Anti-mesothelin antibody and use thereof

Abstract

The present invention relates to a humanized antibody or antigen-binding fragment capable of specifically binding to a mesothelin antigen and various uses thereof. The humanized anti-mesothelin antibody or antigen-binding fragment provided in the present invention exhibits high affinity and binding ability to the mesothelin antigen while demonstrating low immunogenicity when administered to humans. Therefore, it can be advantageously applied to the treatment or diagnosis of cancer or tumors and can also be used to develop various antibody-drug conjugates, bispecific antibodies, or chimeric antigen receptors (CAR).

Description

Anti-mesothelin antibodies and uses thereof

[0001] The present invention relates to an antibody or humanized antibody having excellent binding affinity to a mesothelin antigen and various uses thereof.

[0002] Mesothelin (MSLN) is expressed at relatively low levels in mesothelial cells of the pleura, peritoneum, and pericardium of healthy individuals (Hassan et al., American Journal of Clinical Pathology, Volume 124, Issue 6, December 2005, Pages 838-845), but is known to be highly expressed in various cancers, including mesothelioma, squamous cell carcinomas, pancreatic cancer, lung cancer, gastric cancer, breast cancer, endometrial cancer, and ovarian cancer. The normal biological function of mesothelin is unknown. However, in ovarian cancer, cholangiocarcinoma, lung adenocarcinoma, and triple-negative breast cancer, where mesothelin is overexpressed, the expression level of mesothelin was found to be associated with a poor prognosis of the patients.

[0003] Taking advantage of the fact that mesothelin is highly expressed in tumor cells compared to normal cells, several antibody-based therapies targeting MSLN are being developed, and the therapeutic activity of mesothelin-specific antibodies has been tested primarily in mesothelioma, pancreatic, and non-small cell lung cancer (Hassan et al., J Clin Oncol. 2016 Dec;34(34):4171-4179.). More specifically, these include direct tumor cell killing through the use of anti-MSLN antibodies with antibody-dependent cellular cytotoxicity (ADCC) activity, such as amatuximab, as well as antibody-drug conjugates (ADCs) comprising antibodies or antibody fragments conjugated to toxins, such as SS1P-PE38 and anetumab-rabtansine. Additionally, anti-MSLN binding Fv fragments have been used in chimeric antigen receptor T cell therapies, and bispecifics, such as ABBV-428, are emerging from preclinical studies through clinical trials.

[0004] Given the significant unmet clinical need for cancer treatment, continued development of drugs targeting mesothelin is essential. While several monoclonal antibodies against mesothelin have been developed in the past, most of these antibodies suffer from limitations such as immunogenicity, low affinity, and low specificity. Therefore, the development of novel anti-mesothelin antibodies with stronger binding affinity for mesothelin, superior specificity, potent ADCC and CDC activities, and superior antitumor activity remains essential.

[0005] Meanwhile, monoclonal antibodies are mainly produced using mice. However, non-human antibodies, such as mouse-derived monoclonal antibodies, are considered foreign antigens in the human body, triggering an immune response and having a short half-life, which limits their therapeutic efficacy. To overcome these problems, humanized antibodies have been developed, in which all but the antigen-binding CDR region of the antibody is replaced with a human antibody. The currently used method for replacing a mouse antibody with a humanized antibody involves selecting the most similar human antibody gene to the antibody to be replaced, and then replacing only the CDR region of the mouse antibody with the human antibody CDR position through a method called CDR grafting. Since most of the genes of this type of humanized antibody are humanized, it has the advantage of reducing the immune response in the body.

[0006] One object of the present invention is to provide an antibody or antigen-binding fragment capable of specifically binding to mesothelin.

[0007] Another object of the present invention is to provide a novel humanized antibody or binding fragment thereof, which is a humanized antibody developed based on the above-mentioned antibody, and which can bind to the mesothelin antigen with high affinity while exhibiting low immunogenicity when administered to the human body.

[0008] Another object of the present invention is to provide a nucleic acid molecule encoding the above-described antibody or antigen-binding fragment, an expression vector comprising the same, and a host cell transfected with the vector.

[0009] Another object of the present invention is to provide a use for diagnosing or treating mesothelin overexpression diseases as applicable uses thereof.

[0010] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0011] 1. Antibody or antigen-binding fragment thereof

[0012] According to one embodiment of the present invention, it relates to an antibody or an antigen-binding fragment thereof that specifically binds to mesothelin (MSLN).

[0013] As used herein, the term "mesothelin (MSLN)" refers to a human protein encoded by the MSLN gene, a 40 kDa protein expressed in mesothelial cells. Mesothelin is known to be overexpressed in various human tumors, including mesothelioma, ovarian cancer, pancreatic adenocarcinoma, lung adenocarcinoma, and cholangiocarcinoma. Mesothelin is known to contribute to tumor implantation and peritoneal spread through cell adhesion by binding to and interacting with MUC16. In the present invention, the mesothelin may be composed of an amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto.

[0014] The term "antibody" as used herein refers to a protein molecule that acts as a ligand that specifically recognizes an antigen, including an immunoglobulin molecule that immunologically has reactivity with a specific antigen, and includes polyclonal antibodies, monoclonal antibodies, whole antibodies, and antibody fragments. In addition, the antibody in the present invention includes a chimeric antibody, a human antibody, a humanized antibody, a bivalent, a bispecific molecule, a minibody, a domain antibody, a bispecific or multispecific antibody, an immune cell engaging bispecific or multispecific antibody, an antibody mimetic, a unibody, a diabody, a triabody, or a tetrabody. The term further encompasses single-chain antibodies, scabs, derivatives of antibody constant regions, and artificial antibodies based on protein scaffolds, which possess binding function to the neonatal Fc receptor (FcRn). The whole antibody has a structure comprising two full-length light chains (LC) and two full-length heavy chains (HC), each light chain being linked to a heavy chain by a disulfide bond. The whole antibody comprises IgA, IgD, IgE, IgM, and IgG, and IgG comprises subtypes such as IgG1, IgG2, IgG3, and IgG4. In one embodiment, the antibody provided in the present invention may be an IgG antibody.

[0015] The term "monoclonal antibody" as used herein refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, wherein such monoclonal antibody exhibits a single binding specificity and affinity for a particular epitope. Typically, immunoglobulins have heavy and light chains, each of which comprises a constant region and a variable region (also known as a domain). The variable regions of the light and heavy chains comprise three variable regions and four structural regions, designated complementarity-determining regions (CDRs).

[0016] As used herein, the term "complementarity determining region" or "CDR region" or "CDR" refers to a highly variable region of sequence within an antibody variable region, which forms structurally defined loops ("hypervariable loops") and / or comprises antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigenic epitopes. The complementarity determining regions are sandwiched between relatively conserved constant regions, called framework regions (FRs). Each heavy chain variable region (VH) and light chain variable region (VL) consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The CDRs of the heavy chain variable region may be referred to as HCDR1, HCDR2, HCDR3, the CDRs of the light chain variable region may be referred to as LCDR1, LCDR2, LCDR3, the FRs of the heavy chain variable region may be referred to as HFR1, HFR2, HFR3, HFR4, and the FRs of the light chain variable region may be referred to as LFR1, LFR2, LFR3, LFR4, with the numbers being sequential from the N-terminus. The exact amino acid sequence boundaries of each CDR in a given light chain variable region and heavy chain variable region amino acid sequence can be determined, for example, by Chothia (Chothia et al. (1989) Nature 342: 877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., US Department of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability, AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (imgt.cines.fr / on the World Wide Web), and North CDR definition based on affinity propagation clustering using multiple crystal structures, or a combination thereof.

[0017] The antibody of the present invention may comprise a constant region, wherein the constant region may be, but is not limited to, a constant region derived from IgG, IgA, IgD, IgE, IgM, or a combination thereof or a hybrid thereof. Here, the term "combination" means that a polypeptide encoding a single-chain immunoglobulin constant region of the same origin forms a bond with a single-chain polypeptide of a different origin when forming a dimer or multimer. For example, a dimer or multimer may be formed from two or more constant regions selected from the group consisting of constant regions of IgG, IgA, IgD, IgE, and IgM. Additionally, the term "hybrid" or "hybrid" means that within a single-chain immunoglobulin heavy chain constant region, there are sequences corresponding to immunoglobulin heavy chain constant regions of two or more different origins, for example, a hybrid of domains consisting of one to four domains selected from the group consisting of CH1, CH2, CH3 and CH4 of IgG, IgA, IgD, IgE and IgM is possible.

[0018] The term "full-length antibody" as used herein refers to a structure having two full-length light chains and two full-length heavy chains, each light chain being linked to a heavy chain by a disulfide bond, and includes IgA, IgD, IgE, IgM, and IgG. The IgG subtypes include IgG1, IgG2, IgG3, and IgG4.

[0019] As used herein, the terms "fragment," "binding fragment of polypeptide," and "antibody fragment" are used interchangeably to refer to any fragment of an antibody of the present invention that retains the antigen-binding activity of the antibody. Exemplary antibody fragments include, but are not limited to, single chain antibodies, Fd, Fab, Fab', F(ab')2, dsFv, or scFv. The Fd refers to the heavy chain portion included in a Fab fragment. As used herein, the term "Fab" typically refers to a fragment comprising a heavy chain variable domain and a light chain variable domain, and further comprising a light chain constant domain and a heavy chain first constant domain (CH1). The term "Fab" typically refers to a fragment that differs from a Fab by the addition of a few residues (including one or more cysteines from the antibody hinge region) to the carboxy terminus of the heavy chain CH1 domain. The term "F(ab')2" typically refers to a dimeric antibody fragment of Fab' comprising two Fab fragments linked by a disulfide bridge at the hinge region. The term "Fv" typically refers to the smallest antibody fragment containing a complete antigen recognition and binding site. In certain cases, the fragment may be configured as a dimer in which the heavy chain variable region and the light chain variable region are closely non-covalently associated. The term "dsFv" typically refers to an Fv fragment in which the disulfide bond is stabilized, wherein the linkage between a single light chain variable region and a single heavy chain variable region is a disulfide bond. The term "dAb fragment" typically refers to an antibody fragment consisting of a VH domain. In the present invention, the term "scFv" typically refers to a monovalent molecule formed by covalent pairing of the heavy chain variable domain and the light chain variable domain of an antibody via a flexible peptide linker. These scFv molecules can have the general structure NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.

[0020] The term "chimeric antibody" as used herein refers to an antibody in which the variable region of a mouse antibody and the constant region of a human antibody are recombined, and which has a significantly improved immune response compared to a mouse antibody.

[0021] Additionally, the term "humanized antibody" as used herein refers to an antibody in which the protein sequence of an antibody derived from a non-human species has been modified to resemble a naturally produced antibody variant in humans. For example, the humanized antibody can be produced by recombining a mouse-derived CDR with a human antibody-derived FR to produce a humanized variable region, which can then be recombined with a desired human antibody constant region.

[0022] Additionally, the term "human antibody" as used herein is intended to encompass antibodies or antibody variable domains having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody or antibody variable domain contains a constant region, the constant region is also derived from such human sequences, e.g., human germline sequences, or mutant versions of human germline sequences.

[0023] The terms "anti-mesothelin antibody," "anti-mesothelin," "mesothelin antibody," or "antibody that binds to mesothelin," as used herein, refer to an antibody that can bind to mesothelin with sufficient affinity such that the antibody can be used as a therapeutic agent targeting mesothelin. In one embodiment of the present invention, the mesothelin antibody binds to mesothelin with high affinity in vitro or in vivo. Here, the binding can be determined by, but is not limited to, radioimmunoassay (RIA), biolayer interferometry (BLI), MSD analysis, surface plasmon resonance (SPR), or flow cytometry (FACS).

[0024] The mesothelin-binding antibody or antigen-binding fragment of the present invention may comprise a heavy chain CDR1 comprising at least one amino acid sequence among SEQ ID NOs: 3 and 17 shown in Table 1 below; a heavy chain CDR2 comprising at least one amino acid sequence among SEQ ID NOs: 5 and 19; and a heavy chain CDR3 comprising at least one amino acid sequence among SEQ ID NOs: 7 and 21.

[0025] The mesothelin-binding antibody or antigen-binding fragment of the present invention may comprise a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 10 shown in Table 1 below; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12; and a light chain CDR3 comprising at least one amino acid sequence of SEQ ID NOs: 14 and 26.

[0026] The mesothelin-binding antibody or antigen-binding fragment of the present invention may comprise a heavy chain variable region comprising a heavy chain CDR1 comprising at least one amino acid sequence of SEQ ID NOs: 3 and 17; a heavy chain CDR2 comprising at least one amino acid sequence of SEQ ID NOs: 5 and 19; and a heavy chain CDR3 comprising at least one amino acid sequence of SEQ ID NOs: 7 and 21; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 10; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12; and a light chain CDR3 comprising at least one amino acid sequence of SEQ ID NOs: 14 and 26.

[0027] The mesothelin-binding antibody or antigen-binding fragment of the present invention may comprise at least one of the following antibodies or antigen-binding fragments:

[0028] 1) an antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 12, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 14; and

[0029] 2) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 17, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 19, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 21; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 12, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 26.

[0030] Clone identification sequence information Sequence number #19VHHCDR1GYSFTDYN3HCDR2INPNYGTT5HCDR3AKGDYDGAGFAY7VLLCDR1SSVSY10LCDR2DTS12LCDR3QQWSSNPLT14 #45VHHCDR1GYTFTSYW17HCDR2IHPNSGGT19HCDR3ARYYYGNYVWYFDV21VLLCDR1SSVSY10LCDR2DTS12LCDR3QQWSSYPLT26

[0031] The mesothelin-binding antibody or antigen-binding fragment thereof of the present invention may comprise a heavy chain variable region comprising a heavy chain FR1 comprising at least one amino acid sequence among SEQ ID NOs: 2 and 16 shown in Table 2 below; a heavy chain FR2 comprising at least one amino acid sequence among SEQ ID NOs: 4 and 18; a heavy chain FR3 comprising at least one amino acid sequence among SEQ ID NOs: 6 and 20; and a heavy chain FR4 comprising at least one amino acid sequence among SEQ ID NOs: 8 and 22.

[0032] The mesothelin-binding antibody or antigen-binding fragment thereof of the present invention may comprise a light chain variable region comprising a light chain FR1 comprising at least one amino acid sequence among SEQ ID NOs: 9 and 23 shown in Table 2 below; a light chain FR2 comprising at least one amino acid sequence among SEQ ID NOs: 11 and 24; a light chain FR3 comprising at least one amino acid sequence among SEQ ID NOs: 13 and 25; and a light chain FR4 comprising at least one amino acid sequence among SEQ ID NOs: 15 and 27.

[0033] The mesothelin-binding antibody or antigen-binding fragment thereof of the present invention may comprise a heavy chain variable region comprising a heavy chain FR1 comprising at least one amino acid sequence of SEQ ID NOs: 2 and 16; a heavy chain FR2 comprising at least one amino acid sequence of SEQ ID NOs: 4 and 18; a heavy chain FR3 comprising at least one amino acid sequence of SEQ ID NOs: 6 and 20; and a heavy chain FR4 comprising at least one amino acid sequence of SEQ ID NOs: 8 and 22; and a light chain variable region comprising a light chain FR1 comprising at least one amino acid sequence of SEQ ID NOs: 9 and 23; a light chain FR2 comprising at least one amino acid sequence of SEQ ID NOs: 11 and 24; a light chain FR3 comprising at least one amino acid sequence of SEQ ID NOs: 13 and 25; and a light chain FR4 comprising at least one amino acid sequence of SEQ ID NOs: 15 and 27.

[0034] Clone identification sequence information sequence Number#19VHHFR1QVQLQQSGPELVKPGASVKISCKAS2HFR2MNWVKQSNGKSLEWIGV4HFR3SYNQKFKGKATITADTSSNTAYLQLSSLTSEDSAVYYC6HFR4WGQGTLVTVSA 8VLLFR1DIVITQSPAIMSASPGEKVTMTCSAS9LFR2MHWYQQKSGTSPKRWIY11LFR3KLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYC13LFR4FGAGTKLEIKR15# 45VHHFR1QVQLQQPGAELVKPGASVKLSCKAS16HFR2MHWVKQRPGQGLEWIGM18HFR3NYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYC20HFR4WGTGTTVTVSA 22VLLFR1DIVLTQSPAIMSTSPGEKVTMTCTAS23LFR2MHWYQQKSGTSPKRWIY24LFR3KLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYC25LFR4FGAGTKLEMKR27

[0035] The mesothelin-binding antibody or antigen-binding fragment thereof of the present invention may comprise a heavy chain variable region comprising a heavy chain FR1 comprising at least one amino acid sequence of SEQ ID NOs: 2 and 16, a heavy chain CDR1 comprising at least one amino acid sequence of SEQ ID NOs: 3 and 17, a heavy chain FR2 comprising at least one amino acid sequence of SEQ ID NOs: 4 and 18, a heavy chain CDR2 comprising at least one amino acid sequence of SEQ ID NOs: 5 and 19, a heavy chain FR3 comprising at least one amino acid sequence of SEQ ID NOs: 6 and 20, a heavy chain CDR3 comprising at least one amino acid sequence of SEQ ID NOs: 7 and 21, and a heavy chain FR4 comprising at least one amino acid sequence of SEQ ID NOs: 8 and 22.

[0036] The mesothelin-binding antibody or antigen-binding fragment thereof of the present invention may comprise a light chain variable region comprising a light chain FR1 comprising at least one amino acid sequence among SEQ ID NOs: 9 and 23, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, a light chain FR2 comprising at least one amino acid sequence among SEQ ID NOs: 11 and 24, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 12, a light chain FR3 comprising at least one amino acid sequence among SEQ ID NOs: 13 and 25, a light chain CDR3 comprising at least one amino acid sequence among SEQ ID NOs: 14 and 26, and a light chain FR4 comprising at least one amino acid sequence among SEQ ID NOs: 15 and 27.

[0037] The mesothelin-binding antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region comprising a heavy chain FR1 comprising at least one amino acid sequence among SEQ ID NOs: 2 and 16, a heavy chain CDR1 comprising at least one amino acid sequence among SEQ ID NOs: 3 and 17, a heavy chain FR2 comprising at least one amino acid sequence among SEQ ID NOs: 4 and 18, a heavy chain CDR2 comprising at least one amino acid sequence among SEQ ID NOs: 5 and 19, a heavy chain FR3 comprising at least one amino acid sequence among SEQ ID NOs: 6 and 20, a heavy chain CDR3 comprising at least one amino acid sequence among SEQ ID NOs: 7 and 21, and a heavy chain FR4 comprising at least one amino acid sequence among SEQ ID NOs: 8 and 22; And a light chain variable region comprising a light chain FR1 comprising at least one amino acid sequence among SEQ ID NOs: 9 and 23, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, a light chain FR2 comprising at least one amino acid sequence among SEQ ID NOs: 11 and 24, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 12, a light chain FR3 comprising at least one amino acid sequence among SEQ ID NOs: 13 and 25, a light chain CDR3 comprising at least one amino acid sequence among SEQ ID NOs: 14 and 26, and a light chain FR4 comprising at least one amino acid sequence among SEQ ID NOs: 15 and 27.

[0038] The mesothelin-binding antibody or antigen-binding fragment of the present invention may comprise at least one of the following antibodies or antigen-binding fragments:

[0039] 3) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 3, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 6, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 7, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 8; and an antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 9, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 11, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 12, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 13, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 14, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 15; and

[0040] 4) A heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 16, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 17, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 18, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 19, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 20, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 21, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 22; An antibody or antigen-binding fragment comprising a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 12, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 25, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 26, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 27.

[0041] The mesothelin-binding antibody or antigen-binding fragment of the present invention may comprise a heavy chain variable region comprising at least one amino acid sequence among SEQ ID NOs: 38 and 42 as shown in Table 3 below; and a light chain variable region comprising at least one amino acid sequence among SEQ ID NOs: 39 and 43.

[0042] The mesothelin-binding antibody or antigen-binding fragment of the present invention may comprise at least one of the following antibodies or antigen-binding fragments:

[0043] 5) an antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 38 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 39; and

[0044] 6) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 42 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 43.

[0045] Clone identification sequence information sequence Number#19VHQVQLQQSGPELVKPGASVKISCKASGYSFTDYNMNWVKQSNGKSLEWIGVINPNYGTTSYNQKFKGKATITADTSSNTAYLQLSSLTSEDSAVYYCAKGDYDGAGFAYWGQG TLVTVSA38VLDIVITQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLEIKR39 #45VHQVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGMIHPNSGGTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARYYYGNYVWYFDVWGTG TTVTVSA42VLDIVLTQSPAIMSTSPGEKVTMTCTASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLTFGAGTKLEMKR43

[0046] In one embodiment, the antibody provided in the present invention may be a monoclonal antibody.

[0047] In one embodiment, the antigen-binding fragment provided in the present invention may be a scFv.

[0048] The antibody or antigen-binding fragment of the present invention may further comprise a linker between the heavy chain variable region and the light chain variable region. The linker may be a peptide linker and have a length of about 10-25 amino acids. For example, the linker may include, but is not limited to, hydrophilic amino acids such as glycine and / or serine.

[0049] Specifically, in the present invention, the linker may include, for example, (GS)n, (GGS)n, (GSGGS)n or (GnS)m (n and m are each integers from 1 to 10), preferably GGGGS, and more preferably GGGGSGGGGSGGGGS represented by SEQ ID NO: 50, but is not limited thereto.

[0050] The mesothelin binding antigen binding fragment of the present invention may be a scFv and may comprise at least one amino acid sequence of SEQ ID NOs: 51 and 53.

[0051] The antibody or antigen-binding fragment of the present invention may include not only the sequence of the anti-mesothelin antibody or antigen-binding fragment of the present invention described herein, but also biological equivalents thereof, as long as it can specifically recognize mesothelin. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody or antigen-binding fragment. Such amino acid mutations are made based on the relative similarity of amino acid side-chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side-chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine can be considered biologically functional equivalents.

[0052] Considering the mutations having the above-described biological equivalent activity, the antibody or antigen-binding fragment of the present invention or the nucleic acid molecule encoding them is interpreted to also include a sequence showing substantial identity with the sequence described in the sequence number. The substantial identity means a sequence showing at least 90% homology, most preferably at least 95% homology, 96% or more, 97% or more, 98% or more, or 99% or more homology when the sequence of the present invention is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are known in the art. NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI, etc., and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the Internet. BLAST is available at www.ncbi.nlm.nih.gov / BLAST / . Instructions for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov / BLAST / blast_ help.html.

[0053] Based on this, the antibody or antigen-binding fragment of the present invention may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology to the specified sequences or the entirety of the sequences described in the specification. Such homology may be determined by sequence comparison and / or alignment using methods known in the art. For example, the percent sequence homology of the nucleic acids or proteins of the present invention may be determined using sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection.

[0054] The binding affinity of the anti-mesothelin antibody or antigen-binding fragment provided in the present invention to mesothelin is 10 -5 M to 10 -12 M range. For example, the binding affinity of an anti-mesothelin antibody or antigen-binding fragment to mesothelin is 10 -5 M to 10 -12 M, 10 -6 M to 10 -12 M, 10 -7 M to 10 -12 M, 10 -8 M to 10 -12 M, 10 -9 M to 10 -12 M, 10 -5 M to 10 -11 M, 10 -6 M to 10 -11 M, 10 -7 M to 10 -11 M, 10 -8 M to 10 -11 M, 10 -9 M to 10 -11 M, 10 -10 M to 10 -11 M, 10 -5 M to 10 -10 M, 10 -6 M to 10 -10 M, 10 -7 M to 10 -10 M, 10 -8 M to 10 -10 M, 10 -9 M to 10 -10 M, 10 -5 M to 10 -9 M, 10 -6 M to 10 -9 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -9 M, 10 -5 M to 10 -8 M, 10 -6 M to 10 -8 M, 10 -7M to 10 -8 M, 10 -5 M to 10 -7 M, 10 -6 M to 10 -7 M or 10 -5 M to 10 -6 M may be, but is not limited to, this.

[0055]

[0056] 2. Humanized antibody or antigen-binding fragment thereof

[0057] The present invention provides a mesothelin-binding humanized antibody or an antigen-binding fragment thereof.

[0058] The humanized antibody provided in the present invention can specifically bind to mesothelin and can have low immunogenicity even when administered to the human body.

[0059] The humanized antibody or antigen-binding fragment thereof of the present invention is produced based on a mouse antibody comprising a heavy chain variable region and a light chain variable region having the amino acid sequences shown in Tables 1 to 3 above.

[0060] The sequence of the heavy chain variable region of the humanized antibody or antigen-binding fragment thereof of the present invention may have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence represented by SEQ ID NO: 38.

[0061] Additionally, the sequence of the light chain variable region of the humanized antibody or antigen-binding fragment thereof of the present invention may have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence represented by SEQ ID NO: 39.

[0062] The mesothelin-binding humanized antibody of the present invention or an antigen-binding fragment thereof may comprise a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 29 as shown in Table 4 below; a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5; and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 32.

[0063] In addition, the mesothelin-binding humanized antibody of the present invention or an antigen-binding fragment thereof may include a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10 as shown in Table 4 below; a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 12; and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 14.

[0064] The mesothelin-binding humanized antibody of the present invention or an antigen-binding fragment thereof may include a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 29, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 32; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10; a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 12; and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 14.

[0065] Sequence information Sequence number VHHCDR1GYTFTDYN29HCDR2INPNYGTT5HCDR3ARGDYDGAGFAY32VLLCDR1SSVSY10LCDR2DTS12LCDR3QQWSSNPLT14

[0066] The mesothelin-binding humanized antibody of the present invention or an antigen-binding fragment thereof may include a heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 28 as shown in Table 5 below; a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 30; a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 31; and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 33.

[0067] In addition, the mesothelin-binding humanized antibody of the present invention or an antigen-binding fragment thereof may include a light chain variable region including a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 34 as shown in Table 5 below; a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 35; a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 36; and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 37.

[0068] The mesothelin-binding humanized antibody of the present invention or an antigen-binding fragment thereof may include a heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 28, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 30, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 31, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 33, and a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 34, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 36, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 37.

[0069] Classification sequence Information sequence NumberVHHFR1QVQLVQSGAEVKKPGASVKVSCKAS28HFR2MNWVRQAPGQGLEWMGV30HFR3SYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYC31HFR4WGQGTLVTVSS 33VLLFR1EIVLTQSPATLSLSPGERATLSCSAS34LFR2MHWYQQKPGQAPRLLIY35LFR3KLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYC36LFR4FGQGTKLEIKR37

[0070] The mesothelin-binding humanized antibody of the present invention or an antigen-binding fragment thereof may include a heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 28, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 29, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 30, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 32, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 33.

[0071] The mesothelin-binding humanized antibody of the present invention or an antigen-binding fragment thereof may include a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 34, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 12, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 36, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 14, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 37.

[0072] The mesothelin-binding humanized antibody of the present invention or an antigen-binding fragment thereof comprises a heavy chain variable region comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 28, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 29, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 30, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 32, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 33; and a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 34, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 35, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 12, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 36, and a light chain comprising an amino acid sequence represented by SEQ ID NO: 14. It may comprise a light chain variable region comprising a light chain FR4 comprising an amino acid sequence represented by CDR3 and SEQ ID NO: 37.

[0073] In addition, the mesothelin-binding humanized antibody of the present invention or an antigen-binding fragment thereof may include a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 46 as shown in Table 6 below; and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 47.

[0074] Classification sequence Information sequence NumberVHQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMNWVRQAPGQGLEWMGVINPNYGTTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDYDGAGFAYWGQGTL VTVSS46VLEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSSNPLTFGQGTKLEIKR47

[0075] In one embodiment, the humanized antibody provided in the present invention may be a monoclonal antibody.

[0076] In one embodiment, the antigen-binding fragment provided in the present invention may be a scFv.

[0077] The humanized antibody of the present invention or an antigen-binding fragment thereof may further comprise a linker between the heavy chain variable region and the light chain variable region. The linker may be a peptide linker, and may have a length of about 10-25 aa, and may include, for example, a hydrophilic amino acid such as glycine and / or serine, but is not limited thereto. Specifically, in the present invention, the linker may include, for example, (GS)n, (GGS)n, (GSGGS)n or (GnS)m (wherein n and m are each an integer from 1 to 10), preferably GGGGS, and more preferably GGGGSGGGGSGGGGS represented by SEQ ID NO: 50, but is not limited thereto.

[0078] The mesothelin-binding humanized antigen-binding fragment of the present invention may be a scFv, and the sequence of such scFv may have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence represented by SEQ ID NO: 51.

[0079] Additionally, the mesothelin binding antigen binding fragment of the present invention may be an scFv comprising the amino acid sequence of SEQ ID NO: 55.

[0080] Classification sequence Information sequence NumberscFvEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSSNPLTFGQGTKLEIKRGGGGSGGGGSG GGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMNWVRQAPGQGLEWMGVINPNYGTTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDYDGAGFAYWGQGTLVTVSS55

[0081] The humanized antibody or antigen-binding fragment thereof of the present invention may include not only the sequence of the anti-mesothelin humanized antibody or antigen-binding fragment of the present invention described herein, but also biological equivalents thereof, as long as it can specifically recognize mesothelin. To avoid overlapping with the previously described sequence and thus overlapping the description of biological equivalents, their description is omitted below.

[0082] The binding affinity of the mesothelin-binding humanized antibody or antigen-binding fragment provided in the present invention to mesothelin is 10 -5 M to 10 -12 M range. For example, the binding affinity of a mesothelin-binding humanized antibody or antigen-binding fragment to mesothelin is 10 -5 M to 10 -12 M, 10 -6 M to 10 -12 M, 10 -7 M to 10 -12 M, 10 -8 M to 10 -12 M, 10 -9 M to 10 -12 M, 10 -5 M to 10 -11 M, 10 -6M to 10 -11 M, 10 -7 M to 10 -11 M, 10 -8 M to 10 -11 M, 10 -9 M to 10 -11 M, 10 -10 M to 10 -11 M, 10 -5 M to 10 -10 M, 10 -6 M to 10 -10 M, 10 -7 M to 10 -10 M, 10 -8 M to 10 -10 M, 10 -9 M to 10 -10 M, 10 -5 M to 10 -9 M, 10 -6 M to 10 -9 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -9 M, 10 -5 M to 10 -8 M, 10 -6 M to 10 -8 M, 10 -7 M to 10 -8 M, 10 -5 M to 10 -7 M, 10 -6 M to 10 -7 M or 10 -5 M to 10 -6 M may be, but is not limited to, this.

[0083] The mesothelin-binding humanized antibody or antigen-binding fragment provided in the present invention may have a binding affinity for the mesothelin antigen that is equal to or greater than that of a mouse antibody defined in Table 3 above, or an antigen-binding fragment (e.g., scFv) comprising the amino acid sequence of SEQ ID NO: 51.

[0084]

[0085] According to another embodiment of the present invention, the present invention relates to a nucleic acid molecule encoding an antibody (including a humanized antibody) or an antigen-binding fragment thereof.

[0086] As used herein, the term "nucleic acid" encompasses DNA (gDNA and cDNA) and RNA molecules. Nucleotides, the basic structural units of nucleic acids, include not only natural nucleotides but also analogues with modified sugar or base moieties. The sequences of nucleic acids encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

[0087] In the present invention, the DNA encoding the antibody or antigen-binding fragment can be easily isolated or synthesized using conventional molecular biological techniques (for example, by using an oligonucleotide probe that can specifically bind to DNA encoding the antibody and the heavy and light chains), and the nucleic acid is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or further expression.

[0088] As an example, a nucleic acid molecule of the present invention may comprise a base sequence represented by SEQ ID NO: 40 encoding a heavy chain variable region represented by SEQ ID NO: 38.

[0089] As an example, a nucleic acid molecule of the present invention may comprise a base sequence represented by SEQ ID NO: 44 encoding a heavy chain variable region represented by SEQ ID NO: 42.

[0090] As an example, a nucleic acid molecule of the present invention may comprise a base sequence represented by SEQ ID NO: 48 encoding a heavy chain variable region represented by SEQ ID NO: 46.

[0091] As an example, a nucleic acid molecule of the present invention may comprise a base sequence represented by SEQ ID NO: 41 encoding a light chain variable region represented by SEQ ID NO: 39.

[0092] As an example, a nucleic acid molecule of the present invention may comprise a base sequence represented by SEQ ID NO: 45 encoding a light chain variable region represented by SEQ ID NO: 43.

[0093] As an example, a nucleic acid molecule of the present invention may comprise a base sequence represented by SEQ ID NO: 49 encoding a heavy chain variable region represented by SEQ ID NO: 47.

[0094] As an example, the nucleic acid molecule of the present invention may comprise a base sequence represented by SEQ ID NO: 40 encoding a heavy chain variable region and a base sequence represented by SEQ ID NO: 41 encoding a light chain variable region.

[0095] As an example, the nucleic acid molecule of the present invention may comprise a base sequence represented by SEQ ID NO: 44 encoding a heavy chain variable region and a base sequence represented by SEQ ID NO: 45 encoding a light chain variable region.

[0096] As an example, the nucleic acid molecule of the present invention may comprise a base sequence represented by SEQ ID NO: 48 encoding a heavy chain variable region and a base sequence represented by SEQ ID NO: 49 encoding a light chain variable region.

[0097] As an example, the nucleic acid molecule of the present invention may comprise a base sequence represented by SEQ ID NO: 52 encoding an scFv represented by SEQ ID NO: 51.

[0098] As an example, the nucleic acid molecule of the present invention may comprise a base sequence represented by SEQ ID NO: 54 encoding an scFv represented by SEQ ID NO: 53.

[0099] As an example, the nucleic acid molecule of the present invention may comprise a base sequence represented by SEQ ID NO: 56 encoding an scFv represented by SEQ ID NO: 55.

[0100]

[0101] According to another embodiment, the present invention relates to a recombinant expression vector comprising a nucleic acid molecule provided in the present invention.

[0102] In the present invention, the above "vector" refers to a recombinant vector that can express a target protein when transfected into a suitable host cell, and a genetic construct that includes essential regulatory elements operably linked to allow the gene insert to be expressed. Here, the term "operably linked" means that a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein are functionally linked to perform a general function. The operably linked vector can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be easily performed using enzymes generally known in the art.

[0103] In the present invention, various types of vectors, such as nanoparticles, plasmids, viruses, and cosmids, can be used as recombinant expression vectors for inserting the foreign gene. The type of recombinant vector is not particularly limited as long as it has the function of expressing the desired gene and producing the desired protein in various host cells of prokaryotic and eukaryotic cells. Specifically, a vector that has a promoter that exhibits strong activity and a strong expression ability while being capable of mass-producing a foreign protein in a form similar to that in the natural state can be used.

[0104] A variety of gene delivery vehicles are known in the art, including both viral and non-viral (e.g., naked DNA, plasmid) vectors. Viral vectors suitable for gene delivery are known to those skilled in the art. Non-limiting examples of the viral vectors may include retroviral vectors (e.g., derived from Moloney murine leukemia virus vector (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenovirus (Ad) vectors including replication competent, replication deficient and emasculated forms thereof, adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus vectors, herpes virus vectors, varicella virus vectors, Harvey murine sarcoma virus vectors, murine mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, poliovirus vectors, vesicular stomatitis virus vectors, Maraba virus vectors and group B adenovirus enadenotuxireb vectors.

[0105] Non-viral vectors for gene transfer include naked DNA, plasmids, transposons, and mRNA. Non-limiting examples include pKK plasmid (Clonetech), pUC plasmid, pET plasmid (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego, Calif.), and pMAL plasmid (New England Biolabs, Beverly, Mass.).

[0106] The vector of the present invention can be introduced into many suitable host cells using methods disclosed or cited herein or otherwise known to those skilled in the art.

[0107] Suitable expression vectors of the present invention may include, in addition to expression regulatory elements such as a promoter, initiation codon, termination codon, polyadenylation signal, or enhancer, a nucleotide sequence encoding a signal peptide for membrane targeting or secretion. The initiation and termination codons are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein, and must be functional in a subject when the genetic construct is administered, and must be in frame with the coding sequence.

[0108] The term "promoter," as used herein, refers to any sequence that regulates the expression of a coding sequence, such as a gene. A promoter may be, for example, constitutive, inducible, repressible, or tissue-specific. A promoter is a control sequence, a region of a polynucleotide sequence where the initiation and rate of transcription are controlled. Non-limiting examples of the promoter in the present invention include Rous sarcoma virus (RSV) LTR promoter (optionally with RSV enhancer), cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, U6 promoter, EF1alpha short form (EFS) promoter, human polypeptide chain elongation factor (EF1a) promoter, P5 promoter, Ubc promoter, CAG promoter, TRE promoter, UAS promoter, Ac5 promoter, polyhedrin promoter, CaMKIIa promoter, Gal1 promoter, TEF1 promoter, GDS promoter, ADH1 promoter, CaMV35S promoter, ubiquitin (Ubi) Promoters such as ubiquitin C (UbiC), H1 promoter, U6 promoter, alpha-1-antitrypsin promoter, spleen focus-forming virus (SFFV) promoter, etc. may be included.

[0109] Additionally, in the present invention, the promoter may be coupled to an enhancer to increase transcription efficiency. Non-limiting examples of the enhancer include, but are not limited to, an RSV enhancer, a CMV enhancer, or an α-fetoprotein MERII enhancer. The vector of the present invention may also be fused with other sequences to facilitate the purification of antibodies expressed therefrom. Examples of the fused sequences include, but are not limited to, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA).

[0110] The vector of the present invention includes an antibiotic resistance gene commonly used in the art as a selection marker, and may include, but is not limited to, resistance genes for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0111]

[0112] According to another embodiment of the present invention, the expression vector provided in the present invention relates to a transfected host cell.

[0113] The term "host cell" as used herein includes an individual cell or cell culture that can be or has been the recipient of a vector(s) for incorporation of a polypeptide insert. Host cells include progeny of a single host cell, which progeny may not necessarily be completely identical (morphologically or in genomic DNA complement) to the original parent cell due to natural, accidental, or intentional mutation. Host cells include cells that have been transfected in vivo with the polypeptide(s) of the present disclosure.

[0114] In the present invention, the host cell may include a mammalian, plant, insect, fungal or cellular cell, and for example, bacterial cells such as Escherichia coli, Streptomyces, and Salmonella Typhimurium; fungal cells such as yeast cells and Pichia pastoris; insect cells such as Drozophylla and Spodoptera Sf9 cells; animal cells such as CHO (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bow melanoma cells, HT-1080, BHK (Baby Hamster Kidney cells), HEK (Human Embryonic Kidney cells) or PERC.6 (human retinal cells); Alternatively, the host cell line may be a plant cell, but is not limited thereto, and any cell known to those skilled in the art that can be used as a host cell line may be used.

[0115] In the present invention, the transfection method is any method for injecting the desired vector into the host cell, and any known method capable of injecting a vector into a host cell can be included, and for example, a method using CaCl2, electroporation, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, gene bombardment, and virus-based transfection can be used, but is not limited thereto.

[0116]

[0117] According to another embodiment, the present invention relates to a method for producing an antibody or antigen-binding fragment that specifically binds to mesothelin, comprising the steps of culturing the host cell to produce an antibody (including a humanized antibody) or antigen-binding fragment according to the present invention; and isolating and purifying the produced antibody.

[0118] In the present invention, the host cells can be cultured in various media. Any commercially available media can be used as culture media. Any other essential supplements known to those skilled in the art may be included at appropriate concentrations. Culture conditions, such as temperature, pH, etc., are already used with host cells selected for expression and will be readily apparent to those skilled in the art.

[0119] In the present invention, the antibody or antigen-binding fragment thereof can be recovered by removing impurities, for example, by centrifugation or ultrafiltration, and purifying the resultant product using, for example, affinity chromatography. Additional purification techniques, for example, anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, etc., can be used.

[0120]

[0121] According to another embodiment of the present invention, the present invention relates to a bispecific or multispecific antibody comprising an antibody (including a humanized antibody) or antigen-binding fragment thereof.

[0122] The term "bispecific antibody" as used herein means an antibody having binding ability or antagonism to more than one target, and means an antibody in which antibodies having binding ability or antagonism to two different targets are combined, or an antibody in which an antibody having binding ability to one target and a substance having antagonism to another target are combined.

[0123] The term "multispecific antibody" as used herein refers to an antibody having binding specificities for at least three different antigens. Multispecific antibodies may include antibodies that target more than three specific targets, such as trispecific antibodies, tetraspecific antibodies, or antibodies that target more than one target.

[0124] In the present invention, antibodies belonging to bispecific or multispecific antibodies can be classified into scFv-based antibodies, Fab-based antibodies, and IgG-based antibodies. In the case of bispecific or multispecific antibodies, since two or more signals can be simultaneously suppressed or amplified, they can be more effective than cases where a single signal is suppressed / amplified. In addition, compared to cases where each signal is treated with a separate signal inhibitor, lower dosage administration is possible, and two or more signals can be suppressed / amplified in the same time and space.

[0125] Methods for producing bispecific or multispecific antibodies are widely known. Traditionally, recombinant production of bispecific antibodies relies on the co-expression of two or more immunoglobulin heavy chain / light chain pairs, where the two or more heavy chains have different specificities.

[0126] In the present invention, in the case of a bispecific or multispecific antibody based on scFv, the VL and VH of different scFvs can be combined with each other to produce a hybrid scFv in a heterodimeric form to produce a diabody, different scFvs can be linked to each other to produce a tendem scFv, CH1 and CL of Fab can be expressed at the ends of each scFv to produce a heterodimeric miniantibody, and some amino acids of the CH3 domain, which is a homodimeric domain of Fc, can be substituted to change it into a heterodimeric structure in the form of a 'knob into hole', and these changed CH3 domains can be expressed at the ends of different scFvs to produce a minibody in the form of a heterodimeric scFv.

[0127] In the present invention, in the case of a bispecific or multispecific antibody based on Fab, individual Fabs for specific antigens can be combined with each other using a disulfide bond or a mediator to produce a heterodimeric Fab form, and by expressing scFvs for different antigens at the terminals of the heavy or light chains of a specific Fab, it can be produced to have two antigen-binding valencies, or by providing a hinge region between the Fab and scFv, it can be produced to have four antigen-binding valencies in a homodimeric form. In addition, a dual-target bibody in which the antigen-binding valencies are made three by fusing scFvs for different antigens to the light and heavy chain terminals of the Fab, and a tri-target bibody in which the antigen-binding valencies are made three by fusing different scFvs to the light and heavy chain terminals of the Fab, respectively, can be obtained by chemically conjugating three different Fabs.

[0128] In the case of IgG-based bispecific or multispecific antibodies in the present invention, a method for producing bispecific antibodies by producing hybrid hybridomas, also known as quadromas, by crossbreeding mouse and rat hybridomas again has been known by Trion Pharma. In addition, bispecific antibodies can be produced in the so-called 'Holes and Knob' form, which is produced in a heterodimeric form by modifying some amino acids in the CH3 homodimeric domain of Fc for different heavy chains while sharing the light chain portion. In addition to the heterodimeric form of bispecific antibodies, two different scFvs can be fused and expressed in the constant domains of the light and heavy chains of IgG instead of the variable domains, thereby producing (scFv)4-IgG in a homodimeric form. In addition, ImClone reported the production of a bispecific antibody by fusing only a single variable domain for mouse platelet-derived growth factor receptor-α to the light chain amino terminus of IMC-1C11, a chimeric monoclonal antibody against human VEGFR-2. In addition, antibodies with multiple antigen-binding affinities for CD20 can be produced through the so-called 'dock and lock (DNL)' method using the dimerization and docking domain (DDD) of the R subunit of protein kinase A (PKA) and the anchoring domain of PKA.

[0129] The present invention also encompasses a wide variety of recombinant antibody formats, such as bivalent, trivalent, or tetravalent bispecific or multispecific antibodies. Bivalent, trivalent, or tetravalent antibodies indicate the presence of two or more binding domains, three or more binding domains, or four or more binding domains in the antibody molecule, respectively.

[0130] The bispecific or multispecific antibody according to the present invention may comprise a mesothelin-binding antibody or antigen-binding fragment according to the present invention, specifically in the form of an IgG complete antibody or a fragment thereof, for example, a single-chain Fv, VH domain and / or VL domain, Fab or (Fab)2.

[0131] In addition, the antibody targeting the mesothelin may include an antibody that binds to a target other than the above-mentioned target, for example, an antibody targeting one or more selected from the group consisting of PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, MARCO, specifically an IgG complete antibody or a fragment form thereof, for example, a single-chain Fv, a VH domain and / or a VL domain, a Fab or (Fab)2.

[0132] The bispecific or multispecific antibodies according to the present invention may provide additional binding specificities induced or mediated by other targets in addition to mesothelin.

[0133] For example, the bispecific antibody according to the present invention binds to mesothelin and PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD3, CD20, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, MARCO, FcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, NKG2D, CD3, CD16a, CD19, CD33, CD22, It can simultaneously target one or more selected from the group consisting of CD79A, CD79B, EDAR, GFRA1, MRP4, RET, Steap1, and TenB2.

[0134] For example, the multispecific antibody according to the present invention binds to mesothelin and PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD3, CD20, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, MARCO, FcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, NKG2D, CD3, CD16a, CD19, CD33, CD22, It can simultaneously target two or more selected from the group consisting of CD79A, CD79B, EDAR, GFRA1, MRP4, RET, Steap1, and TenB2.

[0135]

[0136] According to another embodiment of the present invention, there is provided an immune cell engaging bispecific or multispecific antibody comprising an antigen binding fragment (including an antigen binding fragment of a humanized antibody) provided by the present invention and at least one scFv that binds to an immune cell activating antigen.

[0137] In the present invention, a cytolytic synapse between cytotoxic T cells or NK cells and cancer target cells is temporarily induced through the immune cell engaging bispecific or multispecific antibody, thereby causing the release of toxic substances.

[0138] In the present invention, the T cell activation antigen may be, but is not limited to, CD3, TCRα, TCRβ, TCRγ, TCRξ, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2 or CD226.

[0139] In the present invention, the NK cell activating antigen may be, but is not limited to, NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (eg, CD16a, CD16b), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E or CD160.

[0140] Other immune cell engagers are specifically described in U.S. Patent Publication No. 2017 / 0368169, which may be incorporated herein by reference.

[0141] Specifically, the immune cell engaging bispecific or multispecific antibody comprises a tandem scFv and can bind to the following antigen and a surface antigen on a cancer cell. The surface antigen on the cancer cell is mesothelin, which is targeted by the antibody according to the present invention.

[0142] In the present invention, the immune cell engaging bispecific or multispecific antibody may include a structure in the form of, for example, VL(mesothelin)-VH(mesothelin)-VH(CD3 or CD16A)-VL(CD3 or CD16A), VH(mesothelin)-VL(mesothelin)-VH(CD3 or CD16A)-VL(CD3 or CD16A), VH(CD3 or CD16A)-VL(CD3 or CD16A)-VH(mesothelin)-VL(mesothelin), or VH(CD3 or CD16A)-VL(CD3 or CD16A)-VL(mesothelin)-VH(mesothelin).

[0143] The present invention may further include a linker between the heavy chain variable region and the light chain variable region. The linker may be a peptide linker and have a length of about 10-25 amino acids. For example, the linker may include, but is not limited to, hydrophilic amino acids such as glycine and / or serine.

[0144] Specifically, in the present invention, the linker may include, for example, (GS)n, (GGS)n, (GSGGS)n or (GnS)m (n and m are each 1 to 10), preferably GGGGS, and more preferably GGGGSGGGGSGGGGS represented by SEQ ID NO: 50, but is not limited thereto.

[0145] Examples of the immune cell-engaging bispecific or multispecific antibodies in the present invention include blinatumomab (Amgen), which binds to CD3 and CD19; solitomab (Amgen), which binds to CD3 and EpCAM; MEDI 565 (MedImmune, Amgen), which binds to CD3 and CEA; and BAY2010112 (Bayer, Amgen), which binds to CD3 and PSMA. Exemplary DARTs include MGD006 (Macrogenics), which binds to CD3 and CD123; and MGD007 (Macrogenics), which binds to CD3 and gpA33. Exemplary TandAbs may include AFM11 (Affimed Therapeutics), which binds to CD3 and CD19; and AFM13 (Affimed Therapeutics), which binds to CD30 and CD16A.

[0146]

[0147] immune conjugates

[0148] According to another embodiment of the present invention, the present invention relates to an immunoconjugate in which an antibody (including a humanized antibody) or antigen-binding fragment provided by the present invention is combined with a cytotoxic agent.

[0149] In the present invention, the cytotoxic agent may be at least one selected from the group consisting of a chemotherapeutic agent or a chemotherapeutic drug, a growth inhibitor, a toxin (e.g., a protein toxin, an enzymatically active toxin or fragment thereof derived from bacteria, fungi, plants or animals), and a radioactive isotope.

[0150] As an example of implementation, the immune conjugate provided by the present invention may be an antibody-drug conjugate (ADC) in which the antibody or antigen-binding fragment is bound to a drug.

[0151] The term "antibody-drug conjugate (ADC)" used herein refers to a form in which a drug and an antibody are chemically linked without reducing the biological activity of the antibody and drug. In the present invention, the antibody-drug conjugate refers to a form in which a drug is bound to an amino acid residue at the N-terminus of the heavy chain and / or light chain of an antibody, specifically, a form in which a drug is bound to an α-amine group at the N-terminus of the heavy chain and / or light chain of an antibody.

[0152] In the present invention, the antibody or antigen-binding fragment may be bound to a drug via a linker. The linker serves as a linking moiety between the antibody and the drug. It must be cleavable under intracellular conditions, i.e., enable the drug to be released from the antibody in the intracellular environment. Reflecting the antibody's long half-life, the antibody must be stable in systemic circulation, and the linker-drug binding must not affect the stability or pharmacokinetics of the antibody.

[0153] In the present invention, the linker may include, for example, a cleavable linker or a non-cleavable linker. In the case of a cleavable linker, like a peptide linker, it can be cleaved by an intracellular peptidase or protease enzyme, such as a lysosomal or endosomal protease, and in the case of a non-cleavable linker, for example, a thioether linker, the drug can be released after the antibody is non-selectively degraded by intracellular hydrolysis.

[0154] In the present invention, the cleavable linker may include a peptide linker. The peptide linker has a length of at least two amino acids. For example, it may include a Val-Cit, Val-Ala, or Val-Cit dipeptide, or Phe-Leu or Gly-Phe-Leu-Gly. Examples of linkers are specifically described in International Patent Application Publication No. WO2004 / 010957, which may be incorporated herein by reference.

[0155] In the present invention, the antibody-drug conjugate binds to an antigen of a target cancer cell, forming an ADC-antigen complex, which is then internalized into the cancer cell via the endosome-lysosome pathway. In this case, the intracellular release of the cytotoxic drug is controlled by the internal environment of the endosome / lysosome.

[0156] In the present invention, the cleavable linker is pH-sensitive and may be susceptible to hydrolysis at a specific pH value. Generally, a pH-sensitive linker indicates that it can be hydrolyzed under acidic conditions. For example, it may be an acid-labile linker that can be hydrolyzed in lysosomes, such as a hydrazone, a semicarbazone, a thiosemicarbazone, a cis-aconitic amide, an orthoester, an acetal, a ketal, etc.

[0157] In the present invention, the linker may be cleaved under reducing conditions, and for example, a disulfide linker may correspond to this. Various disulfide bonds may be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene). These disulfide linkers can be cleaved by disulfide exchange with the thiol of intracellular glutathione.

[0158] In the present invention, the drug and / or drug-linker may be randomly conjugated via lysine of the antibody, or conjugated via cysteine ​​exposed when the disulfide bond chain is reduced. In some cases, the linker-drug may be conjugated via cysteine ​​present in a genetically engineered tag, for example, a peptide or a protein. The genetically engineered tag, for example, a peptide or a protein, may include an amino acid motif that can be recognized by, for example, an isoprenoid transferase. The peptide or protein may have a deletion at the carboxyl terminus of the peptide or protein, or may have an addition via covalent bonding of a spacer unit to the carboxyl (C) terminus of the peptide or protein.

[0159] In the present invention, the peptide or protein may be directly covalently linked to the amino acid motif or may be covalently linked to a spacer unit to be linked to the amino acid motif. The amino acid spacer unit is composed of 1 to 20 amino acids, and among them, a glycine unit is preferred.

[0160] In the present invention, the linker may include a beta-glucuronide linker that is recognized and hydrolyzed by beta-glucuronidase, which is present in large numbers in lysosomes or is overexpressed in some tumor cells. Unlike peptide linkers, it has a high hydrophilicity, and thus has the advantage of increasing the solubility of the antibody-drug complex when bound to a drug with a high hydrophobicity. In this regard, the beta-glucuronide linker disclosed in International Patent Application Publication No. WO2015 / 182984, for example, a beta-glucuronide linker including a self-immolative group, may be used, and the above document is incorporated by reference.

[0161] In the present invention, the linker may be, for example, a non-cleavable linker, and the drug is released solely through intracellular antibody hydrolysis, producing, for example, an amino acid-linker-drug conjugate. This type of linker may be a thioether group or maleimidocaproyl group, and may maintain stability in blood.

[0162] In the present invention, when the disulfide bond chain of the antibody or antigen-binding fragment is reduced, the linker-drug can be bound by introducing a random linkage or an antibody-terminal binding peptide having the sequence GGGGGGGCVIM through a cysteine ​​exposed therein.

[0163] In the present invention, the drug is a pharmacologically effective agent that can be bound to an antibody, and may be, specifically, a chemotherapeutic agent, a toxin, microRNA (miRNA), siRNA, shRNA, or a radioactive isotope. The chemotherapeutic agent may be, for example, a cytotoxic agent or an immunosuppressant. Specifically, it may include a microtubulin inhibitor, a mitotic inhibitor, a topoisomerase inhibitor, or a chemotherapeutic agent that can function as a DNA intercalator. In addition, it may include a drug for treating cancer, inflammation, or immune diseases. For example, it may include an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an anthelmintic agent, or a combination thereof.

[0164] In the present invention, the anticancer agent is, for example, maytansinoid, auristatin (including MMAE, MMAF), aminopterin, actinomycin, bleomycin, talisomycin, camptothecin, N8-acetyl spermidine, 1-(2 chloroethyl)-1,2-dimethyl sulfonyl hydrazide, esperamycin, etoposide, 6-mercaptopurine, dolastatin, trichothecene, calicheamicin, taxol, taxane, paclitaxel, docetaxel, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mitomycin A, mitomycin C, chlorambucil, duocarmycin, L-asparaginase, mercaptopurine, Thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustard, cytoxan, etoposide, 5-fluorouracil, bischloroethylnitrosourea, irinotecan, camptothecin, bleomycin, idarubicin, daunorubicin, Dactinomycin, plicamycin, mitoxantrone, asparaginase, vinorelbine, chlorambucil, melphalan, carmustine, lomustine,Busulfan, treosulfan, decarbazine, etoposide, teniposide, topotecan, 9-aminocamptothecin, crisnatol, mitomycin C, trimetrexate, mycophenolic acid, tiazofurin, ribavirin, EICAR (5-ethynyl-1-beta-Dribofuranosylimidazole-4-carboxamide), hydroxyurea, deferoxamine, fluxuridine, doxifluridine, raltitrexed, Cytarabine (ara C), cytosine arabinoside, fludarabine, tamoxifen, raloxifene, megestrol, goserelin, leuprolide acetate, flutamide, bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine, photosensitizer Pe4, demethoxy-hypocrellin A, interferon-α, interferon-γ, tumor necrosis factor, Gemcitabine, Velcade, Revamid, Thalamide, Lovastatin,It may be at least one selected from the group consisting of 1-methyl-4-phenylpyridiniumion, staurosporine, actinomycin D, dactinomycin, bleomycin A2, bleomycin B2, peplomycin, epirubicin, pirarubicin, zorubicin, mitoxantrone, verapamil, and thapsigargin, nucleases, and toxins of bacterial, animal, or plant origin, but is not limited thereto.

[0165] In the present invention, the anti-inflammatory agent may include, but is not limited to, a steroid preparation that reduces inflammation or swelling by binding to a glucocorticoid receptor, a non-steroidal anti-inflammatory drug (NSAID) that relieves pain by responding to cyclic oxygenase (COX) that synthesizes prostaglandins that cause inflammation, or an immunospecific anti-inflammatory drug (ImSAID) that alters the activation and transport of inflammatory cells.

[0166] In the present invention, the immune disease treatment agent may include, but is not limited to, azathioprine, chlorambucil, cyclophosphamide, cyclosporine, mycophenolate, azathioprine, or methotrexate.

[0167] In the present invention, in some cases, the drug may include one or more nucleophilic groups selected from the group consisting of, but not limited to, amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups that can react to form a covalent bond with an electrophilic group on a linker and a linker reagent.

[0168]

[0169] chimeric antigen receptor (CAR)

[0170] According to another embodiment of the present invention, there is provided a chimeric antigen receptor (CAR) comprising an antigen-binding fragment (including a humanized antigen-binding fragment) provided by the present invention.

[0171] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an engineered receptor comprising an extracellular antigen-binding domain and an intracellular signaling domain. Although the most common type of CAR comprises a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to the transmembrane and intracellular domains of a T cell coreceptor, such as the CD3ζ chain, the invention described herein is not limited to these domains. Rather, as used herein, "chimeric antigen receptor" or "CAR" refers to any receptor engineered to express any intracellular signaling molecule and an extracellular antigen-binding domain fused or linked thereto.

[0172] In the present invention, the binding domain may include an antigen binding fragment provided in the present invention, preferably scFv.

[0173] The chimeric antigen receptor of the present invention may further comprise at least one of a hinge region (or spacer) and a signal transduction domain.

[0174] In the present invention, the hinge region is a portion that connects the antigen binding domain and the transmembrane domain, also called a 'spacer', and has the purpose of extending the antigen binding domain from the T cell membrane or NK cell membrane.

[0175] In the present invention, the hinge region may be obtained from any suitable sequence from any genus, including, for example, human or a part thereof, or may include a hinge region of a human protein, including all or part of CD8, CD28, 4-1BB, OX40, CD3 zeta (ζ) chain, T cell receptor α or β-chain, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, or combinations thereof, which are commonly used in the art, but are not limited thereto.

[0176] Additionally, in the present invention, the hinge region may include, but is not limited to, one selected from immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, and IgD).

[0177] In the present invention, the hinge region may be a CD8 hinge region including an amino acid sequence represented by SEQ ID NO: 59 or coated by a base sequence represented by SEQ ID NO: 60, but is not limited thereto.

[0178] In the present invention, the signal transduction domain refers to a portion of a chimeric antigen receptor that is found or engineered to be found within a T cell. In the present invention, the signal transduction domain may or may not include a transmembrane domain that serves to anchor the chimeric antigen receptor to the plasma membrane of the T cell.

[0179] In the present invention, the transmembrane domain and the signal transduction domain may be derived from the same protein (e.g., CD3ζ), or the transmembrane domain and the signal transduction domain may be derived from different proteins (e.g., the transmembrane domain of CD28 and the intracellular signal transduction domain of the CD3ζ molecule, or vice versa).

[0180] In the present invention, the transmembrane domain comprises a hydrophobic polypeptide that spans the cell membrane. In particular, the transmembrane domain can span from one side of the cell membrane (extracellular) to the other side of the cell membrane (intracellular or cytoplasmic).

[0181] In the present invention, the transmembrane domain may be in the form of an alpha helix, a beta barrel, or a combination thereof. Furthermore, in the present invention, the transmembrane domain may comprise a multi-component protein having multiple transmembrane segments, each of which is in the form of an alpha helix, a beta sheet, or a combination thereof.

[0182] In the present invention, the transmembrane domain may include, but is not limited to, for example, T cell receptor α or β chain, all or part of CD3 zeta (ζ) chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, or combinations thereof.

[0183] In the present invention, the transmembrane domain may be, for example, an artificially designed polypeptide comprising primarily hydrophobic residues such as leucine and valine. In one embodiment of the present invention, a triad of phenylalanine, tryptophan, and valine may be found at each end of the synthetic transmembrane domain.

[0184] In the present invention, the transmembrane domain may be a CD8 transmembrane domain that includes an amino acid sequence represented by SEQ ID NO: 61 or is encoded by a base sequence represented by SEQ ID NO: 62, but is not limited thereto.

[0185] In the present invention, at least one costimulatory domain may be further included between the transmembrane domain and the signal transduction domain. In the present invention, the costimulatory domain is a site through which a costimulatory signal is transmitted, and may play a role in transmitting a signal so that CAR-T cells or CAR-NK cells that recognize a specific antigen bound to the antigen binding domain cause an immune response, promote self-proliferation, and increase the duration of their retention in the body.

[0186] In the present invention, the costimulatory domain may include, but is not limited to, a functional signaling domain derived from a polypeptide comprising a ligand that binds to 4-1BB (CD137); OX40; CD27; CD28; CD30; CD40; PD-1; CD2; CD7; CD258; natural killer group 2 member C (NKG2C); natural killer group 2 member (NKG2D); B7-H3; CD83; ICAM-1; LFA-1 (CD11a / CD18) or ICOS; an active fragment thereof; a functional derivative thereof; or a combination thereof.

[0187] In the present invention, the co-stimulatory domain may be 4-1BB, which includes an amino acid sequence represented by SEQ ID NO: 63, or encoded by a base sequence represented by SEQ ID NO: 64, but is not limited thereto.

[0188] In the present invention, the signal transduction domain may comprise a polypeptide that provides activation of an immune cell to stimulate or activate at least some aspect of an immune cell signal transduction pathway.

[0189] In the present invention, the signal transduction domain may include, but is not limited to, a functional signal transduction domain derived from a polypeptide comprising all or part of CD3 zeta (ζ), common FcR gamma (FcER1G), FcgammaRIIIa, FcRbeta (Fc epsilon lip), CD3gamma, CD3delta, CD3epsilon, CD79a, CD79b, DNAX-activating protein 10 (DAP10), DNAX-activating protein 12 (DAP12), an active fragment thereof, a functional derivative thereof, or a combination thereof, and such signal transduction domains are known in the art.

[0190] In the present invention, the signal transduction domain may be CD3 zeta (ζ) encoded by an amino acid sequence represented by SEQ ID NO: 65 or a base sequence represented by SEQ ID NO: 66, but is not limited thereto.

[0191] Additionally, the chimeric antigen receptor of the present invention may further comprise a signal peptide.

[0192] In the present invention, the "signal peptide" comprises a peptide sequence, which is a type of peptide of any secreted or transmembrane protein, and can direct the transport of the chimeric antigen receptor of the present invention to the cell membrane and cell surface, and provide precise localization of the chimeric antigen receptor of the present invention. In particular, in the present invention, the signal peptide supports the chimeric antigen receptor of the present invention to the cell membrane, in which the extracellular portion of the chimeric antigen receptor is displayed on the cell surface, the transmembrane portion spans the plasma membrane, and the active domain is in the cytoplasmic portion, or inside the cell.

[0193] In the present invention, the signal peptide may be used without limitation as long as it is a peptide having the above function, but for example, it may be an n-terminal CD8α signal peptide including an amino acid sequence represented by SEQ ID NO: 57 or encoded by a base sequence represented by SEQ ID NO: 58, but is not limited thereto.

[0194] FIG. 8 schematically illustrates the structure of a chimeric antigen receptor according to one embodiment of the present invention, which may be composed of or include a mesothelin binding domain (scFv according to the present invention (e.g., VL-linker-VH))-CD8 hinge-CD8 transmembrane domain-4-1BB-CD3ζ, or may be composed of or include a signal peptide-mesothelin binding domain (scFv according to the present invention)-CD8 hinge-CD8 transmembrane domain-4-1BB-CD3ζ. However, the structure of the chimeric antigen receptor of the present invention is not limited to the above structure.

[0195] As an example, the chimeric antigen receptor provided in the present invention comprises at least one scFv of SEQ ID NOs: 51 and 53, and may comprise or consist of an amino acid sequence represented by at least one of SEQ ID NOs: 68 and 70, but is not limited thereto.

[0196] As an example, the chimeric antigen receptor provided in the present invention comprises a humanized scFv of SEQ ID NO: 55, and may comprise or consist of an amino acid sequence represented by SEQ ID NO: 72, but is not limited thereto.

[0197] In addition, the chimeric antigen receptor provided by the present invention may further include a sequence in which a CD8α signal peptide including an amino acid sequence represented by SEQ ID NO: 57 is additionally linked to the N-terminus of the amino acid sequence represented by SEQ ID NO: 68, 70, or 72. Specifically, it may include or consist of an amino acid sequence represented by SEQ ID NO: 74, 76, or 78, but is not limited thereto.

[0198]

[0199] According to another embodiment of the present invention, the present invention relates to a nucleic acid molecule encoding a chimeric antigen receptor according to the present invention.

[0200] In the present invention, the nucleic acid molecule encoding the chimeric antigen receptor is readily prepared from the amino acid sequence of the specified chimeric antigen receptor by any conventional method. The base sequence encoding the amino acid sequence can be obtained for each domain from the aforementioned NCBI RefSeq ID or GenBenk accession number, and the nucleic acid of the present disclosure can be produced using standard molecular biological and / or chemical procedures. For example, a polynucleotide based on the base sequence can be synthesized, and the nucleic acid molecule of the present disclosure can be produced by combining DNA fragments obtained from a cDNA library using polymerase chain reaction (PCR).

[0201] The nucleic acid molecule of the present invention may be, but is not limited to, a gene or part of an expression or cloning cassette.

[0202] As an example, the nucleic acid molecule of the present invention may comprise or consist of, but is not limited to, a base sequence represented by SEQ ID NO: 69 encoding a chimeric antigen receptor comprising an amino acid sequence represented by SEQ ID NO: 68.

[0203] As an example, the nucleic acid molecule of the present invention may comprise or consist of, but is not limited to, a base sequence represented by SEQ ID NO: 71 encoding a chimeric antigen receptor comprising an amino acid sequence represented by SEQ ID NO: 70.

[0204] As an example, the nucleic acid molecule of the present invention may comprise or consist of, but is not limited to, a base sequence represented by SEQ ID NO: 73 encoding a chimeric antigen receptor comprising an amino acid sequence represented by SEQ ID NO: 72.

[0205] As an example, the nucleic acid molecule of the present invention may comprise or consist of, but is not limited to, a base sequence represented by SEQ ID NO: 75 encoding a chimeric antigen receptor comprising an amino acid sequence represented by SEQ ID NO: 74.

[0206] As an example, the nucleic acid molecule of the present invention may comprise or consist of, but is not limited to, a base sequence represented by SEQ ID NO: 77 encoding a chimeric antigen receptor comprising an amino acid sequence represented by SEQ ID NO: 76.

[0207] As an example, the nucleic acid molecule of the present invention may comprise or consist of, but is not limited to, a base sequence represented by SEQ ID NO: 79 encoding a chimeric antigen receptor comprising an amino acid sequence represented by SEQ ID NO: 78.

[0208]

[0209] According to another embodiment of the present invention, there is provided an expression cassette comprising a nucleic acid molecule provided by the present invention.

[0210] The expression cassette of the present invention may include expression control sequences such as a promoter, enhancer, polyadenylation signal, transcription terminator or internal ribosome entry site (IRES) capable of regulating the expression of the nucleic acid molecule of the present invention.

[0211] In the present invention, the promoter may include, but is not limited to, for example, the SFFV promoter, the elongation factor 1α (EF 1a) promoter, or the CAG (chicken beta-actin promoter with CMV enhancer) promoter.

[0212] In the present invention, the promoter may be an EF1a promoter including a base sequence represented by SEQ ID NO: 67, but is not limited thereto.

[0213] The expression cassette of the present invention can further include a truncated EGFR gene (EGFRt) as an additional gene, which is an additional sequence useful for monitoring its expression. In the present invention, the EGFRt can be used as a non-immunogenic selection tool, for example, to select transformed T cells or NK cells by immunomagnetic selection using biotinylated mesothelin and anti-biotin microbeads, and can also be used as a tracking marker in flow cytometry for tracking T cells or NK cells, and can be used as a suicide gene through the mesothelin-mediated antibody-dependent cellular cytotoxicity (ADCC) pathway.

[0214] In addition, in the present invention, the truncated EGFR gene (EGFRt) and one end of the Fcγ receptor binding domain of the polynucleotide of the present invention can be linked by a ribosomal skip element, which is a cleavable linker.

[0215] As used herein, the term "ribosome skip" refers to an alternative mechanism of translation in which a specific peptide prevents the cellular ribosome from covalently linking the newly inserted amino acid, instead allowing it to continue to be translated, resulting in cotranslational cleavage of the polyprotein. This process is induced by a "2A ribosomal skip" element or a cis-acting hydrolase element (e.g., a CHYSEL sequence). In some embodiments, this sequence comprises a non-conserved amino acid sequence with a strong alpha helix tendency followed by the consensus sequence -D(V / I)ExNPG P, where x is any amino acid. A distinct cleavage occurs between G and P. In the present invention, the ribosomal skip element may be a 2A ribosomal skip element, preferably, but not limited to, a 5' T2A ribosomal skip element.

[0216]

[0217] According to another embodiment of the present invention, there is provided a recombinant expression vector comprising an expression cassette provided by the present invention.

[0218] In the present invention, the description of the recombinant expression vector overlaps with that described above for the antibody or antigen-binding fragment, and thus the description thereof is omitted below.

[0219]

[0220] According to another embodiment of the present invention, the present invention relates to an immune effector cell transduced with a recombinant expression vector provided by the present invention.

[0221] As used herein, the term "immune effector cell" may be a lymphocyte cell that participates in an immune response, such as promoting an immune effector response.

[0222] As used herein, the term "lymphocytes" refers to cells commonly found in lymph, including natural killer cells (NK cells), T cells, and B cells. Those skilled in the art will appreciate that the immune cell types listed above can be further divided into subtypes.

[0223] The term "natural killer cells (NK cells)" as used herein is defined as large granular lymphocytes (LGL), which constitute three types of cells that differentiate from common lymphoid progenitor-producing B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus and enter the circulation. In the present invention, the NK cells may include any type of NK cell without limitation, and may include, but are not limited to, cultured NK cells, such as primary NK cells, NK cells from a cultured NK cell line, or NK cells obtained from a mammal. When the NK cells are obtained from a mammal, the NK cells may be obtained from a number of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The NK cells may be concentrated or purified. The NK cells may preferably be human NK cells (e.g., isolated from a human). NK cell lines are available from, for example, the American Type Culture Collection (ATCC), and include, for example, NK-92 cells (ATCC CRL-2407), NK92MI cells (ATCC CRL-2408), or derivatives thereof.

[0224]

[0225] According to another embodiment of the present invention, the present invention relates to a polypeptide expressed from an immune effector cell provided by the present invention.

[0226] As an example, the polypeptide expressed in the immune effector cell provided in the present invention may comprise or consist of an amino acid sequence represented by any one of SEQ ID NOs: 68 and 70, but is not limited thereto.

[0227] As an example, the polypeptide expressed in the immune effector cell provided in the present invention may include or consist of an amino acid sequence represented by SEQ ID NO: 72, but is not limited thereto.

[0228]

[0229] 3. Therapeutic uses

[0230] According to another embodiment of the present invention, there is provided a pharmaceutical composition for preventing, improving or treating a mesothelin-related disease, comprising as an active ingredient an antibody (including a humanized antibody) or antigen-binding fragment provided by the present invention; an immunoconjugate; a chimeric antigen receptor; or an immune effector cell expressing a chimeric antigen receptor.

[0231] In the present invention, the mesothelin-related disease is a disease mediated by mesothelin overexpression, and may be, for example, cancer or a tumor.

[0232] As used herein, the terms "cancer" and "tumor" refer to or represent a physiological condition typically characterized by uncontrolled cell growth in mammals. The cancer targeted for prevention, improvement, or treatment in the present invention may be a cancer or tumor that overexpresses mesothelin. The above cancers are not particularly limited, but may include, for example, liver cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, colon cancer, bone cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, anal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma, but are not limited thereto.

[0233] In the present invention, the term "prevention" may include, without limitation, any action that can block, suppress or delay symptoms caused by a mesothelin-related disease by using the composition of the present invention.

[0234] In the present invention, the terms “improvement” and “treatment” may include, without limitation, any act that can improve or benefit symptoms caused by a mesothelin-related disease by using the composition of the present invention.

[0235] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, capsules, tablets, aqueous suspensions, etc., external preparations, suppositories, and sterile injection solutions, according to conventional methods, but is not limited thereto. Preferably, the pharmaceutical composition may be formulated for intravenous administration or inhalation; or for use as an injection, but is not limited thereto.

[0236] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may include a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a colorant, a fragrance, etc. for oral administration. In the case of injections, a buffer, a preservative, an analgesic, a solubilizer, an isotonic agent, a stabilizer, etc. may be mixed and used. In the case of topical administration, a base, an excipient, a lubricant, a preservative, etc. may be used. The dosage form of the pharmaceutical composition of the present invention may be prepared in various ways by mixing it with the pharmaceutically acceptable carrier described above. For example, the composition may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. for oral administration. In the case of injections, the composition may be prepared in the form of unit dose ampoules or multiple doses. In addition, the composition may be formulated as a solution, suspension, tablet, capsule, sustained-release preparation, etc.

[0237] Examples of carriers, excipients, and diluents suitable for the formulation of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like may be additionally included.

[0238] The routes of administration of the pharmaceutical composition of the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. It may be administered orally or parenterally, and is preferably administered parenterally, but is not limited thereto.

[0239] The parenteral route of the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0240] The pharmaceutical composition of the present invention may vary depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, administration route, excretion rate, drug combination, and severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route, and period, but may be appropriately selected by those skilled in the art, and may be administered at 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. The administration may be administered once a day or divided into several times. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, a dragee, a capsule, a liquid, a gel, a syrup, a slurry, or a suspension.

[0241]

[0242] According to another embodiment of the present invention, there is provided a method for preventing, improving or treating a mesothelin-related disease, comprising administering a pharmaceutically effective amount of a composition of the present invention to a subject in need of administration.

[0243] In the present invention, the subject is an subject that has developed or is suspected of developing a mesothelin-related disease (particularly a cancer or tumor in which mesothelin is overexpressed). The subject suspected of developing the disease means all animals including humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs that have developed or can develop the disease, but subjects that can be treated with the effective substance provided in the present invention are included without limitation.

[0244] The term "pharmaceutically effective amount" as used herein is an amount sufficient to stop or alleviate the physiological effects of a subject or patient caused by a mesothelin-related condition. An appropriate effective amount may be determined by a treating physician within the scope of sound medical judgment, and may be administered once or in several divided doses. However, for the purpose of the present invention, it is preferable to apply a specific therapeutically effective amount for a specific patient differently depending on various factors such as the type and degree of response to be achieved, whether other agents are used in some cases, the composition containing the specific effective ingredient, the patient's age, weight, general health condition, sex, and diet, the time of administration, route of administration, number of administrations, and secretion rate of the composition containing the effective ingredient, the treatment period, drugs used together or concurrently with the specific composition, and similar factors well known in the medical field.

[0245] The total effective amount of the composition of the present invention can be administered to a patient as a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. The composition of the present invention may vary in the content of the active ingredient depending on the severity of the disease. Specifically, a preferred total dose of the composition of the present invention may be about 0.0001 mg to 500 mg per kg of patient body weight per day. However, since the dosage of the composition is determined by taking into consideration various factors such as the route of administration of the pharmaceutical composition and the number of treatments, as well as the patient's age, weight, health status, sex, severity of the disease, diet, and excretion rate, a person having ordinary skill in the art will be able to determine an appropriate effective dosage for a specific use of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration, or method of administration, as long as it exhibits the effects of the present invention.

[0246] In addition, the method for preventing, improving or treating the mesothelin-related disease in the present invention may be a combination therapy further comprising administering a compound or substance having therapeutic activity against one or more diseases.

[0247] In the present invention, the term "combination" should be understood to refer to simultaneous, separate, or sequential administration. If the administration is sequential or separate, the interval between administrations of the secondary components should be such that the beneficial effects of the combination are not lost.

[0248]

[0249] 4. Diagnostic Uses

[0250] According to another embodiment of the present invention, there is provided a composition for diagnosing a mesothelin-related disease, comprising an antibody (including a humanized antibody) or antigen-binding fragment provided by the present invention; or the bispecific or multispecific antibody.

[0251] Mesothelin, detected by the antibody or antigen-binding fragment of the present invention, is known to be expressed at high levels in cancer or tumor cells. Therefore, the antibody or antigen-binding fragment of the present invention can be used as a diagnostic marker for diagnosing cancer or tumors, assessing disease progression, and assessing prognosis before and after treatment.

[0252] The antibodies or antigen-binding fragments of the present invention can be used in any known analytical methods, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays.

[0253] The antibody or antigen-binding fragment of the present invention may be labeled with a label that: (i) provides a detectable signal; (ii) reacts with a first or second label, for example, a second label to modify a detectable signal provided by Fluorescence Resonance Energy Transfer (FRET); (iii) affects mobility, for example, electrophoretic mobility, by charge, hydrophobicity, shape, or other physical parameters, or (iv) provides a capture moiety, such as an affinity, antibody / antigen, or ion complex. Suitable labels include fluorescent labels, luminescent labels, chromophore labels, radioactive labels, isotope labels, isobaric labels, enzymatic labels, particulate labels, in particular metal particle labels, magnetic particle labels, polymer particle labels, small organic molecules such as biotin, receptor ligands or binding molecules such as cell adhesion proteins or lectins, label-sequences comprising nucleic acid and / or amino acid residues that are detectable by use of a binding agent, etc.

[0254] In the present invention, the specific type of the label is not particularly limited, but examples thereof include enzymes that utilize hydrogen peroxide to oxidize a dye precursor, such as HRP, lactoperoxidase or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, radioisotopes 32P, 14C, 125I, 3H and 131I linked to stable free radicals, etc., fluorophores, such as rare earth chelates or fluorescein and derivatives thereof, rhodamine and derivatives thereof, dansyl, umbelliferone, luciferases, such as firefly luciferase and bacterial luciferase (see US 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, Glucoamylase, lysozyme, saccharide oxidases such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, but are not limited thereto.

[0255]

[0256] According to another embodiment of the present invention, there is provided a diagnostic kit for mesothelin-related diseases comprising the diagnostic composition of the present invention.

[0257] In the present invention, the "kit" refers to a tool that can evaluate the expression level of a biomarker by labeling a probe or antibody that specifically binds to a biomarker component with a detectable label. It includes not only direct labeling of a detectable substance related to a probe or antibody through reaction with a substrate, but also indirect labeling in which a chromogenic label is conjugated through reactivity with another directly labeled reagent. It may include a chromogenic substrate solution, a washing solution, and other solutions that will react with the label for chromogenic reaction, and may be manufactured including the reagent components used. In the present invention, the kit may be a kit that includes the essential elements required for performing RT-PCR, and may include, in addition to each primer pair specific for a marker gene, a test tube, a reaction buffer, deoxynucleotides (dNTPs), Taq polymerase, reverse transcriptase, DNase, RNase inhibitor, sterile water, etc. In addition, the kit may be a kit for detecting genes for cancer diagnosis that includes the essential elements required for performing a DNA chip. The DNA chip kit comprises a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof. The kit of the present invention is not limited thereto, as long as it is known in the art.

[0258] In the present invention, the kit may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.

[0259] The kit of the present invention may further comprise one or more other component compositions, solutions, or devices suitable for the analysis method. For example, the kit of the present invention may further comprise essential elements necessary for performing a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit comprises a pair of primers specific for a gene encoding a marker protein. The primers are nucleotides having a sequence specific for the nucleic acid sequence of the gene, and may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also comprise a primer specific for the nucleic acid sequence of a control gene. In addition, the reverse transcription polymerase reaction kit may comprise a test tube or other appropriate container, a reaction buffer (with various pH and magnesium concentrations), deoxynucleotides (dNTPs), an enzyme such as Taq polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC-water, sterile water, etc.

[0260] Additionally, the diagnostic kit of the present invention may include essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotides corresponding to a gene or fragment thereof are attached, and reagents, preparations, enzymes, etc. for producing a fluorescently labeled probe. The substrate may also include cDNA or oligonucleotides corresponding to a control gene or fragment thereof.

[0261] Additionally, the diagnostic kit of the present invention may include essential components necessary for performing an ELISA. The ELISA kit includes an antibody specific for the protein. The antibody is an antibody with high specificity and affinity for the marker protein and little cross-reactivity with other proteins, and may be a monoclonal antibody, polyclonal antibody, or recombinant antibody. The ELISA kit may also include an antibody specific for a control protein. In addition, the ELISA kit may include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated to antibodies), and their substrates or other substances capable of binding to antibodies.

[0262] In the present invention, a fixative for the antigen-antibody binding reaction may be a nitrocellulose membrane, a PVDF membrane, a well plate synthesized from polyvinyl resin or polystyrene resin, a glass slide glass, etc., but is not limited thereto.

[0263] In the present invention, the washing solution preferably contains a phosphate buffer solution, NaCl, and Tween 20, and a buffer solution (PBST) composed of 0.02 M phosphate buffer solution, 0.13 M NaCl, and 0.05% Tween 20 is more preferred. After the antigen-antibody binding reaction, the washing solution reacts the secondary antibody with the antigen-antibody complex, and then an appropriate amount is added to the fixative and washed 3 to 6 times. The reaction stopping solution can preferably be a sulfuric acid solution (H2SO4).

[0264]

[0265] According to another embodiment of the present invention, there is provided a method for providing information for diagnosing a mesothelin-related disease, comprising the step of contacting a biological sample isolated from a target individual with an antibody (including a humanized antibody) or antigen-binding fragment provided in the present invention; or a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment.

[0266] In the present invention, the subject is an subject that has developed or is suspected of developing a mesothelin-related disease (particularly cancer or tumor caused by overexpression of mesothelin). The subject suspected of developing the disease means all animals including humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs that have developed or can develop the disease. However, any subject that can be diagnosed with a disease using the effective substance provided in the present invention is included without limitation.

[0267] In the present invention, the biological sample is whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint It may include, but is not limited to, joint aspirate, organ secretions, cells, cell extracts, or cerebrospinal fluid.

[0268] The information providing method of the present invention may include a step of measuring the expression level of mesothelin protein in the biological sample using the antibody or antigen-binding fragment provided in the present invention.

[0269] In the present invention, measurement of the expression level of the protein can be performed by Western blotting, enzyme linked immunosorbent assay (ELISA), competitive binding assay, or a similar method.

[0270] The information providing method of the present invention may include a step of predicting that a mesothelin-related disease has developed or is likely to develop if the expression level of the mesothelin protein measured for the biological sample is increased compared to a control group. Here, the control group may be, but is not limited to, the expression level of mesothelin in a healthy normal control group.

[0271] The anti-mesothelin antibody or antigen-binding fragment of the present invention can bind to mesothelin with high affinity and binding strength.

[0272] In addition, the humanized antibody or antigen-binding fragment provided in the present invention not only has high affinity and binding ability to the mesothelin antigen, but also has low immunogenicity even when administered to the human body.

[0273] Accordingly, the antibody or antigen-binding fragment provided by the present invention can be usefully applied not only to the treatment or diagnosis of mesothelin-mediated diseases such as cancer or tumors, but also to the development of various antibody-drug conjugates, bispecific antibodies, or chimeric antigen receptors (CARs).

[0274] Figure 1 shows the results of ELISA analysis of changes in antibody titer according to the number of weeks for each mouse after mouse immunization in Example 1.

[0275] Figure 2 shows the results of yeast display antigen affinity screening in Example 1.

[0276] Figure 3 shows the results of yeast display antigen affinity biopanning in Example 1.

[0277] Figure 4 shows the results of FACS analysis of the binding affinity of mesothelin-specific clones #19 and #45 selected in Example 1 to mesothelin.

[0278] Figure 5 shows the results of purifying the scFv of antibody clone #19 and antibody clone #45 screened in Example 1.

[0279] Figure 6 shows the results of ELISA analysis of the binding ability of the mesothelin-specific scFv (#19, #45) according to the present invention to the mesothelin antigen in Example 2.

[0280] Figure 7 shows the results of FACS analysis of the binding ability of mesothelin-specific scFv (#19, #45) according to the present invention to mesothelin-expressing K562 cells in Example 2.

[0281] FIG. 8 illustrates a construction construct of a mesothelin-specific chimeric antigen receptor according to one embodiment of the present invention.

[0282] Figure 9 shows the results of FACS analysis on whether or not a mesothelin-specific CAR is expressed after cells capable of binding to a mesothelin antigen are selected after being transfected to express a mesothelin-specific CAR according to the present invention in Example 3.

[0283] Figure 10 shows the results of FACS analysis to determine whether CD56, CD16, and CAR were expressed in selected cells transfected to express a mesothelin-specific CAR according to the present invention in Example 3.

[0284] Figure 11 shows the results of analyzing the death rate of cancer cells through FACS analysis after co-culturing mesothelin-expressing pancreatic cancer cells (Capan-2) and mesothelin-specific CAR-expressing NK-92 cells according to the present invention in Example 4.

[0285] Figure 12 shows the results of analyzing the binding affinity of the mesothelin-specific humanized antibody (#19-1) according to the present invention for the mesothelin antigen in Example 7 using the SPR method.

[0286] FIG. 13 illustrates a vector map comprising a polynucleotide encoding a chimeric antigen receptor targeting mesothelin according to the present invention in Example 8.

[0287] Figure 14 shows the results of analyzing the binding ability of 293T cells transfected to express a mesothelin-specific CAR according to the present invention in Example 8 using FACS for mesothelin.

[0288] Figure 15 shows the results of analyzing the intensity of cell staining (MFI; mean fluorescence intensity) as a measure of binding to mesothelin of 293T cells transfected to express a mesothelin-specific CAR according to the present invention in Example 8.

[0289] Figure 16 shows the results of FACS analysis to determine whether CAR is expressed in NK cells transfected to express a mesothelin-specific CAR according to the present invention in Example 9.

[0290] Figure 17 shows the results of FACS analysis to determine whether CD3, CD56, and CD16 were expressed in NK cells transfected to express a mesothelin-specific CAR according to the present invention in Example 9.

[0291] Figure 18 shows the results of analyzing the death rate of cancer cells through FACS analysis after co-culturing mesothelin-expressing pancreatic cancer cells (Capan-2) and mesothelin-specific CAR-expressing NK cells according to the present invention in Example 10.

[0292] Figure 19 shows the results of analyzing the death rate of cancer cells through FACS analysis after co-culturing mesothelin-expressing liver cancer cells (HepG2) and mesothelin-specific CAR-expressing NK cells according to the present invention in Example 10.

[0293] Figure 20 shows the results of analyzing the death rate of cancer cells through FACS analysis after co-culturing mesothelin-expressing breast cancer cells (MDA-MB-231) and mesothelin-specific CAR-expressing NK cells according to the present invention in Example 10.

[0294] According to one embodiment of the present invention, it relates to an antibody or an antigen-binding fragment thereof that specifically binds to mesothelin (MSLN).

[0295] The mesothelin-binding antibody or antigen-binding fragment of the present invention may comprise a heavy chain variable region comprising a heavy chain CDR1 comprising at least one amino acid sequence of SEQ ID NOs: 3 and 17; a heavy chain CDR2 comprising at least one amino acid sequence of SEQ ID NOs: 5 and 19; and a heavy chain CDR3 comprising at least one amino acid sequence of SEQ ID NOs: 7 and 21; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 10; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12; and a light chain CDR3 comprising at least one amino acid sequence of SEQ ID NOs: 14 and 26.

[0296] The mesothelin-binding antibody or antigen-binding fragment of the present invention may comprise at least one of the following antibodies or antigen-binding fragments:

[0297] 1) an antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 12, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 14; and

[0298] 2) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 17, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 19, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 21; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 12, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 26.

[0299] The mesothelin-binding antibody or antigen-binding fragment of the present invention may comprise a heavy chain variable region comprising at least one amino acid sequence of SEQ ID NOs: 38 and 42; and a light chain variable region comprising at least one amino acid sequence of SEQ ID NOs: 39 and 43.

[0300] The mesothelin-binding antibody or antigen-binding fragment of the present invention may comprise at least one of the following antibodies or antigen-binding fragments:

[0301] 1) an antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 38 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 39; and

[0302] 2) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 42 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 43.

[0303] Additionally, the mesothelin binding antigen binding fragment of the present invention may be a scFv and may include at least one amino acid sequence of SEQ ID NOs: 51 and 53.

[0304] The present invention provides a mesothelin-binding humanized antibody or an antigen-binding fragment thereof. The humanized antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 29; a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5; and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 32.

[0305] The mesothelin-binding humanized antibody of the present invention or an antigen-binding fragment thereof may comprise a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 46; and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 47.

[0306] Additionally, the mesothelin-binding humanized antigen-binding fragment of the present invention may be an scFv comprising the amino acid sequence of SEQ ID NO: 55.

[0307]

[0308] According to another embodiment of the present invention, the present invention relates to a nucleic acid molecule encoding an antibody (including a humanized antibody) or an antigen-binding fragment provided by the present invention, a recombinant expression vector comprising the same, or a host cell transfected with the expression vector.

[0309]

[0310] According to another embodiment of the present invention, the present invention relates to a bispecific or multispecific antibody, or an immunoconjugate, comprising an antibody (including a humanized antibody) or an antigen-binding fragment provided by the present invention.

[0311]

[0312] According to another embodiment of the present invention, there is provided a chimeric antigen receptor (CAR) comprising an antigen-binding fragment (including a humanized antigen-binding fragment) provided by the present invention.

[0313] The chimeric antigen receptor of the present invention may further comprise at least one of a hinge region (or spacer) and a signal transduction domain.

[0314] The chimeric antigen receptor provided in the present invention comprises at least one scFv of SEQ ID NOs: 51 and 53, and may comprise or consist of an amino acid sequence represented by at least one of SEQ ID NOs: 68 and 70, but is not limited thereto.

[0315] In addition, the chimeric antigen receptor provided in the present invention comprises a humanized scFv of SEQ ID NO: 55, and may comprise or consist of an amino acid sequence represented by SEQ ID NO: 72, but is not limited thereto.

[0316]

[0317] According to another embodiment of the present invention, the present invention relates to a nucleic acid molecule encoding a chimeric antigen receptor according to the present invention, an expression cassette comprising the nucleic acid molecule, a recombinant expression vector comprising the expression cassette, or an immune effector cell transduced with the expression vector.

[0318]

[0319] According to another embodiment of the present invention, the present invention relates to a use of an antibody (including a humanized antibody) or antigen-binding fragment; a chimeric antigen receptor; or an immune effector cell expressing a chimeric antigen receptor, for the prevention, improvement, or treatment of a mesothelin-related disease.

[0320] In the present invention, the mesothelin-related disease is a disease mediated by mesothelin overexpression, and may be, for example, cancer or a tumor.

[0321]

[0322] According to another embodiment of the present invention, the present invention relates to an application of the antibody (including a humanized antibody) or antigen-binding fragment thereof, or the bispecific or multispecific antibody, for the detection of mesothelin or the diagnosis of a mesothelin-related disease.

[0323] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0324]

[0325] Example

[0326]

[0327] [Example 1] Screening and binding ability evaluation of mesothelin-specific mouse antibodies

[0328] 1. Mouse immunization

[0329] To obtain immunized mice necessary for the development of mesothelin-specific antibodies, a recombinant human mesothelin protein antigen represented by sequence number 1 and Imject TM Alum Adjuvant (Thermo Scientific TM ) were mixed and injected intraperitoneally into 6-week-old male Balb / c mice (n=4). Specifically, the antigen prepared at a concentration of 0.5 μg / μl and Alum Adjuvant were mixed at a 1:1 ratio (600 μl each) to prepare an emulsion, and the final immune concentration of 200 μl / dose / 1 time of antigen was injected intraperitoneally into the mouse. The immunization reaction was performed three times in total at two-week intervals using the same method as the initial immunization method.

[0330] To determine whether mice were immune, tail blood samples were collected weekly to obtain serum, and the increase in antibody titer against the immunogen was confirmed by ELISA. Specifically, each well of a 96-well ELISA plate was coated with mesothelin mature form-his antigen overnight at 4°C, followed by the addition of 3% BSA in PBS and incubation for 1 hour at room temperature. Mouse serum was collected at 10 -3 After serial dilution of 1 / 10 from the dilution ratio, the cells were incubated at room temperature for 1 hour. After washing three times with PBST (tween20 0.05%), anti-mouse IgG HRP was treated at a dilution ratio of 20,000:1 and incubated at room temperature for 1 hour. After washing three times with PBST (tween20 0.05%), TMB was treated and incubated at room temperature for 20 minutes. After treatment with 2 M H2SO4, the absorbance was measured at 450 nm. As a result, as shown in Figure 1, the antibody titer increased weekly in all four mice, indicating that immunity was sufficiently induced.

[0331] 2. cDNA production

[0332] After euthanizing the immunized mice as described above, the spleen and bone marrow were removed to obtain spleen cells and bone marrow cells, and then total RNA was extracted. For the immune antibody library PCR, the extracted RNA was used as a template using SuperScript TM cDNA was synthesized using III First-Strand Synthesis SuperMix (Invitrogen).

[0333] 3. Construction of an E. coli antibody library

[0334] 1 μl of previously synthesized cDNA, 1 μl of forward primer mixture, 1 μl of reverse primer mixture, 4 μl of dNTP mixture, 10 μl of 5x phusion buffer, and 0.5 μl of phusion polymerase were added, and water was adjusted to 50 μl. After repeating the conditions of 98 °C for 3 minutes, 98 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 40 seconds 10 times, the conditions of 98 °C for 30 seconds, 60 °C for 30 seconds, and 72 °C for 40 seconds were repeated 25 times, and then PCR was performed at 72 °C for 3 minutes to obtain the VH and Vk genes.

[0335] Using the pYDS-H sfiI vector and the pYDS-K sfiI vector as templates, the CH1 and CL genes were obtained by PCR by repeating the conditions of 35 times at 98°C for 3 minutes, 98°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, and then performing PCR at 72°C for 3 minutes.

[0336] After confirming the DNA bands of the VH, Vk, CH1, and CL genes obtained by performing PCR on 1% agarose, gel extraction was performed, and overlapping PCR was performed on VH with CH1 and Vk with CL. At this time, PCR was performed under the same conditions as above. For reference, the primer sequence information used in the above experiment is shown in Tables 8 to 14 below. However, in the present specification, among the primer sequences, 'R' may mean A or G, 'Y' may mean C or T, 'S' may mean G or C, 'W' may mean A or T, 'K' may mean G or T, 'M' may mean A or C, and 'D' may mean A or G or T.

[0337] pYDS-H-SfiI, pYDS-K-SfiI, VH-CHI (HC), and Vk-CL (LC) were each treated with sfiI (NEB), and the bands were confirmed on 1% agarose, gel-extracted, and DNA was obtained. Then, pYDS-H-SfiI vector and HC, and pYDS-k-SfiI vector and LC were ligated using T4 DNA ligase (NEB). The ligated DNA was transformed into DH5α competent cells and recovered for 1 hour. Some of the cells were diluted and colonies were counted, and the rest were plated on ampicillin LB solid medium. The plated colonies were collected and stocked with 20% glycerol. As a result of titration of the transformed E. coli library, the library size was 2.98 X 10 for HC and 2.98 X 10 for LC. 5 , LC is 5.64 X 10 5 was obtained by

[0338] VH 정방향 프라이머효소 부위VH 정방향 영역명칭서열번호GGCCGCTAGGGCCG A G G T T C D S C T G C A A C A G T YVH-for 180C A G G T G C A A M T G M A G S A G T CVH-for 281G A V G T G M W G C T G G T G G A G T CVH-for 382C A G G T T A Y T C T G A A A G A G T CVH-for 483G A K G T G C A G C T T C A G S A G T CVH-for 584C A G A T C C A G T T S G Y G C A G T CVH-for 685C A G R T C C A A C T G C A G C A G Y CVH-for 786G A G G T G M A G C T A S T T G A G W CVH-for 887G A A G T G A A G M T T G A G G A G T CVH-for 988G A T G T G A A C C T G G A A G T G T CVH-for 1089C A G A T K C A G C T T M A G G A G T CVH-for 1190C A G G C T T A T C T G C A G C A G T CVH-for 1291C A G G T T C A C C T A C A A C A G T CVH-for 1392C A G G T G C A G C T T G T A G A G A CVH-for 1493G A R G T G M A G C T G K T G G A G A CVH-for 1594

[0339] VH 역방향 프라이머VH 역방향 영역명칭서열번호C G A G G A G A C G G T G A C M G T G GVH-rev 195C G C A G A G A C A G T G A C C A G A GVH-rev 296C G A G G A G A C T G T G A G A S T G GVH-rev 397

[0340] CH forward primer VH overlapping part CH1 front part name sequence number CCA CKG TCA CCG TCT CCT CGGCCTCCACCAAGGGCCCATCGCH1_for 198 CTC TGG TCA CTG TCT CTG CGCH1_for 299 CCA STC TCA CAG TCT CCT CGCH1_for 3100

[0341] CH reverse primer sequence information name sequence number CGAATTCAGAACCTCTTGGAACTAGCAAGTpYDS_H_CH1_rev101

[0342] Vk 정방향 프라이머명칭상동성 부위 (pYDS-K)Vκ 정방향 영역TM서열번호VL-for κ1GATAAAAGAGAGGCCGCTAGGGCCG A C A W T G T T C T C A C C C A G T C52102VL-for κ2G A C A T C C A G A T G A C A C A G W C52103VL-for κ3G A T R T T G T G A T G A C C C A G W C50104VL-for κ4G A C A T T S T G M T G A C C C A G T C52105VL-for κ5G A T G T T G T G V T G A C C C A A A C50106VL-for κ6G A C A C A A C T G T G A C C C A G T C54107VL-for κ7G A Y A T T K T G C T C A C T C A G T C50108VL-for κ8G A T A T T G T G A T R A C C C A G G M49109VL-for κ9G A C A T T G T A A T G A C C C A A T C48110VL-for κ10G A C A T T G T G A T G W C A C A G T C50111VL-for κ11G A T R T C C A G A T G A M C C A G T C51112VL-for κ12G A T G G A G A A A C A A C A C A G G C52113

[0343] Vk 역방향 프라이머명칭Vk 역방향 영역CL 영역 오버행(overhang)TM서열번호Vk1 / 2_revACAGATGGTGCAGCCACCGTACGG C G T T T B A T T T C C A G C T T G G50114VK3_revACAGATGGTGCAGCCACCGTACGG C G T T T T A T T T C C A A T T T T G44115

[0344] CL Primer Name pYDS_K_CL Sequence Number CL_forCGTACGGTGGCTGCACCATCTGT116 CL_revAATTACATGACTCGAGCTACTTGTCATCGTC117

[0345] 4. Construction of a yeast antibody library

[0346] After preparing a plasmid from an E. coli library, the antibody gene sequence was obtained by performing PCR using primers having the sequences shown in Table 15 below, repeating the conditions of 98°C for 3 minutes, 98°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute 35 times, and then 72°C for 3 minutes.

[0347] Primer name Sequence information Sequence number PYDS_Seq_forccagcattgctgctaaagaagaaggg118 PYDS_HC_Seq_Revcgaattcagaacctcttggaactagcaagt119 PYDS_LC_Seq_Revatgactcgagctacttgtcatcgtcgt120

[0348] 200 ㎕ of competent cells (EBY100 for HC and BJ5464 for LC) were placed in a cuvette, and antibody DNA and pYDS vector treated with sfil were added at a ratio of 1:3. After inserting into an electroporator, a pulse was applied to transform the yeast cells and they were recovered for 1 hour. Some of the cells were diluted and colonies were counted, and the rest were serially diluted on SDCAA+Tryptophan (HC) and SDCAA+Leucine (LC) agar plates. As a result of titration of the transformed yeast library, the library size was 2.98 X 10 for HC. 5 , LC is 5.64 X 10 5 was obtained by

[0349] Yeast HC library cells and yeast LC library cells were cultured at 1 x 10 9The cells were mixed one by one, washed three times with YPD (20 g / L Dextrose, 20 g / L peptone, 10 g / L yeast extract, 14.7 g / L sodium citrate, 4.29 g / L citric acid, pH 4.5) (SIGMA), resuspended in 100 ㎕ of YPD, dropped so as not to spread on a YPD plate, dried, and cultured at 30 ℃ for 6 hours. After culture, the cells were collected in SD medium, centrifuged at 3000 rpm for 3 minutes, and washed. The cells were well resuspended so that the total cell pellet was 5 ml, and serial dilutions were performed on SDCAA+Tryptophan and SDCAA agar plates. Subculture was performed in two Erlenmeyer flasks containing 500 mL SDCAA medium with an initial absorbance of 0.1. Only the crossbred yeasts were selected by culturing at 160 rpm at 30 ℃. The diversity of the crossbred yeasts was 4 X 10 7 It was confirmed as .

[0350] Yeast display antigen affinity screening was performed. Specifically, after inducing the yeast library, the absorbance at 600 nm was measured, and the cell OD 600 nm = 20 (2 X 10) to be used for FACS 8After washing, 200:1 anti-frag (FG4R) and biotinylated MSLN-his were mixed in PBSF (0.1% BSA skim milk 1%) to make 1 μM, and the cells were resuspended in 1 mL. After incubating for 1 hour in a rotator (F1 mode, 25 speed) at room temperature, they were washed. Secondary labeling was performed by adding anti-mouse IgGFITC 200:1 (5 ㎕) and anti-human IgG Fc-PE 200:1 (5 ㎕) in a 1 mL reaction volume (PBSF (BSA 0.1%)). After incubation for 15 minutes on a rotator at 4 °C, the cells were washed and subjected to FACS sorting. When displaying yeast, antibody expression was confirmed by the flag tag of the heavy chain, and the affinity for the antigen was confirmed by SA-PE, which binds to the biotin of the antigen. As a result, as shown in Fig. 2, when Flag expression was confirmed, yeast cells with high antigen reactivity were gated, and 128,500 cells were selected.

[0351] Yeast display antigen affinity biopanning was performed. Specifically, MSLN-positive Aspc1 cells and MSLN-negative Aspc1 cells were seeded at 6 × 10 per well in a 6-well plate. 5 The cells were inoculated and cultured. After 2 days, the cultured cells were fixed and washed for biopanning. For positive selection, yeast was cultured at 1 X 10 per 3 wells. 8 , 10 7 , 2 X 10 7 The cells were prepared. After washing the cells, the yeast pellet was resuspended in 1 mL of PBSF (BSA 0.1%). All PBS from the cells in the 6-well plate was discarded, washed once more with 1 mL of PBSF (BSA 0.1%), and the supernatant was discarded. 0.5 mL of PBSF (BSA 0.1%) was added to the cells, and 0.5 mL of yeast cells were added droplets per well, so that 5 x 10 per well. 7 / mL, 2.5x107 / mL, 1x10 7 / mL cell density. It was cultured for 2 hours while rotating on a rotator at 60 rpm at 4℃. After culture, the supernatant was discarded, 1mL / well of PBSF (BSA 0.1%) was added, and the plate was shaken 25 times and rotated 5 times. The supernatant was discarded again, and 1mL / well of PBSF (BSA 0.1%) was added. The plate was shaken 25 times and rotated 10 times, the supernatant was discarded, and PBSF (BSA 0.1%) was added. The plate was sterilized with quaternary ammonium hydroxide and observed under a microscope. As shown in Fig. 3(a), it was confirmed that yeast was well bound to Aspc1 cells. After that, the PBSF in the well was discarded, 1mL of SDCAA medium was added, and the cells were harvested with a cell scraper. The plate was cultured overnight at 160 rpm at 30℃, and it was confirmed that biopanning was successful as shown in Fig. 3(b).

[0352] Single-cell antigen binding experiments were performed. Single colonies were obtained from the plates and inoculated into 96-well plates containing medium. After overnight incubation at 30°C and 160 rpm, the cells were induced for 48 hours at 20°C and 160 rpm. 4 x 10 cells were used for FACS. 6A number of cells were collected and transferred to a 96-well v-bottom plate. A total of 96 single clones were analyzed for binding to the MSLN mature form-his antigen form. After centrifugation at 3000 rpm for 3 minutes, the supernatant was removed, 180 ㎕ of PBSF (BSA 0.1%) was added to all wells, and resuspended more than 6 times. Centrifugation was performed at 3000 rpm for 3 minutes, and the supernatant was removed. Afterwards, MSLN mature form-his was treated as an antigen at 10 nM, and anti-flag antibody was treated at a ratio of 200:1. The reaction volume was 50 ㎕ of PBSF (BSA 0.1%) and skim milk 1%. After mixing well, 50 ㎕ was added to each well of the cell pellet that had been centrifuged previously, and resuspended more than 6 times. Placed on a rocker, incubated at room temperature at 50 rpm for 1 hour, and washed. For secondary labeling, anti-mouse IgG FITC, 50 ㎕ (200:1), SA-APC, 50 ㎕ (200:1) were added to a total of 10 mL of PBSF (BSA 0.1%), incubated at 4 ℃ for 15 minutes, washed, and FACS analysis was performed. As a result, as shown in Fig. 4, it was confirmed that clones #19 and #45 bind to the mature form of MSLN.

[0353] 5. Purification of scFv from selected single cells

[0354] After FACS analysis, single cells from clones #19 and #45 were placed in 0.2% SDS solution and disrupted at 95°C for 20 min. The cells were spun down and the supernatant was used as a template. A PCR mixture was prepared with 1 μl of this template, 20 pmoles of the forward primer in Table 16 below, 20 pmoles of the reverse primer, 4 μl of 10 mM dNTP, 5 μl of 10x pfu buffer, and 1 μl of Pfu DNA pol (total 40 μl), and the conditions were repeated 35 times at 94 °C for 3 minutes, 94 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 1 minute, and then PCR was performed at 72 °C for 3 minutes. The DNA bands were gel-extracted and DNA sequence information was obtained using the Sanger sequencing technique, and the DNA sequence was converted into an amino acid sequence, and the antibody sequence information was confirmed using the IMGT / DomainGapAlign web program (https: / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi). The results are shown in Table 17 below. (Note that the sequence information below is based on IMGT numbering.)

[0355] Primer name Sequence information Sequence number HC, LC Forward primer pYDSH_colonyPCR_forCACTATCCAACAGCACAAATAACGGGTTATCT121 HC Reverse primer BCRHC_CH1_RevGGAGGAGGGTGCCAGGGGGA122 LC Reverse primer pYDSK_CL_RevTGTACTTTGGCCTCTCTGGGATAGAAGTTATT123

[0356] 클론구분서열 정보서열 번호#19VH아미노산 서열QVQLQQSGPELVKPGASVKISCKASGYSFTDYNMNWVKQSNGKSLEWIGVINPNYGTTSYNQKFKGKATITADTSSNTAYLQLSSLTSEDSAVYYCAKGDYDGAGFAYWGQGTLVTVSA38VLDIVITQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLEIKR39VH핵산 서열CAGGTCCAACTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGCGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGACTACAACATGAACTGGGTGAAGCAGAGCAATGGAAAGAGCCTTGAGTGGATTGGAGTAATTAATCCTAACTATGGTACTACTAGCTACAATCAGAAGTTCAAGGGCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAAGGGTGATTACGACGGGGCCGGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCG40VLGATATTGTGATAACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCACTCACGTTCGGTGCTGGGACCAAGCTGGAAATCAAACGC41#45VH아미노산서열QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGMIHPNSGGTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARYYYGNYVWYFDVWGTGTTVTVSA42VLDIVLTQSPAIMSTSPGEKVTMTCTASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLTFGAGTKLEMKR43VH핵산 서열CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAATGATTCATCCTAATAGTGGTGGTACTAACTACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATACTACTATGGTAACTACGTGTGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCTGCG44VLGACATTGTTCTCACCCAGTCTCCAGCAATCATGTCTACATCTCCGGGGGAGAAGGTCACCATGACCTGCACTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTTACCCACTCACGTTCGGTGCTGGGACCAAGCTGGAAATGAAACGC45

[0357] As above, after sequence analysis, each clone was subjected to PCR so that HC and LC and the pmopac12 vector could overlap each other. After adding the HC, LC, and pmpac12 vector PCR products to the Gibson assembly mixture and reacting them at 50°C for 200 minutes, they were cloned into DH5α competent cells, and the scFv expression vectors of antibody clone #19 and antibody clone #45 were obtained. Co-transformation was performed in BL21 with the pET vector containing the BirA gene. After culturing in a medium supplemented with kanamycin and chloramphenicol, the initial cell density was 5 X 10 5 Starting with cells / mL, 6 to 8 X 10 6After growing to 10 cells / mL, IPTG was added to a concentration of 0.5 mM. Induction was performed at 25°C and 160 rpm for 16 hours. After completion of induction, centrifugation was performed at 5000 × g for 10 minutes at 4°C, the supernatant was discarded, and 20 mg / mL lysozyme, 100 mM Tris, 0.75 M sucrose, pH 7.5 was added to the pellet at a volume of 320 mL per 50 mL of induction volume and resuspended. Afterwards, the lysozyme solution was additionally added, and the pellet was shaken and cultured for 20 minutes at 4°C and 100 rpm. Afterwards, 1 mM EDTA was slowly added dropwise at an amount of 6.25 mL per 50 mL of induction volume, and the pellet was shaken and cultured for 20 minutes at 4°C and 100 rpm. After centrifugation at 12,000 xg for 30 minutes, the supernatant was filtered through a 0.22 μm PES syringe filter. The supernatant was loaded onto Ni-NTA resin, and 15 mL of washing buffer (25 mM imidazole, 300 mM NaCl, 50 mM NaH2PO4, pH 8.0) was added, followed by 15 mL of washing buffer (25 mM imidazole, 300 mM NaCl, 50 mM NaH2PO4, pH 8.0, 0.05% Triton X-114). After flowing 15 mL of washing buffer containing 25 mM imidazole, 300 mM NaCl, 50 mM NaH2PO4, pH 8.0, and 3 mL each of imidazole, 300 mM NaCl, 50 mM NaH2PO4, pH 8.0, and elution buffer, a total of 15 mL was flowed to recover the eluate. The eluate was transferred to an Amicon Ultra 15 mL centrifugal filter (10 kDa) and centrifuged at 3500 rpm for 30 minutes. After concentration, the buffer was replaced and the OD 280 nm value was measured using a nano drop to calculate the actual protein concentration. After that, 1 ㎍ of protein was subjected to SDS-PAGE analysis to measure the purity and then the concentration of the scFv protein was calculated. As a result, as shown in Figure 5, it was confirmed that scFv with a molecular weight of approximately 28-29 kDa was purified.

[0358]

[0359] [Example 2] Evaluation of the binding capacity of mesothelin-specific antibodies

[0360] 1. ELISA-based antigen binding ability evaluation

[0361] Prior to the plate, 100 ng of the purified scFv protein from Example 1 was added per well and refrigerated for 15 hours before coating. After incubation, 200 ㎕ of 3% PBS BSA was added per well and blocked for 1 hour at room temperature. 10 biotin-labeled MSLN-his protein was added. -6 After serially diluting 1 / 5 from M, the antibody was bound to the anti-MSLN scFv coated on the ELISA plate at room temperature for 1 hour. Each well was washed three times with PBST (tween 20 0.05%) washing buffer, and then treated with HRP enzyme-conjugated streptavidin (Streptavidin-HRP; abcam) at a dilution ratio of 40,000:1 for detection of biotin-labeled antibodies and incubated for 1 hour at room temperature. Each well was washed three times with PBST (tween 20 0.05%) washing buffer, treated with TMB, and incubated for 20 minutes at room temperature. After treatment with 2 M H2SO4, the absorbance was measured at 450 nm. As a result, as shown in Fig. 6, it was confirmed that the anti-MSLN scFv antibodies manufactured according to the present invention had excellent binding ability to the MSLN antigen.

[0362] 2. Evaluation of antigen binding ability based on MSLN-expressing cells

[0363] The binding ability of each purified scFv antibody to the MSLN antigen was analyzed using cells expressing MSLN. Specifically, MSLN-positive K562 cells and MSLN-negative K562 cells were prepared and seeded in 96-well plates at 5 x 10 per well. 4The cells were inoculated with 10 cells. After washing with PBSF (1% FBS) washing buffer, the anti-MSLN scFv according to the present invention was added to the cells. -6 The cells were serially diluted 1 / 4 in M ​​and incubated at 4°C for 1 hour. Washed with PBSF (1% FBS) washing buffer. Afterwards, anti-HISx6-FITC was treated at a ratio of 250:1 and incubated at 4°C for 1 hour. After washing with PBSF (1% FBS), FACS analysis was performed. As a result, as shown in Fig. 7, it was confirmed that the anti-MSLN scFv antibodies manufactured according to the present invention did not bind to MSLN-negative K562 cells, but selectively bound only to MSLN-positive K562 cells.

[0364]

[0365] [Example 3] Production of NK cells expressing mesothelin-specific chimeric antigen receptor (MSLN-CAR)

[0366] 1. Construction of a mesothelin-specific chimeric antigen receptor construct expression vector

[0367] 10 μg of pNKLV3 vector to be used for virus production was linearized by treatment with XbaI (NEB, cat##R0145S) and BamHI-HF (NEB, cat##R3136S) restriction enzymes. A polynucleotide encoding a CD8a signal peptide (SEQ ID NO: 58); a polynucleotide encoding a mesothelin-binding scFv (clone #19: SEQ ID NO: 52, or clone #45: SEQ ID NO: 54); a polynucleotide encoding a CD8 hinge region (SEQ ID NO: 60); a polynucleotide encoding a CD8 transmembrane domain (SEQ ID NO: 62); a polynucleotide encoding 4-1BB (costimulatory domain) (SEQ ID NO: 64); And a polynucleotide encoding CD3ζ (intracellular signaling domain) (SEQ ID NO: 66); was used to construct a chimeric antigen receptor construct as shown in Fig. 8, and the CAR construct DNA was synthesized in vitro with the linear pNKLV3 vector and the 5' and 3' ends having approximately 12 bp of DNA identical thereto. 100 ng of the linear pNKLV3 vector and 50 ng of the constructed CAR construct DNA were mixed with 5 x Ez-fusion HT cloning PreMIX (Enzynomics, cat## EZ015TL) and ligated at 50°C to produce the construct.

[0368] 2. Production of mesothelin-specific chimeric antigen receptor construct virus

[0369] Virus production was performed using the LV-MAX transfection kit (Thermofisher, cat##A35348). 4.7 x 10 6After preparing 25.5 mL of HEK293F cells cultured at cell / mL, 1.5 mL of LV-MAX supplement was added and cultured in a shaking incubator at 37°C, 120 rpm, 8% CO2. In a 50 mL tube, 33.33 ㎍, 16.67 ㎍, 16.67 ㎍, and 8.33 ㎍ of the four vectors prepared in 1 above, pMDLg / pRRE (Addgene, cat# #12251), pMD2.G (Addgene, cat##12259), and pRSV-Rev (Addgene, cat##12253) were added, respectively, and the volume was adjusted to 1.5 mL with Opti-MEM medium. A new tube was added with 1320 ㎕ of Opti-MEM (Gibco, Cat##11058021) medium and 180 ㎕ of reagent, and left for 1 minute. The solutions in the tube containing the vector and the tube containing the reagent were mixed, and left for 10 minutes. The HEK293F cells in culture were taken out and added to the mixed reagent, and incubated for 5 hours in a shaking incubator at 37℃, 120 rpm, 8% CO2. After incubation, 1.2 mL of enhancer was added, and the cells were removed and the virus was produced.

[0370] 3. Production of mesothelin CAR-expressing NK cells

[0371] Natural killer cells were used as immune effector cells to be genetically engineered to express the above chimeric antigen receptor. NK-92 cell lines were purchased from ATCC as natural killer cell lines. Both the NK-92 cell lines and the lentivirus-transduced NK-92 cell lines described below were cultured in a medium containing 100 μg / mL streptomycin, 100 units / mL penicillin, 20% fetal bovine serum (FBS), 10% MEM vitamin solution, 1X 2-mercaptoethanol, and 200 IU rhIL-2. In order to activate the NK-92 cell lines, they were cultured in a medium containing 500 IU rhIL-2 and 10 ng / mL rhIL-15 two days before transduction. The 293T cell line was cultured in a medium containing 100 μg / mL streptomycin, 100 units / mL penicillin, and 10% fetal bovine serum (FBS), and all cell cultures were maintained at 37°C in an environment with 5% CO2 (95% air). For mesothelin CAR expression, 5 x 10 5 After adding the NK-92 cell line of the dog and the antigen receptor construct virus produced above to an MOI of 10, centrifugation was performed for 60 minutes at 1000 xg to induce transduction, thereby producing mesothelin CAR-expressing NK cells.

[0372] 4. Selection of mesothelin-specific CAR-expressing cells

[0373] To select only cells expressing mesothelin-specific CAR, biotinylated mesothelin of SEQ ID NO: 1 was used for labeling, and then magnetic anti-biotin microbeads were added. Therefore, unlabeled cells passed through the MACS column in a magnetic field, and magnetically labeled cells were retained in the column, allowing only cells expressing mesothelin-specific CAR to be selected. After culturing the selected cells for a total of 12 days, FACS analysis was performed, and as shown in Figure 9, it was confirmed that more than 97% of the selected cells expressed mesothelin-specific CARs containing scFv #19 or scFv #45.

[0374] 5. Phenotypic analysis of cells expressing mesothelin-specific CAR

[0375] To confirm the phenotype of the cells selected in the above 4., the cells were rinsed twice with FACS buffer, and the rinsed cells were subjected to FACS analysis using PE-conjugated anti-CD56 antibody, PE-conjugated anti-CD16 antibody, PE-conjugated anti-NKG2D antibody, PE-conjugated anti-NKp30 antibody, PE-conjugated anti-LDLR antibody, and MSLN antigen conjugated with a fluorescent dye.

[0376] As a result, as shown in Fig. 10, the cells selected in the above 4. were CD56+CD16- cells and NK-92 cells, and it was confirmed that mesothelin-specific CAR was expressed in more than 99% of the cells. In addition, it was confirmed that NKG2D, an NK cell activation receptor, was expressed at a higher level in the NK cells expressing the above-mentioned CAR than in the negative control group (NK-92 cell line).

[0377]

[0378] [Example 4] Evaluation of the cancer cell killing ability of mesothelin-specific chimeric antigen receptor (CAR)-expressing NK cells

[0379] The cells selected in Example 3, item 4, were co-cultured with the Capan-2 pancreatic cancer cell line, known to highly express mesothelin, at a cell number ratio of 2:1. After 4 hours of co-culture, dead or damaged cells were stained using Propidium Iodide (PI). The killing capacity of CAR-expressing NK cells against cancer cells was then analyzed using FACS.

[0380] As shown in Fig. 11, it was confirmed that the killing ability of the mesothelin-specific CAR expressing NK cells according to the present invention was excellent against mesothelin-expressing pancreatic cancer cells.

[0381]

[0382] [Example 5] Induction of mesothelin-specific humanized antibodies

[0383] A humanized antibody was prepared by changing the mouse #19 scFv selected in Example 1 to a structure corresponding to a human. Specifically, the BioPhi site (https: / biophi.dichlab.org / humanization / humanize / ) was used to perform deep learning or CDR grafting (deep learning on natural antibody repertoires (Sapiens) or CDR grafting) and humanized antibody sequence substitution was performed mainly in the human IGHV1-46 and IGKV3-11 framework regions. The sequence was substituted so that it was 37-40% or higher, which is the humanized antibody sequence standard value, according to the OASis percentile standard (MAbs. 2022; 14(1): 2020203.). The 'OASis prevalence threshold', a parameter for evaluating humanized antibody sequences, was set to 'strict (0% subjects)', and the mouse #19 scFv was obtained as the humanized antibody sequence (OASis percentile value: 58%). At this time, the humanized antibody was named humanized #19-1 antibody, and the amino acid and base sequences of the heavy chain variable region and light chain variable region are shown in Table 18 below. (For reference, the sequence information below is based on the IMGT numbering of the IMGT / DomainGapAlign web program (https: / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi).)

[0384] #19-1 Sequence information Sequence number Heavy chain variable region CDR1 GYTFTDYN29 Heavy chain variable region CDR2 INPNYGTT5 Heavy chain variable region CDR3 ARGDYDGAGFAY32 Light chain variable region CDR1 SSVSY10 Light chain variable region CDR2 DTS12 Light chain variable region CDR3 QQWSSNPLT14 Heavy chain variable region amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMNWVRQAPGQGLEWMGVINPNYGTTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDYDGAGFAYWGQGTLVTVSS46 Light chain variable region amino acid SequenceEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSSNPLTFGQGTKLEIKR47 heavy chain variable region bases SequenceCAGGTCCAACTGGTTCAGTCTGGAGCTGAGGTAAAGAAGCCTGGCGCTTCAGTGAAGGTTTCCTGCAAGGCTTCTGGTTACACCTTCACTGACTACAACATGAACTGGGTGCGACAGGCTCCCGGACAAGGACTTGAGTGGATGGGAGTAATTAATCCTAACTATGGTACTACTAGCTACA ATCAGAAGTTCAAGGGCAGGGTGACTATGACACGCGACACATCCACCTCTACAGTGTACATGGAGCTCAGCAGCCTGCGCTCTGAGGACACGGCAGTCTATTACTGTGCAAGAGGTGATTACGACGGGCCGGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTAGC48 light chain variable region baseSequenceGAAATTGTcttAACCCAGTCTCCAGCAACCCTTTCTTTGTCTCCAGGGGAGCGAGCAACCCTGAGTTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGCCTGGCCAAGCGCCCCGCCTTCTCATTTATGACACATCCAAACTGG CTTCTGGAATACCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCGACTTTACTCTCACAATCAGCAGCTTGGAGCCAGAAGATTTTGCCGTATATTACTGCCAGCAGTGGAGTAGTAACCCACTCACGTTTCGGTCAGGGGACCAAGCTGGAAATCAAACGC49

[0385]

[0386] [Example 6] Expression and purification of mesothelin-specific humanized scFv

[0387] The humanized #19-1 antibody sequence predicted in Example 5 above was expressed in mammals and then purified. First, to optimize expression efficiency, the #19-1 scFv antibody gene sequence was optimized using the codon optimization tool (https: / sg.idtdna.com / CodonOpt), and for purification, the gene was synthesized with a structure in which a human IgG-Fc tag was linked to the 3' end of the scFv sequence. The synthesized gene was inserted into the pCEP4 mammalian expression vector and expressed in the form of #19-1 scFv-Fc using the ExpiCHO Expression System. Subsequently, purification was completed using affinity chromatography (Protein A Affinity Chromatography) and size exclusion chromatography (Size Exclusion Chromatography) techniques.

[0388]

[0389] [Example 7] Evaluation of binding affinity of humanized antibodies to mesothelin

[0390] The binding affinity of the humanized #19-1 antibody purified in Example 6 to mesothelin was evaluated using the surface plasmon resonance (SPR) method. First, recombinant human mature MSLN (SEQ ID NO: 1) was amine-immobilized on the surface of a carboxymethyldextran (CM5) chip at densities of approximately 560 RU and 520 RU. Then, the humanized #19-1 antibody was injected at a constant flow rate according to two-fold serial dilutions. The binding and dissociation rates of the protein complex (complex of humanized #19-1 antibody and mature MSLN) were monitored for 180 s and 540 s, respectively. Referencing was performed against a blank flow cell. Finally, the binding affinity of the humanized #19-1 antibody to mesothelin was determined using a 1:1 depletion corrected binding model. The analysis results are shown in Fig. 12 and Table 19 below.

[0391] MSLN humanized #19-1 antibody 80.8 nM

[0392] As shown in Table 19 above, the dissociation constant (KD) of the humanized #19-1 antibody of the present invention was 80.8 nM, confirming that it had high binding affinity for mesothelin.

[0393]

[0394] [Example 8] Production of mesothelin-specific chimeric antigen receptor (MSLN-CAR)-expressing cells

[0395] As shown in Figure 13, polynucleotides encoding the CD8 signal peptide, mesothelin binding domain (humanized scFv), CD8 hinge, CD8 transmembrane domain, 4-1BB, and CD3 zeta signaling domains were inserted into a lentiviral transfer vector as a chimeric antigen receptor expression cassette. At this time, the recombinant genes were placed under the control of the EF1a single promoter. The base sequence of the EF1a promoter used in the experiment and the base sequences of each other gene are shown in Table 20 below, and the amino acid sequences encoded by these base sequences are shown in Table 21 below.

[0396] 293T cells were used as cells to be genetically engineered to express the chimeric antigen receptor. These 293T cells were purchased from ATCC. Both the 293T cell line and the lentivirus-transduced 293T cell line described below were cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 100 μg / mL of streptomycin, 100 units / mL of penicillin, and 10% fetal bovine serum (FBS). The cells were cultured in an environment maintained at 37°C with 5% CO2 (95% air). For mesothelin CAR expression, 5 x 10 cells were seeded in 24-well plates. 5After adding the 293T cell line and the antigen receptor construct virus prepared above to an MOI of 10, centrifugation was performed for 60 minutes at 1000 xg to induce transduction, and mesothelin CAR-expressing 293T cells were produced and cultured for 2 days. To confirm the binding efficiency and strength of mesothelin CAR-expressing 293T cells to mesothelin, the cells were first rinsed twice with FACS buffer, and biotin-conjugated mesothelin mature antigen was bound to the washed cells. Subsequently, biotin was fluorescently labeled using PE-conjugated streptavidin protein and FACS analysis was performed. As a result, as shown in Figures 14 and 15, approximately 55% of the mesothelin CAR-expressing 293T cells bound to the mesothelin antigen, and the binding strength was also high.

[0397] Gene name Base sequence Sequence number EF1a promoter gggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgatccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacag67CD8 signalpeptideATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG58scFvGAAATTGTCTTAACCCAGTCTCCAGCAACCCTTTCTTTGTCTCCAGGGGAGCGAGCAACCCTGAGTTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGCCTGGCCAAGCGCCCCGCCTTCTCATTTATGACACATCCAAACTGGCTTCTGGAATACCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCGACTTTACTCTCACAATCAGCAGCTTGGAGCCAGAAGATTTTGCCGTATATTACTGCCAGCAGTGGAGTAGTAACCCACTCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAACGCGGAGGTGGGGGCAGTGGAGGCGGTGGTAGTGGAGGCGGAGGAAGTCAGGTCCAACTGGTTCAGTCTGGAGCTGAGGTAAAGAAGCCTGGCGCTTCAGTGAAGGTTTCCTGCAAGGCTTCTGGTTACACCTTCACTGACTACAACATGAACTGGGTGCGACAGGCTCCCGGACAAGGACTTGAGTGGATGGGAGTAATTAATCCTAACTATGGTACTACTAGCTACAATCAGAAGTTCAAGGGCAGGGTGACTATGACACGCGACACATCCACCTCTACAGTGTACATGGAGCTCAGCAGCCTGCGCTCTGAGGACACGGCAGTCTATTACTGTGCAAGAGGTGATTACGACGGGGCCGGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTAGC56CD8 hingeACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT60CD8transmembraneATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC624-1BBAAACGGGGCAGAAAGAAAC TCCTGTATATATTCAAACAACCATTTGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAAGAAGGAGGATGTGAACTG64CD3 zetaAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGA AGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCCGGAGGGGCAAGGGGCACGTGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC66

[0398] Peptide name Amino acid sequence Sequence number CD8 signal peptide MALPVTALLLPLALLLHAARP57scFvEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSSNPLTFGQGTKLEIKRGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMNWVRQAPGQGLEWMGVINPNYGTTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDYDGAGFAYWGQGTLVTVSS55CD8 hingeTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD59CD8 transmembraneIYIWAPLAGTCGVLLLSLVITLYC614-1BBKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL63CD3 zetaRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR65

[0399]

[0400] [Example 9] Production of NK cells expressing mesothelin-specific chimeric antigen receptor (MSLN-CAR)

[0401] After producing a mesothelin-specific chimeric antigen receptor construct virus in the same manner as in Example 8, natural killer cells were used as immune effector cells to be genetically engineered to express the chimeric antigen receptor. As this natural killer cell line, PBMC (Human PB MNC) cell line was purchased from StemCell, cultured for 10 days, and differentiated into PBNK. PBMC and PBNK cells, as well as PBNK cells transduced with the lentivirus described below, were all cultured in NK MACS medium containing 100 μg / mL of streptomycin, 100 units / mL of penicillin, 10 ng / mL of rhIL-15, and 500 IU of rhIL-2. All cell cultures were cultured in an environment maintained at 37°C with 5% CO2 (95% air). For mesothelin CAR expression, 5 x 10 in 24-well plates 5 After adding the PBNK cell line and the antigen receptor construct virus produced above to an MOI of 2, PGE2 (Prostaglandin E2) and LentiBOOST (a lentiviral gene transduction inducer) were used to produce mesothelin CAR-expressing NK cells. To confirm the phenotype of the produced mesothelin CAR-expressing cells, the cells were rinsed twice with FACS buffer, and the washed cells were subjected to FACS analysis using Pacific blue-conjugated anti-CD3 antibody, APC-conjugated anti-CD56 antibody, Bv785-conjugated anti-CD16 antibody, and PE-conjugated MSLN antigen. However, in the figures and tables, mock represents the results of NK cells transfected with an empty vector.

[0402] Mock#19-1NK+(CD3-,CD56+)81.82%83.64%activity(CD16+)85.89%71.65%

[0403] As a result, as shown in Figures 16 and 17 and Table 22, it was confirmed that the cells induced through the above process were CD3-CD56+ cells, which were NK cells, and that 71.65% had CD16+, an NK cell activity marker, and that mesothelin-specific CAR was expressed in more than 25% of the cells.

[0404]

[0405] [Example 10] Evaluation of the cancer cell killing ability of mesothelin-specific chimeric antigen receptor (CAR)-expressing NK cells

[0406] To confirm the cytotoxicity of the mesothelin-specific CAR-expressing NK cells produced in Example 9 against cancer cells, the mesothelin-specific CAR-expressing NK cells were co-cultured with the Capan-2 pancreatic cancer cell line, the HepG2 liver cancer cell line, and the MDA-MB-231 breast cancer cell line, which are known to express high levels of mesothelin. After 4 hours of co-culture, dead or damaged cells were stained using PI (Propidium Iodide). Subsequently, the killing ability of the CAR-expressing NK cells against cancer cells was analyzed using FACS analysis.

[0407] As shown in Figures 18 to 20, it was confirmed that the killing ability of the mesothelin-specific CAR expressing NK cells according to the present invention was excellent against all mesothelin-expressing pancreatic cancer cells, liver cancer cells, and breast cancer cells.

[0408]

[0409] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0410] The present invention relates to an antibody or antigen-binding fragment capable of specifically binding to a mesothelin antigen and various uses thereof, such as diagnosis or treatment of mesothelin-related diseases.

Claims

1. It specifically binds to mesothelin (MSLN). An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising at least one amino acid sequence of SEQ ID NOs: 3 and 17; a heavy chain CDR2 comprising at least one amino acid sequence of SEQ ID NOs: 5 and 19; and a heavy chain CDR3 comprising at least one amino acid sequence of SEQ ID NOs: 7 and 21; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 10; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12; and a light chain CDR3 comprising at least one amino acid sequence of SEQ ID NOs: 14 and 26.

2. In paragraph 1, The antibody or antigen-binding fragment comprises at least one of the following antibodies or antigen-binding fragments: 1) an antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 12, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 14; and 2) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 17, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 19, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 21; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 12, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO:

26.

3. In paragraph 1, An antibody or antigen-binding fragment comprising a heavy chain variable region comprising at least one amino acid sequence of SEQ ID NOs: 38 and 42; and a light chain variable region comprising at least one amino acid sequence of SEQ ID NOs: 39 and 43.

4. In paragraph 1, The antibody or antigen-binding fragment comprises at least one of the following antibodies or antigen-binding fragments: 1) an antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 38 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 39; and 2) An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 42 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO:

43.

5. In paragraph 1, The antigen binding fragment is an antibody or antigen binding fragment comprising a single chain antibody, Fd, Fab, Fab', F(ab')2, dsFv or scFv.

6. In paragraph 1, The antibody or antigen-binding fragment thereof comprises a polyclonal antibody, a monoclonal antibody, a whole antibody, a chimeric antibody, a human antibody, a humanized antibody, a bivalent, a bispecific molecule, a minibody, a domain antibody, a bispecific or multispecific antibody, an immune cell engaging bispecific or multispecific antibody, an antibody mimetic, a unibody, a diabody, a triabody or a tetrabody.

7. It binds specifically to mesothelin (MSLN). An antibody or antigen-binding fragment comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 29; a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5; a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 32; a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10; a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 12; and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO:

14.

8. In paragraph 7, An antibody or antigen-binding fragment further comprising a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 28; a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 30; a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 31; a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 33; a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 34; a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 35; a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 36; and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO:

37.

9. In paragraph 7, An antibody or antigen-binding fragment comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 46; and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO:

47.

10. In paragraph 7, An antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment.

11. In paragraph 7, The antigen binding fragment is an antibody or antigen binding fragment comprising a single chain antibody, Fd, Fab, Fab', F(ab')2, dsFv or scFv.

12. A nucleic acid molecule encoding an antibody or antigen-binding fragment of any one of claims 1 to 11.

13. A recombinant expression vector comprising the nucleic acid molecule of claim 12.

14. A host cell transfected with the expression vector of item 13.

15. An immunoconjugate comprising an antibody or antigen-binding fragment of any one of claims 1 to 11 and a cytotoxic agent.

16. A pharmaceutical composition for preventing or treating a mesothelin-related disease, comprising an antibody or antigen-binding fragment of any one of claims 1 to 11 as an active ingredient.

17. In paragraph 16, A pharmaceutical composition wherein the above mesothelin-related disease is cancer or a tumor.

18. A composition for diagnosing a mesothelin-related disease, comprising an antibody or antigen-binding fragment of any one of claims 1 to 11 as an active ingredient.

19. A chimeric antigen receptor (CAR) comprising an antigen binding domain that specifically binds to mesothelin (MSLN). A chimeric antigen receptor comprising an antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain CDR1 comprising at least one amino acid sequence of SEQ ID NOs: 3 and 17; a heavy chain CDR2 comprising at least one amino acid sequence of SEQ ID NOs: 5 and 19; and a heavy chain CDR3 comprising at least one amino acid sequence of SEQ ID NOs: 7 and 21; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 10; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12; and a light chain CDR3 comprising at least one amino acid sequence of SEQ ID NOs: 14 and 26.

20. A chimeric antigen receptor (CAR) comprising an antigen binding domain that specifically binds to mesothelin (MSLN). A chimeric antigen receptor, wherein the antigen binding domain comprises an antigen binding fragment comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 29; a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5; a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 32; a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 10; a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 12; and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO:

14.

21. An immune effector cell expressing the chimeric antigen receptor of claim 19 or 20.

22. A pharmaceutical composition for preventing or treating a mesothelin-related disease, comprising the immune effector cell of Article 21 as an active ingredient.

23. In paragraph 22, A pharmaceutical composition wherein the above mesothelin-related disease is cancer or a tumor.

24. A method for preventing or treating a mesothelin-related disease, comprising administering a pharmaceutical composition of claim 16 or claim 22 in a pharmaceutically effective amount to a subject in need of administration.

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