved affinity against pancreatic cancer Using the anti-MSLN-CAR-T cells prepared in Experimental Example 3, the cell killing effect on pancreatic cancer cells was confirmed through an incucyte-based real-time cytotoxicity assay.

[0148] The in vitro cancer cell-killing effect of the anti-MSLN-CAR-T cells with increased and improved affinity was assessed using Incucyte GFP particles to evaluate the cytotoxic effect on target cells (AsPC-1 / GFP (pancreatic cancer cells)). The experimental results are shown in Figure 18. Statistical significance was determined using two-way ANOVA (full model, Tukey's, 95% confidence interval) (ns, P > 0.05; *, P ≦ 0.05; **, P ≦ 0.01; ***, P ≦ 0.001 (vs. Mock)). As can be seen in Figure 18, in the results of the primary and secondary experiments using MSLN CAR-T Batch #1&2, at an E:T ratio of 0.5, compared to mock T cells, MSLN34-D31L, MSLN34-S192R cells, and MSLN34-D31L / S192R CAR-T cells all showed a change from an increasing trend to a decreasing trend in GFP particles between 21 and 27 hours, demonstrating a statistically significant cell killing effect on pancreatic cancer cells.

[0149] Experimental Example 6: Confirmation of anti-cancer efficacy in animal models of anti-MSLN-CAR-T cells with increased and improved affinity 6.1. Observation of weight changes and general symptoms Body weight changes and general symptoms were observed in a pancreatic cancer animal model treated with anti-MSLN-CAR-T cells.

[0150] As a result, one animal (G5-2) died during the test period, and body weight decreased in some groups. 6In the MSLN34(WT)-treated group, weight loss was observed and some animals died. Specifically, compared with the vehicle control group (G1), the MSLN34(WT) high-concentration (G3) group showed weight loss from day 38 after administration, and the MSLN34-D31L high-concentration (G5) group showed weight loss from day 24 after administration (p<0.05). Furthermore, compared with the mock-treated group (G2), the MSLN34(WT) high-concentration (G3) group showed weight loss from day 42 after administration, and the MSLN34-D31L high-concentration (G5) group showed weight loss from day 24 after administration (p<0.05) (Figure 19, Tables 19 and 20).

[0151] Table 19 below shows the mortality rate of pancreatic cancer animal models after CAR-T cell treatment, expressed as the number of dead animals / total number of animals. Dead animals are shown on day 42 after CAR-T cell treatment. [Table 19]

[0152] Figure 19 and Table 20 below show the changes in body weight in pancreatic cancer animal models after CAR-T cell treatment. Each value is expressed as the mean ± standard deviation of body weight (%). The day of administration was designated as day 0, and the measured body weight was divided by the body weight on day 0 to express the percentage of body weight (%). The above data also includes data on animals that died during the experiment. Statistical analysis was performed using one-way ANOVA and Tukey's post-hoc test (*: p<0.05, **: p<0.01, ***: p<0.001 vs. vehicle control-treated group (G1), #: p<0.05, ##: p<0.01, ###: p<0.001 vs. mock cell-treated group (G2)). [Table 20]

[0153] 6.2. Assessment of tumor growth / shrinkage The level of tumor growth / shrinkage was evaluated in an animal model of pancreatic cancer treated with anti-MSLN-CAR-T cells.

[0154] As a result, compared with the vehicle control group (G1), the mock-treated group (G2) showed statistically significant tumor shrinkage from day 35 after administration (p<0.01 (vs. G1)). Furthermore, compared with the vehicle control group (G1), all MSLN-CAR-T treated groups (G3-G10) showed statistically significant tumor shrinkage (p<0.05 (vs. G1)) (Figure 24 and Table 19).

[0155] Figure 20 and the following Table 21 show the changes in tumor volume in a pancreatic cancer animal model after CAR-T cell treatment. Each value represents the tumor volume (mm 3 ) are shown as mean ± standard deviation. The absence of detectable tumors is indicated by "0." The above data also include data from animals that died during the experiment. Statistical analysis was performed using one-way ANOVA and Tukey's post-hoc test (*: p<0.05, **: p<0.01, ***: p<0.001 vs. vehicle control-treated group (G1), #: p<0.05, ##: p<0.01, ###: p<0.001 vs. mock cell-treated group (G2)). [Table 21]

[0156] 6.2.1.MSLN34(WT) treatment group Compared with the vehicle control group (G1), tumors in the high-concentration MSLN34(WT) group (G3) began shrinking from day 11 after administration (p<0.05 (vs. G1)), and tumors in the low-concentration MSLN34(WT) group (G4) began shrinking from day 15 after administration (p<0.05 (vs. G1)).

[0157] Compared to the mock-administered group (G2), statistical significance was verified in the MSLN34 (WT) high concentration (G3) group from day 11 to day 28 and day 38 after administration (p<0.05 (vs. G2)), but statistical significance was not verified in the MSLN34 (WT) low concentration (G4) group.

[0158] The high-concentration MSLN34(WT) (G3) group showed a tendency for tumors to shrink, whereas the low-concentration MSLN34(WT) (G4) group showed a tendency for tumors to grow.

[0159] 6.2.2.MSLN34-D31L treatment group Compared with the vehicle control group (G1), tumors in the high-concentration (G5) and low-concentration (G6) MSLN34-D31L groups shrank from day 8 after administration (p<0.05 (vs. G1)). Compared with the mock-administered group (G2), statistical significance was verified in the high-concentration MSLN34-D31L (G5) group from day 8 to day 38 after administration, and statistical significance was verified in the low-concentration MSLN34-D31L (G6) group from day 8 to day 28 after administration (p<0.05 (vs. G1)).

[0160] In the MSLN34-D31L-treated groups (G5 and G6), tumors tended to shrink initially regardless of the dose. However, over time, one individual in the low-dose (G6) group showed a tendency for tumor growth to increase. In the blood analysis of this individual, hCD 3% was observed as negligible, suggesting that T cell proliferation was not occurring in the body.

[0161] 6.3. Measurement of tumor weight Tumor weight was measured / evaluated in a pancreatic cancer animal model treated with anti-MSLN-CAR-T cells.

[0162] As a result, tumor weight was reduced in all treatment groups compared to the vehicle control group (G1) (p<0.01 (vs. G1)).

[0163] Compared with the mock-treated group (G2), the MSLN34(WT) high-concentration (G3) group showed an average 93% reduction in tumor weight, and the MSLN34-D31L high-concentration (G5) group showed an average 91% reduction in tumor weight. Furthermore, complete tumor elimination was observed in some individuals in the MSLN34-D31L high-concentration (G5) group. However, compared with the mock-treated group (G2), the MSLN34(WT) low-concentration (G4) group showed an increase in average tumor weight, and the MSLN34-D31L low-concentration (G6) group showed an 18% reduction in average tumor weight. In the MSLN34-D31L low-concentration (G6) group, tumor growth occurred in one individual, resulting in a large standard deviation. Blood analysis of this individual showed a negligible hCD of 3%, suggesting that T cell proliferation was not occurring in vivo (Figures 21 to 23 and Table 22).

[0164] Figure 23 and the following Table 22 show the tumor weights of pancreatic cancer animal models after CAR-T cell treatment, with each value representing the mean tumor weight (mg) ± standard deviation. The pancreatic cancer animal models were euthanized on day 49 after CAR-T cell treatment, and the tumor weights were measured. If no tumor was found, the weight was indicated as 0 mg. Statistical analysis was performed using one-way ANOVA and Tukey's post-hoc test (*: p<0.05, **: p<0.01, ***: p<0.001 vs. vehicle control treatment group (G1), #: p<0.05, ##: p<0.01, ###: p<0.001 vs. mock cell treatment group (G2)). [Table 22]

[0165] 6.4. Histopathological evaluation We examined histopathological findings in an animal model of pancreatic cancer treated with anti-MSLN-CAR-T cells. Specifically, three animals per group were selected and subjected to immunohistochemistry (IHC) for hCD3ε.

[0166] As a result, in the vehicle control group (G1), almost no T cell infiltration was observed in the tumor. In the mock-administered group (G2), sporadic T cell infiltration was confirmed in the tumor. The highest level of T cell infiltration in the tumor was observed in the MSLN34 (WT) high concentration (G3) group. When the same type of CAR-T cells were administered, the high concentration (1.5x10 6 High T cell infiltration was observed in the MSLN34-D31L scFv-optimized mice (cells / head). When the same costimulatory factors were used, T cell infiltration was not observed to be higher in the MSLN34-D31L scFv-optimized mice than in the conventional MSLN34 mice (Figures 24 and 25).

[0167] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and not limiting.

[0168] This research was supported by Korea Drug Development Fund funded by the Ministry of Science and ICT, the Ministry of Trade, Industry, and Energy, and the Ministry of Health and Welfare (Project ID: RS-2023-00217215, Republic of Korea).

Claims

1. a heavy chain variable region comprising a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 19, a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence of SEQ ID NO: 20, and a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence of SEQ ID NO: 21; a light chain variable region comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 22, a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 23, and a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence of SEQ ID NO: 24; An anti-mesothelin antibody, or an antigen-binding fragment thereof, comprising one or more amino acid substitutions in the heavy chain variable region and the light chain variable region.

2. The anti-mesothelin antibody, or antigen-binding fragment thereof, of claim 1, wherein the one or more amino acid substitutions are at one or more positions selected from the group consisting of position 1 of SEQ ID NO: 19, position 7 of SEQ ID NO: 23, and position 4 of SEQ ID NO:

24.

3. 2. The anti-mesothelin antibody, or antigen-binding fragment thereof, of claim 1, wherein the one or more amino acid substitutions include one or more selected from the group consisting of the following amino acid substitutions: 1) the first amino acid of SEQ ID NO: 19 is substituted from D to K, W, L, or R; 2) The seventh amino acid of SEQ ID NO: 23 is substituted with F or R, and 3) The fourth amino acid of SEQ ID NO:24 is substituted from Y to R.

4. 2. The anti-mesothelin antibody, or antigen-binding fragment thereof, of claim 1, wherein the one or more amino acid substitutions are selected from the group consisting of the following amino acid substitutions: 1) the first amino acid of SEQ ID NO: 19 is substituted from D to L; 2) the seventh amino acid of SEQ ID NO: 23 is substituted from S to R; and 3) The first amino acid of SEQ ID NO: 19 is substituted from D to L, and the seventh amino acid of SEQ ID NO: 23 is substituted from S to R.

5. The anti-mesothelin antibody or antigen-binding fragment thereof of claim 1 is selected from the group consisting of antibodies or antigen-binding fragments thereof comprising a heavy chain variable region containing the following heavy chain CDRs and a light chain variable region containing the following light chain CDRs: 1) an antibody, or an antigen-binding fragment thereof, comprising: a heavy chain variable region comprising HCDR1 comprising the amino acid sequence of SEQ ID NO:27, HCDR2 comprising the amino acid sequence of SEQ ID NO:28, and HCDR3 comprising the amino acid sequence of SEQ ID NO:29; and a light chain variable region comprising LCDR1 comprising the amino acid sequence of SEQ ID NO:30, LCDR2 comprising the amino acid sequence of SEQ ID NO:31, and LCDR3 comprising the amino acid sequence of SEQ ID NO:32; 2) an antibody, or an antigen-binding fragment thereof, comprising: a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 35, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 36, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 38, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 39, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 40; and 3) An antibody, or an antigen-binding fragment thereof, comprising: a heavy chain variable region comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 43, HCDR2 comprising the amino acid sequence of SEQ ID NO: 44, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 45; and a light chain variable region comprising LCDR1 comprising the amino acid sequence of SEQ ID NO: 46, LCDR2 comprising the amino acid sequence of SEQ ID NO: 47, and LCDR3 comprising the amino acid sequence of SEQ ID NO:

48.

6. The anti-mesothelin antibody or antigen-binding fragment thereof of claim 1 is selected from the group consisting of antibodies or antigen-binding fragments thereof comprising the following heavy chain variable region and light chain variable region: 1) an antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 34; 2) an antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 41 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 42; and 3) An antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

50.

7. 2. The anti-mesothelin antibody or antigen-binding fragment thereof of claim 1, selected from the group consisting of antibodies or antigen-binding fragments thereof, including the following antigen-binding fragments: 1) an antibody comprising an antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof; 2) an antibody comprising an antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 3, or an antigen-binding fragment thereof; and 3) An antibody comprising an antigen-binding fragment comprising the amino acid sequence of SEQ ID NO: 4, or an antigen-binding fragment thereof.

8. The anti-mesothelin antibody, or antigen-binding fragment thereof, of claim 1 , wherein the substitution of one or more amino acids increases the affinity of the anti-mesothelin antibody, or antigen-binding fragment thereof, for mesothelin.

9. 9. An isolated nucleic acid encoding the antibody, or antigen-binding fragment thereof, of any one of claims 1 to 8.

10. A vector comprising the nucleic acid of claim 9.

11. An isolated host cell transformed with the vector of claim 10.

12. A method for producing an anti-mesothelin antibody, comprising culturing the host cell of claim 11 to express the antibody.

13. It is a chimeric antigen receptor comprising an antigen-binding domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain, A chimeric antigen receptor, wherein the antigen-binding domain comprises an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

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

15. A polynucleotide encoding the chimeric antigen receptor of claim 13.

16. The polynucleotide of claim 15, wherein the polynucleotide comprises one or more base sequences selected from the group consisting of SEQ ID NOs: 53 to 55.

17. A vector comprising the polynucleotide of claim 15.

18. An isolated cell transformed with the vector of claim 17.

19. 19. The isolated cells of claim 18, wherein the isolated cells are T cells, NK cells, NKT cells, or gamma delta T cells (γδ T cells).

20. A pharmaceutical composition for the prevention or treatment of cancer, comprising the isolated cells of claim 19.

Images