Chimeric antigen receptors with increased affinity for mesothelin and uses thereof

Abstract

The present invention relates to an anti-mesothelin chimeric antigen receptor which has increased affinity to mesothelin and which specifically binds to mesothelin. The anti-mesothelin chimeric antigen receptor according to one aspect has an increased affinity for mesothelin, and exhibits a specific binding ability for mesothelin, and thus can be effectively used for the prevention or treatment of mesothelin overexpression cancers.

Description

Technical Field

[0001] Disclosed herein is an anti-mesothelin chimeric antigen receptor and its use, wherein the anti-mesothelin chimeric antigen receptor has increased affinity for mesothelin and specifically binds to mesothelin.

Background Art

[0002] Recently, the effectiveness of immune anti-cancer agents such as immune checkpoint inhibitors and CAR-T cell therapeutic agents has been demonstrated in various cancers. However, it has been reported that for solid cancers, the therapeutic efficiency of these novel immune anti-cancer agents does not show a significant response. It is speculated that this is because the fibrous tissue surrounding the tumor interferes with the immune therapy response, making drug delivery difficult. Therefore, as a specific and more effective CAR-T cancer treatment method, there is a need to develop an antibody that targets a protein specifically overexpressed on the surface of solid cancer cells as a cancer antigen and has high affinity for it, and to study methods for effectively treating solid cancers by using the antibody.

[0003] On the one hand, mesothelin is a glycoprotein anchored to the cell surface via a glycosylphosphatidylinositol (GPI) domain. Under normal circumstances, mesothelin is expressed at a limited and low level in the mesothelium that surrounds the body cavity and internal organs of the human body. However, it has been reported that it is expressed at a high level in cancers such as pancreatic cancer, mesothelioma, ovarian cancer, and non-small cell lung cancer.

Summary of the Invention

[0004]

Technical Problem

[0005] On the one hand, provided is an anti-mesothelin antibody or an antigen-binding fragment thereof, which has increased affinity for mesothelin.

[0006] On the other hand, provided is an isolated nucleic acid encoding the anti-mesothelin antibody or an antigen-binding fragment thereof, wherein the anti-mesothelin antibody has increased affinity for mesothelin.

[0007] On the other hand, provided is a vector comprising the isolated nucleic acid.

[0008] On the other hand, provided is an isolated host cell transformed with the vector.

[0009] On the other hand, provided is a method for producing an anti-mesothelin antibody with increased affinity for mesothelin, which comprises expressing the antibody by culturing the isolated host cell.

[0010] On the other hand, provided is 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.

[0011] On the other hand, a polynucleotide encoding the chimeric antigen receptor is provided.

[0012] On the other hand, a vector comprising the polynucleotide is provided.

[0013] On the other hand, an isolated cell transformed with the vector is provided.

[0014] On the other hand, provided are: a pharmaceutical composition comprising the isolated cell; the pharmaceutical use of the cell; and a method for preventing or treating cancer, which comprises administering a therapeutically effective amount of the cell to an individual.

[0015] Other objects and advantages of the present application will become more apparent in conjunction with the appended claims, the accompanying drawings and the following detailed description. Regarding the content not described in this specification, as long as it can be fully recognized and analogized by those of ordinary skill in the art to which the present application pertains or those of ordinary skill in similar technical fields, the relevant description thereof is omitted.

[0016]

Technical Solution

[0017] The various descriptions and embodiments disclosed in the present application can also be applied to various other descriptions and embodiments. That is, all combinations of the various elements disclosed in the present application fall within the scope of the present application. And it is not possible to determine that the scope of the present application is limited to the specific descriptions set forth below.

[0018] On the one hand, an anti-mesothelin antibody or an antigen-binding fragment thereof is provided, which comprises: a heavy chain variable region comprising heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3), wherein the HCDR1 comprises the amino acid sequence consisting of SEQ ID NO: 19, the HCDR2 comprises the amino acid sequence consisting of SEQ ID NO: 20, and the HCDR3 comprises the amino acid sequence consisting of SEQ ID NO: 21; and a light chain variable region comprising light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2) and light chain complementarity determining region 3 (LCDR3), wherein the LCDR1 comprises the amino acid sequence consisting of SEQ ID NO: 22, the LCDR2 comprises the amino acid sequence consisting of SEQ ID NO: 23, and the LCDR3 comprises the amino acid sequence consisting of SEQ ID NO: 24, wherein the heavy chain variable region and the light chain variable region comprise one or more amino acid substitutions.

[0019] The "mesothelin (MSLN)" is a cell surface glycoprotein with a total amino acid length of 622 aa (NCBI Gene ID: 10232), which is selectively expressed in certain cells (especially, specific tumor cells), and the amino acids of the mesothelin protein are shown as follows.

[0020] MALPTARPLL GSCGTPALGS LLFLLFSLGW VQPSRTLAGE TGQEAAPLDG VLANPPNISSLSPRQLLGFP CAEVSGLSTE RVRELAVALA QKNVKLSTEQ LRCLAHRLSE PPEDLDALPL DLLLFLNPDAFSGPQACTRF FSRITKANVD LLPRGAPERQ RLLPAALACW GVRGSLLSEA DVRALGGLAC DLPGRFVAESAEVLLPRLVS CPGPLDQDQQ EAARAALQGG GPPYGPPSTW SVSTMDALRG LLPVLGQPII RSIPQGIVAAWRQRSSRDPS WRQPERTILR PRFRREVEKT ACPSGKKARE IDESLIFYKK WELEACVDAA LLATQMDRVNAIPFTYEQLD VLKHKLDELY PQGYPESVIQ HLGYLFLKMS PEDIRKWNVT SLETLKALLE VNKGHEMSPQVATLIDRFVK GRGQLDKDTL DTLTAFYPGY LCSLSPEELS SVPPSSIWAV RPQDLDTCDP RQLDVLYPKARLAFQNMNGS EYFVKIQSFL GGAPTEDLKA LSQQNVSMDL ATFMKLRTDA VLPLTVAEVQ KLLGPHVEGLKAEERHRPVR DWILRQRQDD LDTLGLGLQG GIPNGYLVLD LSMQEALSGT PCLLGPGPVL TVLALLLASTLA(SEQ ID NO: 60)

[0021] Mesothelin was found to be expressed at low levels in normal mesothelial cells, but at high levels in solid cancers (solid tumors), and its overexpression has been confirmed in esophageal cancer, breast cancer, triple-negative breast cancer (TNBC), gastric cancer, cholangiocarcinoma, pancreatic cancer, colon cancer, lung cancer, thymic carcinoma, mesothelioma, ovarian cancer, endometrial cancer, cervical cancer, uterine serous carcinoma (USC), and acute myeloid leukemia (AML) in children, etc. (Cancer Discov. February 2016; 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.).

[0022] In this specification, the term "antibody" refers to the general name of proteins that selectively act on antigens and participate in biological immunity, and there is no particular limitation on its type. The heavy and light chains of an antibody have antigen-binding sites that recognize epitopes including variable regions, and antigen specificity appears according to changes in the variable region sequences. The variable regions of the antigen-binding sites are divided into framework regions (FR) with relatively small variability and complementarity-determining regions (CDR) with relatively large variability. Both the heavy and light chains each have 3 CDR regions (divided into CDR1, CDR2, and CDR3) and 4 FR regions. The CDRs of each chain are usually named CDR1, CDR2, and CDR3 in sequence starting from the N-terminus, and are also identified by the chain where a specific CDR is located.

[0023] In this specification, the term "complementarity-determining region" refers to the site in the variable region of an antibody that confers binding specificity to an antigen.

[0024] In this specification, the term "epitope" refers to a specific three-dimensional molecular structure within an antigen molecule to which an antibody can specifically bind.

[0025] The antibodies include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, and chimeric antibodies (e.g., humanized murine antibodies). Additionally, the antibodies can include diabodies, triabodies, and tetra-bodies.

[0026] In this specification, an antibody includes an "antigen-binding fragment" or "antibody fragment" of an antibody having antigen-binding ability. The antigen-binding fragment can be an antibody fragment including one or more complementarity-determining regions, and for example, can be selected from the group consisting of scFv, (scFv)2, scFv-Fc, Fab, Fab', and F(ab')2. Fab in the antibody fragment is a structure including the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (CH1) of the heavy chain, and it has one antigen-binding site. Fab' is different from Fab in that a hinge region including one or more cysteine residues is located at the C-terminus of the heavy chain CH1 domain. When the cysteine residues in the Fab' hinge region form a disulfide bond, the F(ab')2 antibody is generated. Fv is the smallest antibody fragment, which only has the variable region of the heavy chain and the variable region of the light chain. Two-chain Fv includes the variable region of the heavy chain and the variable region of the light chain connected by non-covalent bonds. Single-chain Fv (scFv) generally has a dimer structure similar to two-chain Fv, in which the variable region of the heavy chain and the variable region of the light chain are covalently connected by a peptide linker or directly connected at the C-terminus.

[0027] In a specific embodiment, the anti-mesothelin antibody or its antigen-binding fragment includes one or more amino acid substitutions, wherein the one or more amino acid substitutions can include about 1 to about 5 amino acid substitutions, about 1 to about 4 amino acid substitutions, about 1 to about 3 amino acid substitutions, or about 1 to about 2 amino acid substitutions among the amino acids of SEQ ID NO: 1.

[0028] The one or more amino acid substitutions can occur at one or more positions selected from the following group: the 1st position of SEQ ID NO: 19 (the 1st 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).

[0029] In a specific embodiment, the one or more amino acid substitutions can include one or more selected from the group consisting of the following 1) to 3):

[0030] 1) The first amino acid of SEQ ID NO: 19 is substituted from D to K, W, L or R,

[0031] 2) The seventh amino acid of SEQ ID NO: 23 is substituted from S to F or R; and

[0032] 3) The fourth amino acid of SEQ ID NO: 24 is substituted from Y to R.

[0033] In the specification of this application, the first amino acid of SEQ ID NO: 19 can be substituted from D to K, W, L or R, and is respectively represented as D31L, D31K, D31W or D31R. The seventh amino acid of SEQ ID NO: 23 can be substituted from S to F or R, and is respectively represented as S192F or S192R. The fourth amino acid of SEQ ID NO: 24 can be substituted from Y to R, and is represented as Y228R.

[0034] In a specific embodiment, the one or more amino acid substitutions can be selected from one or more of the groups consisting of the following 1) to 3):

[0035] 1) The first amino acid of SEQ ID NO: 19 is substituted from D to L,

[0036] 2) The seventh amino acid of SEQ ID NO: 23 is substituted from S to R; and

[0037] 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.

[0038] In a specific embodiment, the anti-mesothelin antibody or its antigen-binding fragment is selected from antibodies or their antigen-binding fragments comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the following heavy chain CDRs, and the light chain variable region comprises the following light chain CDRs:

[0039] 1) An antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence consisting of SEQ ID NO: 27, HCDR2 comprising the amino acid sequence consisting of SEQ ID NO: 28, and HCDR3 comprising the amino acid sequence consisting of SEQ ID NO: 29, and the light chain variable region comprises: LCDR1 comprising the amino acid sequence consisting of SEQ ID NO: 30, LCDR2 comprising the amino acid sequence consisting of SEQ ID NO: 31, and LCDR3 comprising the amino acid sequence consisting of SEQ ID NO: 32,

[0040] 2) An antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence consisting of SEQ ID NO: 35, HCDR2 comprising the amino acid sequence consisting of SEQ ID NO: 36, and HCDR3 comprising the amino acid sequence consisting of SEQ ID NO: 37, and the light chain variable region comprises: LCDR1 comprising the amino acid sequence consisting of SEQ ID NO: 38, LCDR2 comprising the amino acid sequence consisting of SEQ ID NO: 39, and LCDR3 comprising the amino acid sequence consisting of SEQ ID NO: 40, and

[0041] 3) An antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence consisting of SEQ ID NO: 43, HCDR2 comprising the amino acid sequence consisting of SEQ ID NO: 44, and HCDR3 comprising the amino acid sequence consisting of SEQ ID NO: 45, and the light chain variable region comprises: LCDR1 comprising the amino acid sequence consisting of SEQ ID NO: 46, LCDR2 comprising the amino acid sequence consisting of SEQ ID NO: 47, and LCDR3 comprising the amino acid sequence consisting of SEQ ID NO: 48.

[0042] In a specific embodiment, the anti-mesothelin antibody or its antigen-binding fragment is selected from the antibody or its antigen-binding fragment comprising the following heavy chain variable region and light chain variable region:

[0043] 1) An antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence consisting of SEQ ID NO: 33, and the light chain variable region comprises the amino acid sequence consisting of SEQ ID NO: 34,

[0044] 2) An antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence consisting of SEQ ID NO: 41, and the light chain variable region comprises the amino acid sequence consisting of SEQ ID NO: 42, and

[0045] 3) An antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence consisting of SEQ ID NO: 49, and the light chain variable region comprises the amino acid sequence consisting of SEQ ID NO: 50.

[0046] In a specific embodiment, the anti-mesothelin antibody or antigen-binding fragment thereof can be or can include an scFv (single-chain variable fragment), and the anti-mesothelin scFv comprising the one or more amino acid substitutions can include substitutions selected from the group consisting of the following amino acid substitutions:

[0047] 1) The 31st amino acid of SEQ ID NO: 1 is substituted from D to L (D31L),

[0048] 2) The 192nd amino acid of SEQ ID NO: 1 is substituted from S to R (S192R), and

[0049] 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).

[0050] In a specific embodiment, the anti-mesothelin antibody or antigen-binding fragment thereof can be selected from antibodies or antigen-binding fragments thereof comprising the following antigen-binding fragments:

[0051] 1) An antibody or antigen-binding fragment thereof comprising an antigen-binding fragment, wherein the antigen-binding fragment comprises an amino acid sequence consisting of SEQ ID NO: 2,

[0052] 2) An antibody or antigen-binding fragment thereof comprising an antigen-binding fragment, wherein the antigen-binding fragment comprises an amino acid sequence consisting of SEQ ID NO: 3, and

[0053] 3) An antibody or antigen-binding fragment thereof comprising an antigen-binding fragment, wherein the antigen-binding fragment comprises an amino acid sequence consisting of SEQ ID NO: 4.

[0054]

Table 1

[0055]

[0056]

[0057] The anti-mesothelin antibody or antigen-binding fragment thereof can include a heavy-chain variable region comprising a sequence having 80% or higher sequence homology, preferably 90% or higher sequence homology, more preferably 95% or higher sequence homology, and even more preferably 100% homology with the amino acid sequence consisting of SEQ ID NO: 33, 41, or 49.

[0058] The anti-mesothelin antibody or antigen-binding fragment thereof may include a light chain variable region, and the light chain variable region includes a sequence having a sequence homology of 80% or higher, preferably 90% or higher, more preferably 95% or higher, still more preferably 100% homology with the amino acid sequence consisting of SEQ ID NO: 34, 42 or 50.

[0059] Taking into account mutations with biologically equivalent activities, an antibody or nucleic acid molecule encoding the same on the one hand may be interpreted as including a sequence having substantial identity with the sequence shown in the sequence number. When the said sequence is aligned with any other sequence to correspond to each other to the greatest extent and the aligned sequences are analyzed by using algorithms commonly used in the art, the substantial identity may refer to a sequence showing a homology of at least 61%, more preferably 70%, still more preferably 80%, still more preferably 90%, still more preferably 95%, still more preferably 98%, most preferably 99%. Alignment methods for sequence comparison are known in the art.

[0060] When including the one or more amino acid substitutions, the affinity for mesothelin may increase, and thus improved mesothelin affinity may be shown. When including the one or more amino acid substitutions, the binding force to the target mesothelin may increase compared with the wild type that does not include such substitutions.

[0061] On the other hand, there is provided an anti-mesothelin antibody or antigen-binding fragment thereof, which includes one or more amino acid substitutions in the amino acid sequence consisting of SEQ ID NO: 1. The same parts as above also apply to the antibody or antigen-binding fragment thereof.

[0062] In a specific embodiment, the anti-mesothelin antibody or antigen-binding fragment thereof may be an anti-mesothelin scFv, which includes one or more amino acid substitutions in the amino acid sequence consisting of SEQ ID NO: 1. The one or more amino acid substitutions may include about 1 to about 5, about 1 to about 4, about 1 to about 3, or about 1 to about 2 amino acid substitutions in the amino acids of SEQ ID NO: 1.

[0063] The one or more amino acid substitutions may occur at one or more positions selected from the group consisting of the 31st position, the 192nd position and the 228th position of SEQ ID NO: 1. For example, the one or more amino acid substitutions of the anti-mesothelin antibody or antigen-binding fragment thereof may be selected from one or more of the following groups (group a to group c).

[0064] a) D31K, D31W, D31L, and D31R;

[0065] b) S192F and S192R; and

[0066] c) Y228R.

[0067] When including the one or more amino acid substitutions, the affinity for mesothelin can be increased, and thus improved mesothelin affinity can be shown. When including the one or more amino acid substitutions, the binding ability to mesothelin as a target can be increased compared to the wild type that does not include such substitutions.

[0068] The anti-mesothelin scFv including the one or more amino acid substitutions can be, for example, one or more selected from the group consisting of SEQ ID NO: 2, 3, and 4.

[0069] On the other hand, there is provided an isolated nucleic acid encoding the antibody or its antigen-binding fragment. The same parts as those described above also apply to the nucleic acid.

[0070] In the present specification, the term "nucleic acid" broadly includes DNA and RNA molecules, and nucleotides, which are the basic structural units in nucleic acids, include not only natural nucleotides but also analogues in which the sugar or base positions are modified. The nucleic acid sequences encoding the heavy and light chain variable regions of one aspect can be modified. The modifications include addition, deletion, non-conservative substitution, or conservative substitution of nucleotides.

[0071] The nucleic acid is interpreted to further include nucleotide sequences showing substantial identity to the nucleotide sequence of the nucleic acid. Substantial identity means a nucleotide sequence showing a homology of at least 80%, more preferably at least 90%, and most preferably at least 95% when the nucleotide sequence of one aspect is aligned with any other sequence to correspond to each other to the maximum extent and the aligned sequences are analyzed using algorithms commonly used in the art.

[0072] On the other hand, there is provided a vector including the isolated nucleic acid. The same parts as those described above also apply to the vector.

[0073] For expressing an antibody or an antibody fragment thereof in a suitable host cell, the vector can be obtained from DNA encoding a partial or full-length light chain and heavy chain by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma expressing the target antibody), and the vector can include the necessary regulatory factors operably linked to enable the expression of the DNA (gene) insert. "Operably linked" refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a target protein or RNA to perform general functions, and refers to the connection being made such that the gene can be expressed through the expression control sequence.

[0074] The "expression control sequence" refers to a DNA sequence that regulates the expression of an operably linked DNA sequence in a specific host cell. This regulatory sequence includes a promoter for achieving transcription, any operator sequences for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, sequences regulating the termination of transcription and translation, a start codon, a stop codon, a polyadenylation signal, and an enhancer, etc. Those skilled in the art will recognize that the design of an expression vector can vary by selecting different regulatory sequences according to factors (e.g., the choice of host cell to be transformed and the expression level of the protein, etc.).

[0075] There is no particular limitation on the type of the vector as long as it is a vector commonly used in the fields of cloning and antibody production. For example, it includes plasmid vectors, cosmid vectors, phage vectors, and viral vectors, etc., but is not limited thereto. The plasmid can include plasmids derived from Escherichia coli (pBR322, pBR325, pUC118, pUC119, and pET-21b(+)), plasmids derived from Bacillus subtilis (pUB110 and pTP5), and plasmids derived from yeast (YEp13, YEp24, and YCp50), etc. And as the virus, animal viruses such as retroviruses, adenoviruses, or vaccinia viruses and insect viruses such as baculoviruses can be used. Commonly used pComb3 series vectors such as phage display can also be used. For expressing an antibody in mammalian cells, vectors commonly used for expressing proteins in mammalian cells, such as pcDNA or pVITRO, etc., can be used.

[0076] On the other hand, there is provided an isolated host cell transformed with the vector. The same parts as those described above also apply to the host cell.

[0077] In this specification, the term "transformation" refers to a molecular biology technique that permeates a DNA strand fragment or plasmid containing foreign genes of a different type from the original cell between cells to bind to the DNA present in the original cell, thereby changing the genetic inheritance of the cell. The vector is transfected into the host cell. For transfection, various techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells can be used, such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection, etc.

[0078] Considering the applicability to microorganisms such as bacteria (Escherichia coli, E. coli) or yeast (Yeast) or mammalian cells, an antibody or an antigen-binding fragment thereof according to one aspect can be expressed in eukaryotic cells (preferably, mammalian host cells). The mammalian host cell can be, for example, any one selected from the group consisting of Chinese Hamster Ovary (CHO) cells, NSO myeloma cells, COS cells, SP2 cells, F2N cells, HEK293 cells, and antibody-producing hybridoma cells, but is not limited thereto.

[0079] On the other hand, a method for producing an anti-mesothelin antibody with increased affinity for mesothelin is provided, which includes expressing the antibody by culturing the isolated host cell. The same parts as described above also apply to the method.

[0080] The method may include the step of transforming a host cell for producing an antibody or an antigen-binding fragment thereof according to one aspect with a vector operably linked to DNA encoding the antibody or antigen-binding fragment. The types of the selected host cell and recombinant expression vector are as described above, and this step can be implemented by selecting a suitable transformation method. 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 of time sufficient for the antibody to be expressed in the host cell or, more preferably, for a period of time sufficient for the antibody to be secreted into the culture medium of the cultured host cell.

[0081] In addition, the method may further include producing a polypeptide of an antibody or an antigen-binding fragment thereof according to one aspect from a recombinant expression vector introduced into a host cell by culturing the transformed and isolated host cell. The composition of the culture medium, the culture conditions, the culture time, etc. for culturing the selected host cell can be appropriately selected, and the antibody molecule produced in the host cell can accumulate in the cytoplasm, can be secreted outside the cell or into the culture medium through an appropriate signal sequence, or can be targeted to the periplasm, etc. In addition, methods known in the art can be used to cause protein refolding to occur and have a functional structure so that the antibody according to one aspect maintains the binding specificity for mesothelin. Furthermore, when producing an antibody in the Ig form, the heavy chain and the light chain can be expressed in separate cells, and the heavy chain and the light chain can be brought into contact with each other in a separate step to form a complete antibody, or the heavy chain and the light chain can be expressed in the same cell to form a complete antibody intracellularly.

[0082] In addition, the method may further include obtaining an antibody or an antigen-binding fragment thereof produced in an isolated host cell. The obtaining method can be appropriately selected and regulated by considering the characteristics of the polypeptide of the antibody or its antigen-binding fragment produced in the host cell, the characteristics of the host cell, the expression method, or whether the polypeptide is targeted, etc. For example, an antibody or an antigen-binding fragment thereof secreted into the culture medium can be recovered by a method of obtaining the culture medium for culturing the host cell and centrifuging to remove impurities, etc., and if necessary, in order to release and recover the antibody present in a specific organelle or cytoplasm in the cell, the cell can be lysed within the range that does not affect the functional structure of the antibody or its antigen-binding fragment.

[0083] Through methods such as filtration, dialysis using chromatography or filters, etc., the obtained antibody can also be further processed to remove impurities and concentrate. The separation or purification of the obtained antibody can be carried out by common separation and purification methods for proteins (for example, chromatography). The chromatography may include, for example, affinity chromatography, ion exchange chromatography, or hydrophobic chromatography, which includes protein A columns, protein G columns, and protein L columns. In addition to the chromatography, the antibody can also be separated and purified by combining filtration, ultrafiltration, salting out, dialysis, etc.

[0084] On the other hand, a chimeric antigen receptor is provided, which includes an antigen-binding domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain, wherein the antigen-binding domain includes the anti-mesothelin antibody or an antigen-binding fragment thereof. The same parts as the above apply to the chimeric antigen receptor. Since the chimeric antigen receptor has the characteristic of specifically binding to mesothelin, the chimeric antigen receptor includes an antigen-binding domain that specifically binds to mesothelin.

[0085] The antigen-binding domain may comprise an anti-mesothelin antibody or an antigen-binding fragment thereof, which comprises: a heavy-chain variable region comprising heavy-chain complementarity determining region 1 (HCDR1), heavy-chain complementarity determining region 2 (HCDR2), and heavy-chain complementarity determining region 3 (HCDR3), wherein the HCDR1 comprises an amino acid sequence consisting of SEQ ID NO: 19, the HCDR2 comprises an amino acid sequence consisting of SEQ ID NO: 20, and the HCDR3 comprises an amino acid sequence consisting of SEQ ID NO: 21; and a light-chain variable region comprising light-chain complementarity determining region 1 (LCDR1), light-chain complementarity determining region 2 (LCDR2), and light-chain complementarity determining region 3 (LCDR3), wherein the LCDR1 comprises an amino acid sequence consisting of SEQ ID NO: 22, the LCDR2 comprises an amino acid sequence consisting of SEQ ID NO: 23, and the LCDR3 comprises an amino acid sequence consisting of SEQ ID NO: 24, wherein the heavy-chain variable region and the light-chain variable region comprise one or more amino acid substitutions.

[0086] In a specific embodiment, the one or more amino acid substitutions may include one or more selected from the group consisting of the following 1) to 3):

[0087] 1) The 1st amino acid of SEQ ID NO: 19 is substituted from D to K, W, L, or R,

[0088] 2) The 7th amino acid of SEQ ID NO: 23 is substituted from S to F or R; and

[0089] 3) The 4th amino acid of SEQ ID NO: 24 is substituted from Y to R.

[0090] In the specification of the present application, the 1st amino acid of SEQ ID NO: 19 may be substituted from D to K, W, L, or R, and are respectively represented as D31L, D31K, D31W, or D31R, the 7th amino acid of SEQ ID NO: 23 may be substituted from S to F or R, and are respectively represented as S192F or S192R, and the 4th amino acid of SEQ ID NO: 24 may be substituted from Y to R, and is represented as Y228R.

[0091] In a specific embodiment, the one or more amino acid substitutions may be selected from one or more of the group consisting of the following 1) to 3):

[0092] 1) The first amino acid of SEQ ID NO: 19 is substituted from D to L,

[0093] 2) The seventh amino acid of SEQ ID NO: 23 is substituted from S to R; and

[0094] 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.

[0095] When including the one or more amino acid substitutions, the affinity for mesothelin can be increased, and thus improved mesothelin affinity can be shown. When including the one or more amino acid substitutions, the binding ability to mesothelin as a target can be increased compared to the wild type that does not include such substitutions.

[0096] When including the one or more amino acid substitutions, the affinity for mesothelin can be increased, and thus improved mesothelin affinity can be shown. When including the one or more amino acid substitutions, the binding ability to mesothelin as a target can be increased compared to the wild type that does not include such substitutions. Additionally, when including the one or more amino acid substitutions, excellent killing ability can be shown against cancer cells expressing mesothelin (e.g., mesothelioma, ovarian cancer, pancreatic cancer, etc.).

[0097] In the present specification, the term "chimeric antigen receptor (CAR)" refers to the structure of a chimeric antigen receptor constituted by including an antigen-binding (recognition) domain, a transmembrane domain, and an intracellular signaling domain.

[0098] In a specific embodiment, the antigen-binding fragment may be scFv (single chain variable fragment).

[0099] In a specific embodiment, the antigen-binding domain may be selected from antibodies or antigen-binding fragments thereof including the following antigen-binding fragments:

[0100] 1) An antibody or antigen-binding fragment thereof including an antigen-binding fragment, wherein the antigen-binding fragment includes the amino acid sequence consisting of SEQ ID NO: 2,

[0101] 2) An antibody or antigen-binding fragment thereof including an antigen-binding fragment, wherein the antigen-binding fragment includes the amino acid sequence consisting of SEQ ID NO: 3, and

[0102] 3) An antibody or antigen-binding fragment thereof including an antigen-binding fragment, wherein the antigen-binding fragment includes the amino acid sequence consisting of SEQ ID NO: 4.

[0103] The hinge domain, transmembrane domain, and intracellular signaling domain included in the chimeric antigen receptor are well known in the art.

[0104] The hinge domain is a domain that connects the anti-mesothelin antibody or its antigen-binding fragment to the transmembrane domain, also known as a spacer, and its purpose is to extend the antigen-binding domain from the T cell membrane. The hinge domain can be a CD8 hinge domain, IgG1 hinge domain, IgG4 hinge domain, extracellular region of CD28, extracellular region of killer immunoglobulin-like receptor (KIR), or a combination thereof, but is not limited thereto. As the hinge domain, hinge domains commonly used in the art can be used.

[0105] The transmembrane domain serves as a support for the chimeric antigen receptor molecule and can simultaneously connect the hinge domain and the intracellular signaling domain. The transmembrane domain can penetrate the cell membrane, such 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 inside the cell. The transmembrane domain can be the transmembrane region of CD3-zeta (CD3z), CD4, CD8, CD28, or KIR protein. Preferably, it can be the transmembrane domain of CD8 or CD28, but commonly used transmembrane domains for manufacturing chimeric antigen receptors can be used herein without limitation.

[0106] The intracellular signaling domain receives the signal transmitted through the anti-mesothelin antibody or its antigen-binding fragment and transmits it into the cell to which the chimeric antigen receptor binds. The type of the intracellular signaling domain is not particularly limited as long as it is a part that transmits a signal capable of inducing T cell activation when the antibody binds to the antigen-binding site present outside the cell, and various intracellular signaling domains can be used. The intracellular signaling domain can be, for example, a tyrosine-based activation motif or ITAM, and the ITAM includes those derived from CD3-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CDS, CD22, CD79a, CD79b, CD66d, or FcεRIγ, but is not limited thereto.

[0107] In addition, the chimeric antigen receptor according to one aspect may further include a costimulatory domain and an intracellular signaling domain.

[0108] The co-stimulatory domain is a part that transmits a signal to a T cell in addition to the signal transmitted through the intracellular signaling domain, and refers to the intracellular part of a chimeric antigen receptor, which includes the intracellular domain of a co-stimulatory molecule.

[0109] The co-stimulatory molecule is a cell surface molecule, which refers to a molecule required for lymphocytes to produce a sufficient response to an antigen. It can be, for example, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C or B7-H3, but is not limited thereto. The co-stimulatory domain can be the intracellular part of a molecule selected from the group consisting of such co-stimulatory molecules and combinations thereof.

[0110] Each domain of the chimeric antigen receptor including the transmembrane domain and the intracellular signaling domain can optionally be connected by a short oligopeptide or polypeptide linker. The length of the linker is not particularly limited, and linkers known in the art can be used as long as they can induce T cell activation through the intracellular domain when an antigen binds to an antibody located extracellularly.

[0111] In addition, the chimeric antigen receptor can include a modified form of the antibody and domain as described above. At this time, the modification can be carried out by substituting, deleting or adding one or more amino acids in the amino acid sequence of the wild-type antibody and domain without changing the function of the antibody and domain. Generally, the substitution can be alanine, or can be carried out by conservative amino acid substitution that does not affect the charge, polarity or hydrophobicity of the whole protein.

[0112] On the other hand, a polynucleotide encoding the chimeric antigen receptor is provided. The same parts as those described above also apply to the polynucleotide.

[0113] Those skilled in the art will understand that for the polynucleotide, due to the degeneracy of codons or considering the preferred codons in the organism expressing the antigen receptor, within the range of not changing the amino acid sequence of the antigen receptor expressed by the coding region, various modifications can be made to the coding region, and parts outside the coding region can also be variously changed or modified without affecting gene expression, and these modified genes are also included within the scope of the present invention. In other words, the polynucleotide according to one aspect can be mutated by substituting, deleting or inserting one or more nucleic acid bases or combinations thereof as long as the polynucleotide encodes a protein with equivalent activity, and these polynucleotides can also be included within the scope of the present invention.

[0114] On the other hand, there is provided a vector comprising the polynucleotide and an isolated cell transformed with the vector. The same parts as those described above also apply to the cell.

[0115] For the vector, various vectors known in the art can be used, and depending on the type of host cell used for producing the antigen receptor, expression regulatory sequences such as a promoter, a terminator, and an enhancer, or sequences for membrane targeting or secretion can be appropriately selected and variously combined according to the purpose. The vectors of the present invention include plasmid vectors, cosmid vectors, phage vectors, viral vectors, etc., but are not limited thereto. Suitable vectors can include, in addition to expression regulatory factors such as a promoter, an operator, a start codon, a stop codon, a polyadenylation signal, and an enhancer, a signal sequence or a leader sequence for membrane targeting or secretion, and can be prepared in various ways according to the purpose.

[0116] In addition, the vector can be introduced into a cell to transform the cell, and the isolated cell can be a T cell, an NK cell, an NKT cell, or a gamma-delta (γδ) T cell, but is not limited thereto. The isolated cell can be obtained or prepared from bone marrow, peripheral blood, peripheral blood mononuclear cells, or cord blood.

[0117] On the other hand, there is provided a pharmaceutical composition comprising the isolated cell; the pharmaceutical use of the isolated cell; and a method for preventing or treating cancer, which comprises administering a therapeutically effective amount of the isolated cell to an individual. The same parts as those described above also apply to the composition and the method.

[0118] Since the pharmaceutical composition utilizes the above-described isolated cell, a common description of the two will be omitted to avoid overcomplicating this specification.

[0119] The pharmaceutical composition or the pharmaceutical use can be used for preventing or treating cancer.

[0120] In this specification, the term "prevention" refers to all actions of inhibiting cancer (tumor) or delaying its onset by administering the pharmaceutical composition according to the present invention.

[0121] In this specification, the term "treatment" refers to all actions of improving or beneficially changing the symptoms of cancer (tumor) by administering the pharmaceutical composition according to the present invention.

[0122] In this specification, an "individual" refers to a subject in need of treatment for a disease, and more specifically, to a mammal such as a human or non-human primate, or a rodent (such as a rat, mouse, and guinea pig), mouse, dog, cat, horse, cow, sheep, pig, goat, camel, and antelope.

[0123] In this specification, the term "cancer" refers to the general term for diseases caused by cells that have aggressive characteristics in which the cells ignore normal growth restrictions and divide and grow, invasive characteristics in which the cells penetrate into surrounding tissues, and metastatic characteristics in which the cells spread to other parts of the body. In this specification, the cancer is used in the same meaning as a malignant tumor, and preferably, it can be a cancer that is mesothelin-positive or mesothelin-overexpressing.

[0124] Preferably, the cancer can be a solid cancer. For example, more preferably, it can be a solid cancer that is mesothelin-positive or mesothelin-overexpressing. For example, the solid cancer can be any one selected from the group consisting of esophageal cancer, breast cancer, triple-negative breast cancer (TNBC), gastric cancer, cholangiocarcinoma, pancreatic cancer, colon cancer, lung cancer, thymic carcinoma, mesothelioma, ovarian cancer, endometrial cancer, cervical cancer, uterine serous carcinoma (USC), non-small cell lung cancer, and acute myeloid leukemia (AML) in children, but is not limited thereto.

[0125] Relative to the total weight of the pharmaceutical composition, the pharmaceutical composition may include 10% to 95% by weight of cells on the one hand, which are the active ingredients. In addition, the pharmaceutical composition of the present invention may further contain one or more active ingredients that exhibit the same or similar functions in addition to the above active ingredients.

[0126] The dosage of the cells can be regulated according to various factors, including the type of disease, the severity of the disease, the types and contents of the active ingredient and other ingredients contained in the pharmaceutical composition, the type of dosage form, the age, weight, general health condition, gender and diet of the patient, the administration time, the administration route, the treatment period, and the drugs used simultaneously, etc. However, in order to achieve the preferred effect, the effective amount of the cells included in the pharmaceutical composition according to the present invention can be 1×10 5 cells / kg to 1×10 11 cells / kg. At this time, the administration can be once a day, or can be administered several times separately per day. The effective amount of the cells or pharmaceutical composition proposed in the present application can be determined empirically without excessive experimentation.

[0127] The pharmaceutical composition can be a preparation having a dosage form suitable for its purpose according to the common methods in the pharmaceutical field. In addition, the composition can be formulated and administered into a unit dosage form suitable for administration in the patient's body according to the common methods in the pharmaceutical field. In addition to the active ingredient, the pharmaceutical preparation can further include one or more pharmaceutically acceptable inert carriers. For example, in the case of an injection, it can include preservatives, analgesics, solubilizers or stabilizers, etc., and in the case of a preparation for topical administration, it can include a base, excipients, lubricants or preservatives, etc.

[0128] In addition, the cells or the pharmaceutical composition including the cells can be administered to an individual by various methods known in the art. For example, it can be administered by intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, pulmonary administration, rectal administration, etc., but not limited thereto.

[0129]

Beneficial effects

[0130] The anti-mesothelin chimeric antigen receptor according to one aspect has increased affinity for mesothelin and exhibits specific binding ability to mesothelin. Therefore, it can be effectively used for preventing or treating cancers with overexpression of mesothelin.

[0131]

Brief description of the drawings

[0132] Figure 1 A figure showing the polymerase chain reaction (PCR) conditions for cloning a group of antibody candidates with improved affinity.

[0133] Figure 2 A figure for confirming the three-dimensional structure of the antibody MSLN 34 calculated in Discovery Studio 2021.

[0134] Figure 3 A figure showing the representative docking models of each cluster in various docking models confirmed by Discovery Studio.

[0135] Figure 4 A figure showing the structure of the docking models of the finally selected MSLN and MSLN34 antibodies.

[0136] Figure 5 A figure for confirming the SDS-PAGE results after purification of 7 mutant antibody candidates (D31K, D31R, D31W, D31L, S192F, S192R, and Y228R) including wild-type (WT).

[0137] Figures 6 to 8 A figure showing the results of measuring the ELISA-based affinity for mesothelin for WT and 5 mutant antibodies (D31W, D31L, S192F, S192R, and Y228R).

[0138] Figure 9 and Figure 10 A figure for confirming the affinity of WT, D31L, and S192R single mutants of MSLN34 and the mutant including both D31L and S192R mutations for mesothelin.

[0139] Figure 11 A figure for confirming and showing the amino acid sequences of the scFv of the anti-MSLN chimeric antigen receptor vector with increased and improved affinity, which includes WT, D31L, and S192R single mutants of MSLN34 and the mutant including both D31L and S192R mutations.

[0140] Figure 12 and Figure 13 A figure for the characterization analysis of mutant MSLN CAR-T with increased affinity confirmed by Batch #1 experiment.

[0141] Figure 14 and Figure 15 A figure for the characterization analysis of mutant MSLN CAR-T with increased affinity confirmed by Batch #2 experiment.

[0142] Figure 16 A figure for confirming the apoptosis effect of mutant MSLN CAR-T with increased affinity on mesothelioma and ovarian cancer cells by calcein release assay (Calcein-AM) method in Batch #1 experiment.

[0143] Figure 17A figure for confirming the apoptosis effect of mutant MSLN CAR-T with increased affinity on mesothelioma and ovarian cancer cells using the Calcein-AM method through batch #2 experiments.

[0144] Figure 18 A figure for confirming the in vitro pancreatic cancer cell apoptosis effect of anti-MSLN-CAR-T cells with increased and improved affinity through Incucyte-based real-time cytotoxicity testing.

[0145] Figure 19 A figure showing the body weight change of a pancreatic cancer animal model after receiving CAR-T cell treatment.

[0146] Figure 20 A figure showing the tumor volume change of a pancreatic cancer animal model after receiving CAR-T cell treatment.

[0147] Figure 21 A figure for visually observing the pancreatic cancer animal model on the 49th day after receiving CAR-T cell treatment.

[0148] Figure 22 A figure for visually observing the isolated tumor of the pancreatic cancer animal model on the 49th day after receiving CAR-T cell treatment.

[0149] Figure 23 A figure showing the tumor weight of a pancreatic cancer animal model after receiving CAR-T cell treatment.

[0150] Figure 1 A figure showing the immunohistochemical staining results of tumor sections of a pancreatic cancer animal model after receiving CAR-T cell treatment (magnification: 5 times). Staining was performed using a human CD3ε antibody.

[0151] Primer A figure showing the immunohistochemical staining results of tumor sections of a pancreatic cancer animal model after receiving CAR-T cell treatment (magnification: 20 times). Staining was performed using a human CD3ε antibody.

Detailed Description of the Invention

[0152] Hereinafter, one aspect will be described in more detail by way of examples. However, these examples are only used to exemplarily describe one aspect, and the scope of one aspect is not limited to these examples, and the examples of one aspect are provided to more completely describe one aspect to those of ordinary skill in the art.

[0153]

Materials and Methods

[0154] 【1. Materials, Equipment, and Experimental Methods Used in the Experiment for Constructing an Antibody with High Affinity for Mesothelin】

[0155] 【1.1 Materials and Equipment】

[0156] In the experiment for constructing an anti-MSLN chimeric antigen receptor with improved affinity for mesothelin, the anti-mesothelin antibody, i.e., MSLN34 antibody, was used as a control group.

[0157] In addition, the specific reagents and equipment used in the experiment are shown in Tables 2 and 3 below.

[0158]

Table 2

[0159]

[0160]

Table 3

[0161]

[0162]

[0163] 【1.2 Design of Antibodies with Improved Affinity for Mesothelin】

[0164] To develop antibodies with improved affinity for mesothelin, Discovery studio 2021 was used.

[0165] Specifically, the Model Antibodies function of Discovery studio 2021 was used to calculate the three-dimensional structure of the anti-mesothelin antibody, i.e., MSLN34. By using the docking function (ZDOCK) of Discovery studio 2021 to calculate the antigen-antibody docking model, and by considering the binding force, etc., the best docking model was selected from the calculated docking models. Next, by using the Mutation (binding ability) function of Discovery studio 2021, the selected docking model was used to design antibodies with improved affinity.

[0166] 【1.3 Selection of Antibodies with Improved Affinity for MSLN】

[0167] 【1.3.1 Cloning of the Group of Antibody Candidates with Improved Affinity】

[0168] Based on the mutation information calculated from Discovery Studio 2021, primers were designed to be able to clone into the gene sequence that can encode the designed amino acids. The primer sequences for mutation cloning are shown in Table 4 below, and the polymerase chain reaction (PCR) conditions used are as Sequence (5'→3′) shown.

[0169]

Table 4

[0170] SEQ ID NO. D31K_F AAATATGGTA TGCACTGGGT TCG D31K_R ATACCATATT TAGAGAAAGT AAAACCCGAG 5 D31W_F CTCTTGGTAT GGTATGCACT GGGTTCG 6 D31W_R ATACCATACC AAGAGAAAGT AAAACCCGAG 7 D31L_F CTCTCTGTAT GGTATGCACT GGGTTCG 8 D31L_R ATACCATACA GAGAGAAAGT AAAACCCGAG 9 D31R_F CTCTCGTTAT GGTATGCACT GGGTTCG 10 D31R_R ATACCATAAC GAGAGAAAGT AAAACCCGAG 11 S192F_F GCAGTTTGGT GTACCGTCCC GT 12 S192F_R GGTACACCAA ACTGCAGAGA GGAAG 13 S192R_F GCAGCGTGGT GTACCGTCCC GT 14 S192R_R GGTACACCAC GCTGCAGAGA GGAAG 15 Y228R_F CGCTCTTTTC CGTTTACGTT CGG 16 Y228R_R AAACGGAAAA GAGCGAGATT GCTGACAAT 17 Cell line information Organism 18

[0171] The PCR products of each mutation were cloned using Gibson assembly method and transformed into Escherichia coli (E. coli) DH5α to obtain single clones. Nucleotide sequence analysis was performed by sequencing to confirm the amino acid sequences of the final candidate group.

[0172] 【1.3.2 Production of mutant antibody candidate group】

[0173] The cloned gene was transformed into the protein expression strain, Escherichia coli TOP10F′, and spread on LB solid medium containing the antibiotic ampicillin, and cultured at 37 °C for 20 hours to obtain the transformed gene organism. For pre-culture, the colonies of the transformed gene organism were cultured with shaking at 37 °C for 16 hours in LB medium containing 10 mL of ampicillin, and 5 mL of the cultured cells were inoculated into LB medium containing 500 mL of ampicillin. When the cell density (O.D600) reached 0.6 or higher during culture at 37 °C, 0.5 mM IPTG was added, the cells were expressed at 30 °C, and harvested after 16 hours. The harvested Escherichia coli was reacted in 1×TES buffer (50 mM tris-HCl, 1 mM EDTA, 20% sucrose, pH 8.0) for 1 hour, and additionally reacted in 0.2×TES buffer for 1 hour to completely lyse. The lysed Escherichia coli was centrifuged (15,000 rpm, 40 minutes, 4 °C) to recover the supernatant, and passed through a column equilibrated with 1×PBS to bind to the internal resin. After washing the resin with 1×PBS, the antibody was recovered with 1×BXT buffer. The recovered protein was concentrated using a centrifugal filter (3 kDa).

[0174] 【1.3.3 ELISA-based affinity measurement】

[0175] 1 μg / mL of MSLN antigen was aliquoted at 30 μL per well into each well of a 96-well ELISA plate coated with polystyrene and reacted at 4 °C for 16 hours. After the reaction, the MSLN antigen was removed and treated with a blocking solution of 5% MPBS (5% w / v non-fat dry milk in PBS), then the antibody at 1 mg / mL was diluted to 1 / 3, 1 / 9, 1 / 27, 1 / 81 and 1 / 243 and reacted at room temperature for 1 hour. Washed 4 times again with PBST buffer, then reacted with HRP-anti-streptococcus at 37 °C for 1 hour. After washing 4 times with PBST buffer, reacted with TMB substrate at room temperature for 8 minutes, and the reaction was terminated with 2N H2SO4. The absorbance value at OD450nm was confirmed using an enzyme-linked immunosorbent assay reader.

[0176] 【2. Materials and Equipment Used in the Experiment for Confirming the Killing Ability of CAR-T Anticancer Cells Comprising a Newly Discovered, High-Affinity MSLN34 Variant scFv】

[0177] 【2.1 Materials, Cell Lines, Reagents and Equipment Used】

[0178] Experiments were conducted to evaluate the killing ability of CAR-T against pancreatic cancer, mesothelioma and ovarian cancer cell lines, where the CAR-T was equipped with MSLN34 scFv variants (MSLN34-D31L, MSLN34-S192R and MSLN34-D31L / S192R), which were obtained by in silico affinity maturation of the scFv of the anti-mesothelin antibody, i.e., MSLN34. The materials used in the experiments are as follows.

[0179] The lentiviral vector (pLV) was used as the vector for expressing the anti-MSLN CAR, and the cell lines used are shown in Tables 5 to 7 below.

[0180]

Table 5

[0181] Homo sapiens, human 293T Tissue Embryonic kidney Disease Normal cell Characteristic Adhesion Culture condition Supplier ATCC (CRL-3216) <![CDATA[DMEM, 10% FBS, 1% penicillin and streptomycin, 5% CO2, 37 °C]]> Cell line information AsPC-1 / GFP

[0182]

Table 6

[0183] Organism Homo sapiens, human Tissue Pancreas Disease Adenocarcinoma Characteristic Adhesion Culture condition Supplier In-house <![CDATA[RPMI1640, 10% FBS, 1% penicillin and streptomycin, 5% CO2, 37°C]]> Cell line information OVCAR-3

[0184]

Table 7

[0185]

[0186]

[0187]

Table 8

[0188] Organism Homo sapiens, human Tissue Ovary Disease Adenocarcinoma Characteristic Adhesion Culture condition Supplier ATCC (HTB-161) <![CDATA[RPMI1640, 20% FBS, 1% penicillin and streptomycin, 5% CO2, 37°C]]> No. Category

[0189] Information on the lentiviral production vectors used is shown in Table 9 below.

[0190]

Table 9

[0191] Vector type Catalog number Manufacturing company Comment Packaging vector pMDLg / pRRE 1 Addgene Packaging vector 12251 pRSV-Rev - 2 Addgene Packaging vector 12253 pMD2.G - 3 Addgene Expression vector 12259 pLV-MSLN - 4 KBIO Equipment - Model number <![CDATA[2 nd G.KBIO CAR]]>

[0192] In addition, other reagents and materials are shown in Table 10 below.

[0193]

Table 10

[0194]

[0195]

[0196] In addition, the equipment used is shown in Table 11 below.

[0197]

Table 11

[0198] Manufacturing company Biosafety cabinet ThermoFisher Centrifuge 1367 Eppendorf Galaxy 170S 5810R Eppendorf <![CDATA[CO2 Incubator]]> CountessTMII Automated Cell Counter AMQAX1000 ThermoFisher Ultracentrifuge Optima XE-100 Beckman Coulter Incucyte Incucyte zoom Essen bioscience BD ND-2000 <![CDATA[FACSCanto TM II]]> 338960 ​ <![CDATA[NanoDrop TM 2000 Spectrophotometer]]> ​ ThermoFisher Multimode microplate reader FilterMax F5 Molecular devices FLOWJO Single Cell Analysis Software V10 663335 FlowJo, LLC. GraphPad Prism (Version 9) - GraphPad Software

[0199] 【2.2 Preparation of pLV Lentiviral Expression Vector Expressing Anti-MSLN CAR】

[0200] Using three kinds of MSLN34-D31L, MSLN34-S192R, and MSLN34-D31L / S192R obtained from the Artificial Intelligence Structure Design Team of the New Drug Development Support Center, which are affinity-matured scFv sequences based on MSLN34 scFv, the vector was prepared according to the following method. Using the KOD plus mutagenesis kit, affinity-matured anti-MSLN scFv-loaded CAR vectors (MSLN34-D31L CAR, MSLN34-S192R CAR, MSLN34-D31L / S192R CAR) based on the MSLN34 CAR vector (REP-RD21-011) manufactured in the second-generation KBIO CAR vector were prepared. The preparation method refers to the instruction manual of the KOD plus mutagenesis kit. The prepared vector was confirmed by gene sequence analysis to have no abnormality in the entire anti-MSLN scFv gene composition sequence.

[0201] 【2.3 Plasmid DNA Extraction for Lentivirus Production】

[0202] The lentiviral packaging plasmids (pMDLg / pRRE, pRSV-Rev, pMD2.G) and pLV MSLN DNA vector were introduced into Escherichia coli (DH5α) strain by heat shock transformation. According to Maxi EF kit and Xtra Midi Plasmid DNA Purification Instruction Manual to extract plasmid DNA. The concentration and purity of the extracted plasmid DNA were measured by using a NanoDrop TM 2000 spectrophotometer.

[0203] 【2.4 Production and Concentration / Purification of Lentivirus】

[0204] The 293T cells were seeded at 6.0×10 6Cells were inoculated into 100 mm cell culture dishes at a concentration of [number] cells / dish and cultured for 1 day. The third-generation lentiviral packaging plasmids pMDLg / pRRE, pRSV-Rev, pMD2.G DNA, and MSLN CAR vector DNA were respectively diluted in Opti-MEM at a predetermined ratio and then transfected using Lipofectamine 3000. The transfection was carried out according to the Lipofectamine 3000 user manual. The produced lentiviral culture solution was purified / concentrated by using 20% sucrose gradient purification method and finally stored at -80 °C.

[0205] [2.5 Measuring the infectious titer of lentivirus using FACS analysis]

[0206] HeLa cells were inoculated into 6-well plates at a concentration of 1.5×10 5 cells / well and cultured for 1 day. On the next day, the lentivirus with the infectious titer to be measured and polybrene were added to each well of HeLa cells at a final concentration of 8 μg / mL to perform transduction. After 48 hours of transduction, the cells were harvested for FACS analysis.

[0207] Using recombinant MSLN protein as an antigen, the number of cells binding to the anti-MSLN scFv antibody site was measured by performing FACS analysis. The conversion formula for the infectious titer is as follows.

[0208] Transducing Unit (TU) / mL = [(number of inoculated cells) × (frequency of PE+ cells) × 1000] / (μL of lentiviral vector)

[0209] [2.6 Preparation of MSLN CAR-T]

[0210] [2.6.1 Performing T cell activation]

[0211] Human PBMCs were dissolved and diluted in 9 mL of T cell medium (RPMI-1640 medium + 10% FBS + 1% penicillin-streptomycin + IL-2 200 U / mL) and centrifuged at room temperature and 300 g for 7 minutes. Then, the supernatant was removed and resuspended in 10 mL of fresh T cell medium. The differentiation process from human PBMCs to T cells was carried out according to the instructions provided by the manufacturer of the TransAct bead reagent.

[0212] [2.6.2 MSLN CAR transduction]

[0213] One day after the start of T cell activation, all activated T cells in the culture were harvested and centrifuged at room temperature and 300 g for 7 minutes. Prepare to place the activated T cells into a new 24-well plate at a concentration of 5.0×10 5 cells / well and culture them in a final volume of 0.5 mL. For each experimental group, lentivirus was added at 5 MOI based on the infectious titer, and protamine sulfate was added to a final concentration of 1 μg / mL and inoculated into a new 24-well plate. The 24-well plate was subjected to spin infection at 300 g and 32 °C for 90 minutes, and then cultured in an incubator at 37 °C and 5% CO2. The next day, all T cells were harvested, centrifuged at 300 g for 7 minutes, the supernatant was removed, and fresh medium was added for culture.

[0214] 【2.6.3 MSLN CAR-T Characterization Analysis】

[0215] The MSLN CAR-T cells in the culture were adjusted using a cell counter to obtain 1×10 6 cells. After washing the cells with wash buffer (PBS + 2% FBS), biotin-MSLN antigen was added and then stored at 4 °C for 20 minutes. The cells were washed again with wash buffer, then PE-biotin antibody and APC-CD3 antibody were added and stored at 4 °C in the dark for 20 minutes. Finally, the cells were washed with wash buffer and then resuspended in 100 μL of wash buffer, and finally FACS analysis of the differentiated CAR-T was performed.

[0216] 【2.7 MSLN CAR-T In Vitro Efficacy Evaluation: Calcein Release Assay】

[0217] 【2.7.1 Preparation of Target Cells Stained with Calcein-AM】

[0218] 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 and stained at 37 °C for 1 hour. Centrifuge at 1,200 rpm for 5 minutes at room temperature and wash the cells 3 times with 1 mL of medium. The cells stained with calcein-AM were inoculated into a 96-well plate (type R) at a concentration of 1.0×10 4 cells / well.

[0219] 【2.7.2 Preparation of Effector Cells (MSLN CAR-T)】

[0220] The ratio of effector cells that will react with target cells starts from E:T = 10:1, and a 2-fold serial dilution method is performed to form a total of 4 E:T ratios, which are corrected and calculated based on the number of T cells expressing CAR according to the expression ratio of each CAR. However, for mock T cells, the cell number is calculated to be the same as the maximum cell number when corrected to the CAR expression rate. For the spontaneous release of calcein-AM, RPMI-1640 medium is treated. For the maximum release of calcein-AM, 2% Triton X-100 is treated.

[0221] 【2.7.3 Perform calcein release assay】

[0222] The target cells and effector cells are mixed and co-cultured at 37 °C and 5% CO2 for 4 hours. Centrifugation is performed at room temperature and 100 g for 5 minutes. To measure the amount of calcein-AM leaked extracellularly due to apoptosis, 100 μL of the co-culture medium is transferred to a black optical plate (type F). Using an enzyme-linked immunosorbent assay reader that can measure fluorescence, the value is measured within the wavelength range of an excitation wavelength of 485 mm and an emission wavelength of 530 nm. The calculation formula for obtaining the cytotoxic effect value is as follows.

[0223] Percentage of specific lysis = [(test release - spontaneous release) / (maximum release - spontaneous release)] × 100%

[0224] 【2.8 MSLN CAR-T in vitro efficacy evaluation: Incucyte-based real-time cytotoxicity assay】

[0225] 【2.8.1 Preparation of target cells】

[0226] Seed AsPC-1 / GFP cells into a 96-well plate at 100 μL per well to achieve 1×10 4 cells / well.

[0227] 【2.8.2 Preparation of effector cells】

[0228] Mock T (non-transduced T) and MSLN CAR-T (MSLN34, MSLN34-D31L, MSLN34-S192R, and MSLN34-D31L / S192R) cells were tested. The ratio of effector cells to react with target cells was adjusted to E:T = 0.5:1 and corrected and calculated based on the number of CAR-expressing T cells according to the expression ratio of each CAR. However, for mock T cells, the cell number was calculated to be the same as the maximum cell number when corrected for the CAR expression rate.

[0229] [2.8.3 Real-time cytotoxicity test]

[0230] The target cells and effector cells were mixed and co-cultured. The Incucyte was set to measure GFP every 3 hours in a 96-well plate for 72 hours and then loaded into the plate in the co-culture. After all GFP measurements of the Incucyte were completed, the GFP measurement values were analyzed.

[0231] [2.9 Experimental data and statistical analysis]

[0232] For Incucyte analysis and calcein release analysis, each experimental group consisted of 4 independent E+T co-culture wells (Tetra-plicated assay) to obtain experimental data. All experimental data were plotted using GraphPad Prism 9.0 software. The two-way analysis of variance (Two-Way ANOVA) (full model, Tukey, 95% confidence interval) included in GraphPad Prism 9.0 software was used to determine the statistical significance of all experimental data. (ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001)

[0233] [3. Materials, equipment, and experimental methods used in the experiment to confirm the anti-cancer effect of CAR-T including the newly discovered, high-affinity MSLN34 scFv on a pancreatic cancer animal model]

[0234] [3.1 Establishing a pancreatic cancer (AsPC-1) animal model]

[0235] [3.1.1 Breeding mice]

[0236] To establish a pancreatic cancer animal model, NOG (NOD / Shi-scid / IL-2Rγ nullSix-week-old (15.0 g to 25.0 g) male specific pathogen-free (SPF) mice of the strain. The mice were bred and the related experiments were conducted under the conditions of a temperature of 22 ± 2 °C, a relative humidity of 50 ± 10%, a ventilation frequency of 10 to 20 times per hour, a lighting time of 12 hours (lights on at 8:00 am to lights off at 8:00 pm), and an illuminance of 150 to 300 Lux, and were carried out in the animal room of the Wusong Advanced Medical Industry Revitalization Foundation Experimental Animal Center. The mice were allowed to freely ingest feed and water. The experiments related to the mice were conducted in accordance with the regulations of the Wusong Advanced Medical Industry Revitalization Foundation Experimental Animal Management Committee.

[0237] [3.1.2 Cell culture and transplantation]

[0238] To prepare a pancreatic cancer animal model, the AsPC-1 cell line was used. The cell line was tested for Mycoplasma pneumoniae, Murine coronavirus (Mouse hepatitis virus: MHV), and Murine respirovirus (Sendai virus, SeV), and was used after being confirmed negative. Using a medium composed of RPMI-1640, 10% FBS, and 1% P / S (penicillin / streptomycin), the cell line was cultured in a CO2 incubator at 37 °C and 5% CO2. After adjusting the concentration of the AsPC-1 cells using PBS, 200 μL was subcutaneously transplanted into each mouse.

[0239] [3.2 Composition of the experimental groups for the pancreatic cancer animal model]

[0240] The pancreatic cancer animal models prepared above were grouped by the random allocation method according to tumor size, and the experimental groups were formed as shown in the following table.

[0241] [Table 12]

[0242]

[0243] During the experiment, the individual identification of the experimental groups was carried out using the ear-punch method, and the identification cards of each group were attached to the breeding box. After grouping, the test substance was administered once intravenously (I.V).

[0244] [3.4 Measurement of body weight and tumor size]

[0245] The body weight and tumor size of the experimental groups were measured twice a week from the start of administration. Based on the body weight on the start day of administration (day 0), the change in body weight was observed until the end day of the experiment. The following formula was used to calculate the body weight (%).

[0246] Body weight (%) = (body weight / body weight on day 0) × 100

[0247] Tumor size (mm) was calculated by measuring the short axis (A) and long axis (B) of the tumor using calipers and using the following formula 3 )

[0248] Tumor volume (mm 3 ) = ([A (mm)] 2 × B (mm)) / 2

[0249] 【3.5 Dissection】

[0250] Anesthesia was induced by intraperitoneal injection of Zoletil TM (50 mg / kg) and Rompun (10 mg / kg), the abdominal cavity was opened, blood was collected from the abdominal vein, and then exsanguination was performed for euthanasia. Serum was separated from the blood and then cryopreserved (-80 °C or lower). A part of the isolated tumor was cryopreserved (-80 °C or lower), and a part was fixed with a fixative (10% neutral formalin), and then histopathological examination was performed. Slides were prepared and hematoxylin & eosin (H&E) staining was performed. The stained slides were photographed using PANNORAMIC SCAN II (3DHISTECH, Hungary), and analysis was performed using 3DHISTECH software

[0251] 【3.6 Data analysis】

[0252] SPSS 10.1 was used to analyze the statistical comparison of the data. The data were expressed as mean ± standard deviation (SD). For the analysis, Tukey's post hoc analysis was performed after one-way analysis of variance (One-way ANOVA) for multiple analysis. (*: 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))

[0253]

Results

[0254]

Experimental Example 1: Calculation of the docking model of antigen (MSLN)-antibody (MSLN34)

[0255] First, the three-dimensional structure of MSLN34, an antibody that specifically binds to mesothelin as an antigen, was calculated. MSLN34 was calculated by the Homology Model function of Discovery studio 2021, and the calculated structure was asFigure 2 As shown. As in Figure 2 it can be confirmed that the CDR regions of the antibody are shown in pink (light chain) and blue (heavy chain).

[0256] Next, the antigen-antibody docking model of mesothelin and MSLN34 was calculated using the ZDOCK program of Discovery Studio 2021. As a result, as confirmed in Figure 3 various docking models that bind to the inner surface of the MSLN bend from the N-terminus to the C-terminus can be seen, and various binding positions can be confirmed. Based on the results of the docking program of Discovery studio 2021, approximately 2000 docking models were calculated, and the structures of approximately 200 models were confirmed in descending order of the predicted binding force value. Considering whether there is specific binding between mesothelin and MSLN34, etc., finally Figure 4 the docking model in

[0257] As confirmed from the above Figure 4 the selected docking model binds to amino acids 385 to 569 of mesothelin, and this binding position was confirmed as the position where the most docking models were calculated in the docking results of Discovery studio 2021. In addition, it was also confirmed that all 6 variant regions were used for the amino acids involved in the binding of MSLN34 to the target mesothelin, so it was confirmed that its structure formed a stable binding.

[0258]

Experimental Example 2: Selection of MSLN34 antibodies with increased affinity

[0259] 【2.1 Design of a candidate group of mutations with increased affinity based on the silicon-based model structure】

[0260] An experiment was designed to confirm antibodies that can increase the binding force based on the docking model selected in Experimental Example 1. The calculation was performed using the Mutagenesis (binding) function in Discovery studio 2021, and the calculation results are shown in Table 13 below.

[0261]

Table 13: Cluster-3 calculation of mutation energy (binding)

[0262] Mutation Mutation energy Mutation effect VDW term Electrostatic term Entropy term Non-polar term B: ASP31>ARG -3.24 Stable -4.66 -4.26 1.53 0 B: ASP31>LYS -3.22 Stable -6.14 -2.55 1.4 0 B: ASP31>TRP -3.1 Stable -4.37 -2.52 0.43 0 B: ASP31>LEU -3.05 Stable -3.8 -2.69 0.24 0 B: SER192>ARG -3.04 Stable -6.8 -0.8 0.95 0 B: ASP31>HIS -2.73 Stable -3.89 -2.17 0.38 0 B: SER192>PHE -2.73 Stable -4.98 -0.28 -0.12 0 B: ASP31>PHE -2.62 Stable -2.83 -2.68 0.17 0 B: ASP31>ILE -2.57 Stable -2.7 -2.57 0.08 0 B: TYR102>ARG -2.46 Stable -4.6 -1.89 0.98 0 B: ASP31>TYR -2.44 Stable -3.15 -2.49 0.48 0 B: HIS100>ARG -2.33 Stable -3.45 -1.83 0.39 0 B: TYR104>ARG -2.14 Stable -5.04 -1.08 1.15 0 B: TYR228>ARG -0.87 Stable -1.6 -1.58 0.9 0

[0263] As confirmed from Table 13 above, the candidate group with the highest affinity in the docking model was selected in descending order of the mutation energy.

[0264] Specifically, since induced mutations at positions D31, S192, and Y228 were judged to be most likely to increase the affinity of MSLN34 for the target mesothelin in the docking model, mutations were induced in the anti-mesothelin antibody or its antigen-binding fragment. Thus, the following mutations were designed: in the scFv (SEQ ID NO: 1) of the anti-mesothelin antibody (MSLN34), aspartic acid (D) at the 31st amino acid (the 1st amino acid of HCDR1) was replaced with lysine (K), tryptophan (W), leucine (L), and arginine (R); serine (S) at the 192nd amino acid (the 7th amino acid of LCDR2) was replaced with phenylalanine (F) and arginine (R), and tyrosine (Y) at the 228th amino acid (the 4th amino acid of LCDR3) was replaced with arginine (R). However, although the mutation energy was low, positions Y102 and Y104 were excluded because they are amino acids directly involved in binding in the docking model. The sequences of the mutant candidate group of the anti-mesothelin antibody or its antigen-binding fragment with increased affinity designed in this way were analyzed. The sequences of the mutants are scFv sequences in which the amino acids at each position in the MSLN34 scFv of SEQ ID NO: 1 are replaced with specific amino acids.

[0265] [2.2 Confirm the affinity of the antibody candidate group including single mutations with increased and improved affinity for mesothelin]

[0266] Experiments were conducted to confirm whether the antibody candidate substances in Experimental Example 2.1 could actually be confirmed as anti-mesothelin antibodies or their antigen-binding fragments with increased and improved affinity. To produce the anti-mesothelin antibody or its antigen-binding fragment confirmed in Experimental Example 2.1, the DNA nucleic acid sequence encoding the mutant amino acid sequence of the MSLN34 scFv was expressed in the Escherichia coli strain Top10F' and purified by affinity chromatography using a strep tag. The purity and yield of a total of 8 mutant antibody candidate substances including the wild type (WT) were confirmed on an SDS-PAGE gel, and the confirmation results are shown in Figure 5 . As confirmed from Figure 5 , D31K and D31R were not expressed or purified, while WT, D31W, D31L, S192F, S192R, and Y228R were confirmed to be purified to a purity of 95% or higher.

[0267] Next, the ELISA-based affinity of 6 antibodies including the purified WT was measured. Mesothelin (MSLN) was attached to a 96-well plate, the purified antibodies were serially diluted 1 / 3-fold and reacted, and the graph regarding the affinity is shown in Figures 6 to 8 . As confirmed from Figures 6 to 8As confirmed, compared with WT, the EC in the D31L, S192F, and S192R candidate substances is lower, so it is confirmed that their affinity increases. 50 The value is lower, so it is confirmed that their affinity increases.

[0268] 【2.3 Confirm the affinity of the antibody candidate group including the double mutation with increased and improved affinity for mesothelin】

[0269] For the D31L and S192R mutant candidate groups with increased affinity among the single mutant antibodies confirmed by the above experimental examples, experiments were conducted to confirm whether the affinity for mesothelin would further increase when double mutations occurred.

[0270] The specific experimental method was carried out in the same way as the method for measuring the affinity of the single mutant antibody in Experimental Example 2.2. The results of confirming the affinity of WT, D31L, and S192R single mutants of MSLN34 without mutations and the mutant including both D31L and S192R mutations are shown in Figure 9 and Figure 10 . As confirmed from Figure 9 and Figure 10 , according to the ELISA-based affinity measurement results, it can be confirmed that the affinity of the double mutant antibody is significantly increased compared with WT.

[0271] The amino acid sequences of the single mutants of MSLN34 scFv with increased affinity confirmed above, namely D31L (the first amino acid of HCDR1 is replaced from D to L) or S192R (the seventh amino acid of LCDR2 is replaced from D to R), and the mutant including both D31L and S192R mutations (D31L / S192R) are shown in Figure 11 .

[0272] In addition, the amino acid sequences of the light chain CDR sequence, heavy chain CDR sequence, light chain variable region, and heavy chain variable region of the MSLN34 and its modified mutants are recorded in Table 14 below. In addition, the parts to be replaced in the MSLN34 mutant are indicated in bold and underlined.

[0273]

Table 14

[0274]

[0275]

[0276]

Experimental Example 3: Construction of an anti-MSLN chimeric antigen receptor with increased and improved affinity

[0277] 【3.1 Clone the lentiviral vector of anti-MSLN-CAR】

[0278] To construct a chimeric antigen receptor comprising the improved anti-mesothelin antibody or antigen-binding fragment confirmed in Experimental Example 2, the anti-MSLN-CAR lentiviral vector was cloned.

[0279] As the vector, it belongs to the second-generation CAR lentiviral vector (pLV lentiviral vector) system held by the New Drug Development Support Center, and the system includes pMDLg / pRRE (addgene) encoding gag / pol, the envelope plasmid pRSV-Rev (addgene) encoding the Rev protein, and the envelope plasmid pMD2.G (addgene) encoding the VSV-G protein.

[0280] First, gene cloning was performed on MSLN34 scFv (antigen-binding domain) and its mutants, which were confirmed to have excellent efficacy in Experimental Example 2. Each anti-MSLN scFv and the lentiviral vector were digested with XhoI (R0146S, NEB) and EcoRI (R0101, NEB) at 37 °C for 2 hours, followed by agarose gel electrophoresis, and then the confirmed products were purified using the FavorPrep Gel / PCR Purification Mini Kit (Favorgen). Each purified anti-MSLN scFv (100 ng) was reacted with the vector (50 ng) at a ratio of 2:1 at 16 °C for 16 hours for ligation, and then transformed into Stbl3 competent cells to obtain colonies. The colonies were obtained, cultured in 5 mL of LB medium (ampicillin), and plasmid DNA was obtained using the DNA plasmid mini-prep method. It was confirmed whether each anti-MSLN scFv inserted by cutting the plasmid DNA with XhoI and EcoRI was successfully cloned into the vector. Then, sequencing was performed, and finally the DNA sequence was confirmed.

[0281] For the MSLN scFv, the CD8 hinge region and CD8 transmembrane (TM) as transmembrane regions, the cytoplasmic region of 4-1BB as a signal transduction domain, and the intracellular domain of CD3-zeta (CD3z) as a T cell activation domain are successively connected to constitute an anti-MSLN CAR. Specifically, the anti-MSLN CAR is constituted of a CD8 signal sequence (signal peptide, SP) (SEQ ID NO: 51), MSLN34 scFv and its mutants (SEQ ID NOs: 52 to 55), CD8 hinge region (SEQ ID NO: 56), CD8 transmembrane region (SEQ ID NO: 57), 4-1BB signal transduction domain (SEQ ID NO: 58), and CD3-zeta signal transduction domain (SEQ ID NO: 59). Each domain is successively connected using corresponding restriction enzymes. The specific base sequence information corresponding to each domain is organized in the following table.

[0282]

Table 15

[0283]

[0284]

[0285]

[0286] Cloning was completed, and the amino acid sequences of the scFv of the anti-MSLN chimeric antigen receptor vector with increased and improved affinity were confirmed, including the WT of MSLN 34, single mutants of D31L and S192R respectively, and mutants both including D31L and S192R mutations.

[0287] 【3.2 Production of lentivirus loaded with anti-MSLN-CAR and measurement of infectious titer】

[0288] The results of the infectious titer of the CAR-loaded lentivirus confirmed using HeLa cells are shown in Table 16 [Infectious titer measurement results (FACS analysis)].

[0289]

Table 16

[0290] Sample Measured titer (TU / mL) MSLN34 <![CDATA[1.76×10 7 > MSLN34-D31L <![CDATA[1.74×10 7 > MSLN34-S192R <![CDATA[2.20×10 7 > MSLN34-D31L / S192R <![CDATA[1.65×10 7 >

[0291] 【3.3 Preparation of cells transfected with anti-MSLN-CAR】

[0292] Experiments (batch #1 and batch #2) were conducted to produce MSLN CAR-T cells transfected with the vector of Experimental Example 3.1, and their characteristics were analyzed by FACS. The results of the analysis of the characteristics of MSLN CAR-T in the produced batch #1 are shown in Figure 12and Figure 13 , and the results of the analysis of the characteristics of MSLN CAR-T in production batch #2 are shown in Figure 14 and Figure 15 . In addition, the results of the analysis of the characteristics of MSLN CAR-T in batches #1 and 2 are summarized in Table 17.

[0293] As confirmed from Figure 12 and Figure 13 , the CAR-expressing cells in MSLN CAR-T batch #1 were 46.1%, 39.3%, 54.6%, and 43.3% in 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%).

[0294] In addition, as confirmed from Figure 14 and Figure 15 , the CAR-expressing cells in MSLN CAR-T batch #2 were 43.9%, 46.9%, 37.9%, and 52.9% in MSLN34, MSLN34-D31L, MSLN34-S192R, and MSLN34-D31L / S192R, respectively. Most of the CAR-expressing cells were confirmed to be CD3 + T cells (≥99.0%).

[0295]

Table 17

[0296]

[0297]

[0298]

Experimental Example 4: Confirmation of the apoptosis effect of anti-MSLN-CAR-T cells with increased and improved affinity on mesothelioma and ovarian cancer

[0299] Using the anti-MSLN-CAR-T cells prepared in Experimental Example 3 above, the apoptosis effect on mesothelioma and ovarian cancer cells was confirmed by calcein release assay (Calcein-AM).

[0300] First, for the in vitro efficacy of CAR-T pairs in NCI-H2052, a malignant pleural mesothelioma cell line, and OVCAR-3, an ovarian cancer cell line, the cytotoxic effect on target cells was evaluated using Calcein-AM through experiments in Batch #1 and Batch #2. For the evaluation results, two-way analysis of variance (Two-Way ANOVA) (full model, Tukey, 95% confidence interval) was used to determine statistical significance (ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 vs. mock). Additionally, the experimental results of the cytotoxic effect of anti-MSLN-CAR-T cells on target cells using Calcein-AM are shown in Table 18.

[0301]

Table 18

[0302]

[0303]

[0304] The results of the first Calcein-AM using MSLN CAR-T Batch #1 are shown in Figure 16 . As confirmed from Figure 11 , based on the E:T = 10:1 experimental group, compared with MSLN34 CAR-T cells, the cytotoxic effects of MSLN34-D31L CAR-T and MSLN34-D31L / S192R CAR-T on the NCI-H2052 target cell line were 24.7% and 6.0% higher, respectively, while the cytotoxic effect of MSLN34-S192R CAR-T was 10.7% lower. Based on the E:T = 10:1 experimental group, compared with MSLN34 CAR-T cells, the cytotoxic effects of MSLN34-D31L CAR-T and MSLN34-D31L / S192R CAR-T on the OVCAR-3 target cell line were 19.3% and 9.3% higher, respectively, while the cytotoxic effect of MSLN34-S192R CAR-T was 10.5% lower.

[0305] The results of the second Calcein-AM using MSLN CAR-T Batch #2 are shown in Figure 17 . As confirmed from Figure 17As confirmed, based on the E:T = 10:1 experimental group, compared with MSLN34 CAR-T cells, the cytotoxic effects of MSLN34-D31L CAR-T and MSLN34-D31L / S192R CAR-T on the NCI-H2052 target cell line were 16.7% and 7.5% higher, respectively, while the cytotoxic effect of MSLN34-S192R CAR-T was 4.4% lower. Based on the E:T = 10:1 experimental group, compared with MSLN34 CAR-T cells, the cytotoxic effects of MSLN34-D31L CAR-T and MSLN34-D31L / S192R CAR-T on the OVCAR-3 target cell line were 25.3% and 19.5% higher, respectively, while the cytotoxic effect of MSLN34-S192R CAR-T was 1.0% lower.

[0306] Therefore, it was confirmed that in mesothelioma and ovarian cancer cell lines, MSLN34-D31L and MSLN34-D31L / S192R exhibited higher cancer cell killing ability compared with MSLN34 CAR-T.

[0307]

Experimental Example 5: Confirmation of the apoptosis effect of anti-MSLN-CAR-T cells with increased and improved affinity on pancreatic cancer

[0308] Using the anti-MSLN-CAR-T cells prepared in the above Experimental Example 3, the apoptosis effect on pancreatic cancer cells was confirmed by an Incucyte based real-time cytotoxicity assay.

[0309] Regarding the in vitro cancer cell apoptosis effect of anti-MSLN-CAR-T cells with increased and improved affinity, GFP particles were analyzed using Incucyte to evaluate the cytotoxic effect on target cells (AsPC-1 / GFP, pancreatic cancer cells), and the experimental results are shown in Figure 18 . For the evaluation results, two-way ANOVA (full model, Tukey, 95% confidence interval) was used to determine statistical significance (ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 vs. mock).

[0310] As shown in Figure 18As confirmed, according to the results of the first and second experiments using MSLN CAR-T batches #1 and #2, at an E:T ratio of 0.5, compared with mock T cells, MSLN34-D31L, MSLN34-S192R, and MSLN34-D31L / S192R CAR-T cells all showed a statistically significant apoptotic effect on pancreatic cancer, where the GFP particles changed from an increasing trend to a decreasing trend within 21 to 27 h.

[0311]

Experimental Example 6: Confirming the anti-cancer efficacy of anti-MSLN-CAR-T cells with increased and improved affinity on an animal model

[0312] 【6.1 Observation of body weight changes and general symptoms】

[0313] Body weight changes and general symptoms were observed in a pancreatic cancer animal model treated with anti-MSLN-CAR-T cells.

[0314] As a result, during the test period, a total of 1 animal (G5-2) died, and the body weights of the animals in some groups decreased. In the high-concentration (1.5×10 6 cells / animal) administration group, weight loss was observed and there was individual death. Specifically, compared with the excipient control group (G1), the MSLN34(WT) high-concentration group (G3) showed weight loss starting from the 38th day after administration, while the MSLN34-D31L high-concentration group (G5) showed weight loss starting from the 24th day after administration (p < 0.05). In addition, compared with the mock administration group (G2), the MSLN34(WT) high-concentration group (G3) showed weight loss starting from the 42nd day after administration, while the MSLN34-D31L high-concentration group (G5) showed weight loss starting from the 24th day after administration (p < 0.05)( Figure 19 , Tables 19 and 20).

[0315] Table 19 below is a table showing the mortality rate of a pancreatic cancer animal model after treatment with CAR-T cells, expressed as the number of dead individuals / total number of individuals. Dead individuals appeared on the 42nd day after CAR-T cell treatment.

[0316]

Table 19

[0317] Day G1 G2 G3 G4 G5 G6 0 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 42 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 45 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 49 0 / 5 0 / 5 0 / 5 0 / 5 1 / 5 0 / 5

[0318] Figure 19Table 20 below shows the changes in body weight of pancreatic cancer animal models after treatment with CAR-T cells. Each value is expressed as the mean ± standard deviation of body weight (%). The administration day was set as day 0, and the measured body weight was expressed as a percentage (%) of the body weight divided by the body weight on day 0. The data also include data of individuals who died during the experiment. One-way ANOVA and Tukey's post hoc test were used for statistical analysis (*: 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)).

[0319] [Table 20]

[0320] Day G1 G2 G3 G4 G5 G6 0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 4 103.4±0.7 103.8±0.6 102.7±1.9 105.6±3.5 105.3±1.7 104.2±2.4 8 107.0±2.8 105.0±1.6 106.9±1.5 105.7±2.5 107.4±2.4 108.2±2.8 11 105.5±4.8 102.7±1.7 104.6±2.8 106.0±3.6 104.8±1.1 105.9±4.0 15 111.3±6.1 106.3±1.8 109.0±5.8 108.4±5.0 105.8±6.6 108.2±6.3 18 110.5±5.2 108.3±3.1 111.3±3.3 111.7±4.2 101.2±9.4 109.0±5.9 21 110.9±8.2 108.5±5.0 108.0±6.2 110.8±4.4 99.1±10.8 111.7±6.0 24 112.2±6.4 107.9±4.4 105.8±3.8 111.7±4.8 <![CDATA[93.8±12.3 ***,# > 108.4±6.4 28 110.2±8.9 109.8±3.8 102.6±5.1 112.1±5.2 <![CDATA[88.5±12.4 ***.## > 109.0±4.4 31 107.2±8.0 109.6±3.2 100.6±7.5 112.8±4.5 <![CDATA[83.9±10.7 ***,### > 108.6±4.4 35 109.6±8.9 108.8±3.1 96.2±9.1 115.8±4.3 <![CDATA[82.5±14.3 ***,## > 109.0±5.2 38 109.8±9.0 108.5±0.5 <![CDATA[92.7±10.4 * > 116.7±4.3 <![CDATA[82.2±13.6 ***,## > 107.9±6.8 42 112.1±10.9 110.6±2.3 <![CDATA[90.4±11.2 **,# > 117.8±3.5 <![CDATA[80.2±12.3 ***,### > 108.7±4.7 45 107.2±9.4 114.0±2.8 <![CDATA[87.4±11.2 **,## > 119.6±5.6 <![CDATA[78.9±11.2 ***,### > 109.1±3.8 49 106.5±13.4 112.2±2.9 <![CDATA[83.8±9.2 **,### > 118.5±5.5 <![CDATA[77.0±12.0 ***,### > 106.7±3.6

[0321] [6.2 Evaluation of tumor growth / reduction]

[0322] The level of tumor growth / reduction was evaluated in pancreatic cancer animal models treated with anti-MSLN-CAR-T cells.

[0323] As a result, compared with the vehicle control group (G1), the tumor in the mock administration group (G2) significantly decreased from day 35 after administration (p < 0.01 vs. G1). In addition, compared with the vehicle control group (G1), the tumors in all MSLN-CAR-T administration groups (G3 to G10) significantly decreased (p < 0.05 vs. G1)( Figure 24 and Table 19).

[0324] Figure 20 and Table 21 below are graphs showing the changes in tumor volume of pancreatic cancer animal models after treatment with CAR-T cells. Each value is expressed as the mean ± standard deviation of tumor volume (mm 3 ). In the case of no detectable tumor, it was marked as "0". The data also include data of individuals who died during the experiment. One-way ANOVA and Tukey's post hoc test were used for statistical analysis (*: 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)).

[0325] [Table 21]

[0326]

[0327] [6.2.1 MSLN34(WT) treatment group]

[0328] Compared with the excipient control group (G1), the tumor began to decrease in the high-concentration MSLN34(WT) group (G3) from the 11th day after administration (p < 0.05 vs. G1), while the tumor began to decrease in the low-concentration MSLN34(WT) group (G4) from the 15th day after administration (p < 0.05 vs. G1).

[0329] Compared with the mock administration group (G2), statistical significance was verified in the high-concentration MSLN34(WT) group (G3) from the 11th day to the 28th day and on the 38th day after administration (p < 0.05 vs. G2), while no statistical significance was verified in the low-concentration MSLN34(WT) group (G4).

[0330] A decreasing trend of the tumor was shown in the high-concentration MSLN34(WT) group (G3), while an increasing trend of the tumor was observed in the low-concentration MSLN34(WT) group (G4).

[0331] [[6.2.2 MSLN34-D31L treatment group]]

[0332] Compared with the excipient control group (G1), the tumor began to decrease in both the high-concentration (G5) and low-concentration (G6) MSLN34-D31L groups from the 8th day after administration (p < 0.05 vs. G1).

[0333] Compared with the mock administration group (G2), statistical significance was verified in the high-concentration MSLN34-D31L group (G5) from the 8th day to the 38th day after administration, while statistical significance was verified in the low-concentration MSLN34-D31L group (G6) from the 8th day to the 28th day after administration (p < 0.05 vs. G1).

[0334] For the MSLN34-D31L administration groups (G5 and G6), a decreasing trend of the tumor was observed regardless of the administration concentration. However, over time, an increasing trend of the tumor was observed in one individual in the low-concentration (G6) group, and a lower level of hCD3% was observed in the blood analysis of the corresponding individual, which was regarded as no T cell proliferation occurring in vivo.

[0335] [[6.3 Measuring tumor weight]]

[0336] The tumor weight was measured / evaluated in a pancreatic cancer animal model treated with anti-MSLN-CAR-T cells.

[0337] As a result, compared with the excipient control group (G1), the tumor weight decreased in all administration groups (p < 0.01 vs. G1).

[0338] Compared with the mock administration group (G2), the average tumor weight in the high-concentration group of MSLN34(WT) (G3) decreased by 93%, while the average tumor weight in the high-concentration group of MSLN34-D31L (G5) decreased by 91%. Additionally, individuals with completely removed tumors were found in the high-concentration group of MSLN34-D31L (G5). However, compared with the mock administration group (G2), the average tumor weight increased in the low-concentration group of MSLN34(WT) (G4), while the average tumor weight in the low-concentration group of MSLN34-D31L (G6) decreased by 18%. Specifically, in the low-concentration group of MSLN34-D31L (G6), tumor growth occurred in one individual, resulting in a relatively large standard deviation. A lower level of hCD3% was observed in the blood analysis of this individual, which was regarded as no T cell proliferation occurring in vivo ( Figures 21 to 23 and Table 22).

[0339] Figure 23 And Table 22 below is a table showing the tumor weights of pancreatic cancer animal models after treatment with CAR-T cells. Each value is represented as the mean ± standard deviation of the tumor weight (mg). The pancreatic cancer animal models were euthanized on the 49th day after treatment with CAR-T cells, and the tumor weights were measured. When no tumors were found, it was represented as 0 mg. One-way ANOVA and Tukey's post hoc test were used for statistical analysis (*: 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)).

[0340]

Table 22

[0341]

[0342] 【6.4 Histopathological Evaluation】

[0343] The histopathological level was observed in pancreatic cancer animal models treated with anti-MSLN-CAR-T cells. Specifically, 3 animals were selected from each group and immunohistochemistry (IHC) staining for hCD3ε was performed.

[0344] As a result, in the vehicle control group (G1), almost no T cell infiltration was observed in the tumors. In the mock administration group (G2), sporadic T cell infiltration was confirmed in the tumors. In the high-concentration group of MSLN34(WT) (G3), the highest level of T cell infiltration was confirmed in the tumors. When the same type of CAR-T cells was administered, at a relatively high concentration (1.5×10 6T cell infiltration was confirmed at (cells per animal). When using the same co-stimulatory factors, no higher T cell infiltration was observed for MSLN34-D31L with an optimized scFv region compared to the existing MSLN34 ( Figure 24 and Figure 25 ).

[0345] The description of the present invention is provided only by way of example, and those of ordinary skill in the art should be able to 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, the embodiments described above should be understood as exemplary in all aspects and not restrictive.

[0346] This study was conducted with the support of the Korean New Drug Development Project funded by the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, and the Ministry of Health and Welfare (Project Identification Number: RS-2023-00217215).

[0347] This study was conducted with the support of the Korean New Drug Development Project funded by the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, and the Ministry of Health and Welfare (Project Identification Number: RS-2023-00217215).

Claims

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

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

24.

3. The anti-mesothelin antibody or antigen-binding fragment thereof according to claim 1, wherein, The one or more amino acid substitutions comprise one or more selected from the group consisting of the following amino acid substitutions: (1) The 1st 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 4th amino acid of SEQ ID NO: 24 is substituted from Y to R.

4. The anti-mesothelin antibody or antigen-binding fragment thereof according to claim 1, wherein, The one or more amino acid substitutions are selected from the group consisting of the following amino acid substitutions: (1) The 1st amino acid of SEQ ID NO: 19 is substituted from D to L, (2) The 7th amino acid of SEQ ID NO: 23 is substituted from S to R; and (3) The 1st amino acid of SEQ ID NO: 19 is substituted from D to L, and the 7th amino acid of SEQ ID NO: 23 is substituted from S to R.

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

48.

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

50.

7. The anti-mesothelin antibody or antigen-binding fragment thereof according to claim 1, wherein, The anti-mesothelin antibody or an antigen-binding fragment thereof is selected from antibodies or antigen-binding fragments thereof comprising the following antigen-binding fragments: (1) An antibody or an antigen-binding fragment thereof comprising an antigen-binding fragment, wherein the antigen-binding fragment comprises the amino acid sequence consisting of SEQID NO: 2, (2) An antibody or antigen-binding fragment thereof comprising an antigen-binding fragment, wherein the antigen-binding fragment comprises the amino acid sequence consisting of SEQ ID NO: 3, and (3) An antibody or antigen-binding fragment thereof comprising an antigen-binding fragment, wherein the antigen-binding fragment comprises the amino acid sequence consisting of SEQ ID NO:

4.

8. The anti-mesothelin antibody or antigen-binding fragment thereof according to claim 1, wherein The one or more amino acid substitutions increase the affinity of the anti-mesothelin antibody or antigen-binding fragment thereof for mesothelin.

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

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

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

12. A method of producing an anti-mesothelin antibody, comprising expressing the antibody by culturing the host cell of claim 11.

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

14. The chimeric antigen receptor according to 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 according to 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. The isolated cell according to claim 18, wherein the isolated cell is a T cell, NK cell, NKT cell, or gamma delta (γδ) T cell.

20. A pharmaceutical composition for preventing or treating cancer, comprising the isolated cell of claim 19.

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