Antibody against human mesothelin and use thereof

Abstract

The present invention relates to an antibody that specifically binds to mesothelin and use thereof. In an aspect of the present invention, it was confirmed that an anti-MSLN antibody or antigen-binding fragment thereof comprising a CDR consisting of a specific amino acid sequence binds to MSLN, which is an antigen, with very high specificity, and the anti-MSLN antibody or an antigen-binding fragment thereof can be used not only as a therapeutic agent capable of delivering a drug through cell internalization, but also as an anti-MSLN antibody-drug conjugate capable of killing cancer cells by targeting MSLN. Therefore, the anti-MSLN antibody or an antigen-binding fragment thereof, according to an aspect of the present invention, and a composition comprising same have an excellent effect of preventing or treating cancer caused by MSLN overexpression or predicting or diagnosing the cancer.

Description

Antibody against human mesothelin and its uses

[0001] The present specification discloses an antibody that specifically binds to human mesothelin, a nucleic acid encoding said antibody, a recombinant expression vector comprising said nucleic acid, a cell transformed with said recombinant expression vector, a method for producing said antibody, an antibody-drug conjugate comprising said antibody and a drug, a bispecific antibody comprising said antibody, a composition for preventing or treating cancer comprising said antibody, a composition for predicting or diagnosing cancer comprising said antibody, a kit, and a method for providing information.

[0002] The present invention relates to an antibody against human mesothelin (MSLN) or an antigen-binding fragment thereof, a nucleic acid encoding the same, a vector comprising said nucleic acid, a cell transformed with said vector, a method for preparing said antibody or said antigen-binding fragment, an antibody-drug conjugate (ADC) comprising the same, a pharmaceutical composition for treating cancer, a composition for diagnosing cancer, a chimeric antigen receptor (CAR) comprising the same, and a bispecific antibody.

[0003] Mesothelin (MSLN) is a tumor differentiation antigen present in normal mesothelial cells and overexpressed in various human tumors, including mesothelioma, ovarian, and pancreatic adenocarcinomas. Mesothelin has a molecular weight of 40 kDa and is attached to the cell membrane by a glycosylphosphatidyl inositol linkage (GPI), forming a precursor protein with a shed fragment of about 31 kDa called megakaryocyte-potentiating factor (MPF) at its N-terminal region. There are soluble forms of SMRP (soluble mesothelin-related peptides) that are not bound to cells, and since antibodies targeting mesothelin may bind to SMRP, anti-MSLN antibodies are required to have excellent specificity that binds specifically only to cell membrane-expressed mesothelin (MSLN) rather than SMRP.

[0004] Meanwhile, a common problem encountered in cancer treatment is that the limited therapeutic index of chemotherapy drugs restricts their therapeutic utility as they cause significant toxicity to normal tissues. One approach to achieving higher specificity for targeting cancer cells is to use antibodies to deliver cytotoxic effects to cells expressing specific tumor-specific antigens, while preserving normal cells that do not express the specific tumor-specific antigens at all or express them at much lower levels. By utilizing this tumor-specific targeting, anti-tumor activity can be increased, or the off-target cytotoxicity of the therapeutic agent can be reduced. Antibodies targeting tumor-specific antigens can deliver cytotoxic effects through various mechanisms, including inhibition of the biological activity of the antigen, induction of immune effector activity, and / or induction of cytotoxicity in antibody-dependent cells.

[0005] The selection of tumor-specific antigens for antibody-based therapeutic approaches may involve the specific expression of the antigen by tumor cells and the potent killing of the antigen-expressing tumor cells. While mesothelin is expressed in limited normal tissues, it is overexpressed in the aforementioned mesothelioma, ovarian cancer, and pancreatic cancer, as well as several other solid tumors, and its clinical relevance has been reported, leading to its consideration as a potential target for anticancer therapeutics.

[0006] Therefore, antibodies that specifically bind to human mesothelin are required.

[0007] In one aspect, the object of the present invention is to provide an anti-MSLN antibody that specifically binds to human mesothelin (MSLN) or an antigen-binding fragment thereof.

[0008] In another aspect, the object of the present invention is to provide a nucleic acid encoding the anti-MSLN antibody or its antigen-binding fragment.

[0009] In another aspect, the object of the present invention is to provide a recombinant expression vector comprising the nucleic acid.

[0010] In another aspect, the object of the present invention is to provide cells transformed with the recombinant expression vector.

[0011] In another aspect, the object of the present invention is to provide a method for producing the anti-MSLN antibody or its antigen-binding fragment.

[0012] In another aspect, the object of the present invention is to provide an antibody-drug conjugate comprising the anti-MSLN antibody or its antigen-binding fragment and a drug.

[0013] In another aspect, the object of the present invention is to provide a bispecific antibody comprising the anti-MSLN antibody or an antigen-binding fragment thereof.

[0014] In another aspect, the object of the present invention is to provide a composition for the prevention or treatment of cancer comprising the anti-MSLN antibody or its antigen-binding fragment, the antibody-drug conjugate or the bispecific antibody.

[0015] In another aspect, the object of the present invention is to provide a composition for cancer prediction or diagnosis comprising the anti-MSLN antibody or an antigen-binding fragment thereof.

[0016] In another aspect, the object of the present invention is to provide a kit for cancer prediction or diagnosis comprising the above composition.

[0017] In another aspect, the object of the present invention is to provide a method for providing information for cancer prediction or diagnosis, comprising the step of treating a sample of a subject with the anti-MSLN antibody or an antigen-binding fragment thereof.

[0018] In another aspect, the object of the present invention is to provide a chimeric antigen receptor (CAR) comprising the anti-MSLN antibody or an antigen-binding fragment thereof.

[0019] In one aspect, the present invention provides an anti-MSLN antibody or an antigen-binding fragment thereof comprising complementarity determining regions (CDRs) of a heavy chain CDR1 having an amino acid sequence of SEQ ID NO. 2 or 16, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO. 4 or 18, a heavy chain CDR3 having an amino acid sequence of SEQ ID NO. 6 or 20, a light chain CDR1 having an amino acid sequence of SEQ ID NO. 9 or 23, a light chain CDR2 having an amino acid sequence of SEQ ID NO. 11 or 25, and a light chain CDR3 having an amino acid sequence of SEQ ID NO. 13 or 27.

[0020] In another aspect, the present invention provides a nucleic acid encoding the anti-MSLN antibody or its antigen-binding fragment.

[0021] In another aspect, the present invention provides a recombinant expression vector comprising the nucleic acid.

[0022] In another aspect, the present invention provides a cell transformed with the recombinant expression vector.

[0023] In another aspect, the present invention provides a method for producing an anti-MSLN antibody or an antigen-binding fragment thereof, comprising the steps of: culturing the cells; and recovering an antibody that specifically binds to MSLN or an antigen-binding fragment thereof from the cultured cells.

[0024] In another aspect, the present invention provides an antibody-drug conjugate comprising the anti-MSLN antibody or its antigen-binding fragment and a drug.

[0025] In another aspect, the present invention provides a bispecific antibody comprising the anti-MSLN antibody or an antigen-binding fragment thereof.

[0026] In another aspect, the present invention provides a composition for the prevention or treatment of cancer comprising the anti-MSLN antibody or its antigen-binding fragment, the antibody-drug conjugate or the bispecific antibody.

[0027] In another aspect, the present invention provides a composition for cancer prediction or diagnosis comprising the anti-MSLN antibody or an antigen-binding fragment thereof.

[0028] In another aspect, the present invention provides a method for providing information for cancer prediction or diagnosis, comprising the step of treating a sample of a subject with the anti-MSLN antibody or an antigen-binding fragment thereof.

[0029] In another aspect, the present invention provides a chimeric antigen receptor (CAR) comprising the anti-MSLN antibody or an antigen-binding fragment thereof.

[0030] In one aspect, the present invention has confirmed that an anti-MSLN antibody or an antigen-binding fragment thereof comprising a complementary determining region (CDR) having a heavy chain CDR1 with an amino acid sequence of SEQ ID NO. 2 or 16, a heavy chain CDR2 with an amino acid sequence of SEQ ID NO. 4 or 18, a heavy chain CDR3 with an amino acid sequence of SEQ ID NO. 6 or 20, a light chain CDR1 with an amino acid sequence of SEQ ID NO. 9 or 23, a light chain CDR2 with an amino acid sequence of SEQ ID NO. 11 or 25, and a light chain CDR3 with an amino acid sequence of SEQ ID NO. 13 or 27 binds to MSLN, which is an antigen, with very high specificity, and that the anti-MSLN antibody or the antigen-binding fragment thereof can be utilized not only as a therapeutic agent capable of delivering a drug through cell internalization, but also as an anti-MSLN antibody-drug conjugate capable of killing cancer cells by targeting MSLN. Accordingly, the anti-MSLN antibody or the antigen-binding fragment thereof according to one aspect of the present invention, and the antibody comprising The composition has excellent effects in preventing or treating cancer associated with the expression or overexpression of MSLN, or in predicting or diagnosing said cancer.

[0031] Figure 1 shows the results of confirming the purity of purified proteins of recombinant human, mouse, and monkey mesothelin (MSLN) prepared for use as antigens according to one embodiment of the present invention using SDS-PAGE (Figure 1A) and SEC-HPLC (Figure 1B) (Reducing condition; Non-reducing condition). In addition, after transducing (hMSLN, rhMSLN) or temporarily expressing (mMSLN) these three types of proteins into human pancreatic cancer cell lines (MIA PaCa-2) without MSLN expression, the results of confirming MSLN expression on the cell surface using a flow cytometer (Figure 1C).

[0032] Figure 2 is a representative result confirming the binding ability of monophage clones expressing scFv to human MSLN according to one embodiment of the present invention to human MSLN using an ELISA method.

[0033] Figure 3 shows the results of confirming the purity of the antibodies purified after converting the anti-MSLN monoclone according to one embodiment of the present invention into an IgG form using SDS-PAGE (Figure 3A) and SEC-HPLC (Figure 3B), where Amatuximab and Anetumab are control antibodies.

[0034] Figure 4 shows the results of confirming the cell binding specificity of a selected anti-MSLN monoclonal antibody according to one embodiment of the present invention using a flow cytometer with an MIA PaCa-2 cell line that does not express human MSLN (Fig. 4A) and an MIA PaCa-2 / hMSLN cell line that overexpresses human MSLN (Fig. 4B).

[0035] FIG. 5 shows the binding affinity (K) of a selected anti-MSLN monoclonal antibody to human MSLN according to one embodiment of the present invention. D This is the result of confirming ) by ELISA.

[0036] Figure 6 shows the results of confirming the interspecies cross-linking and cell binding affinity of a selected anti-MSLN monoclonal antibody according to one embodiment of the present invention using a flow cytometer with human (Fig. 6A), mouse (Fig. 6B), monkey (Fig. 6C), and human pancreatic cancer cell line (MIA PaCa-2 / MSLN) of Fig. 1C, which is overexpressed with MSLN.

[0037] Figure 7 is a result showing the amount of intracellular antibody inflow over time (Figure 7A) and the degree of intracellular antibody accumulation over 24 hours (Figure 7B) using a real-time image analysis device for the cellular internalization of a selected anti-MSLN monoclonal antibody according to one embodiment of the present invention in a human MSLN overexpressing cell line (MIA PaCa-2 / hMSLN).

[0038] Figure 8 shows the results of confirming the toxin-induced cancer cell death effect of a selected anti-MSLN monoclonal antibody according to one embodiment of the present invention in MIA PaCa-2 cells without human MSLN expression (Fig. 8A) or MIA PaCa-2 / hMSLN cell lines with MSLN overexpression (Fig. 8B).

[0039] In one aspect, the present invention provides an anti-MSLN antibody or an antigen-binding fragment thereof comprising complementarity determining regions (CDRs) of a heavy chain CDR1 having an amino acid sequence of SEQ ID NO. 2 or 16, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO. 4 or 18, a heavy chain CDR3 having an amino acid sequence of SEQ ID NO. 6 or 20, a light chain CDR1 having an amino acid sequence of SEQ ID NO. 9 or 23, a light chain CDR2 having an amino acid sequence of SEQ ID NO. 11 or 25, and a light chain CDR3 having an amino acid sequence of SEQ ID NO. 13 or 27.

[0040] The present invention will be described in detail below.

[0041] In one aspect of the present invention, "mesothelin (MSLN)" is a collective term for any variants, isoforms, and species homologs of MSLN that are naturally expressed by cells. Specifically, it may refer to human MSLN, but is not limited thereto, and may include MSLN of other mammalian animals.

[0042] According to one aspect of the present invention, an anti-MSLN antibody or an MSLN protein acting as an antigen of its antigen-binding fragment is attached to the cell membrane of a mesothelial cell, and the MSLN may be derived from mammals such as humans, primates such as monkeys, rodents such as mice and rats.

[0043]

[0044] In one aspect, the present invention provides an anti-MSLN antibody or an antigen-binding fragment thereof comprising the following complementarity determining regions (CDRs): a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO. 2 or 16, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO. 4 or 18, a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO. 6 or 20, a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO. 9 or 23, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO. 11 or 25, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO. 13 or 27.

[0045] An anti-MSLN antibody or an antigen-binding fragment thereof according to one aspect of the present invention may specifically bind to the amino acid sequence or a part thereof of a human MSLN protein (Uniprot accession No. Q13421-3), a mouse MSLN (Uniprot accession No. Q61468-1), or a monkey MSLN (Uniprot accession No. F6Q1U7), but is not limited thereto.

[0046] An antibody according to one aspect of the present invention refers to an anti-MSLN antibody that specifically binds to MSLN. An anti-MSLN antibody according to one aspect of the present invention comprises not only a complete antibody form that specifically binds to MSLN, but also an antigen-binding fragment of said antibody molecule.

[0047] A complete antibody according to one aspect of the present invention has a structure having two full-length light chains and two full-length heavy chains, each light chain being connected to the heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and has gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2) subclasses. The light chain constant region has kappa (κ) and lambda (λ) types.

[0048] According to one aspect of the present invention, an "antigen-binding fragment" or "antibody fragment" of an antibody refers to a fragment possessing an antigen-binding function, and said antibody or antigen-binding fragment may be scFv, (scFv)2, Fab, Fab', or F(ab')2. Among said antibody fragments, Fab is a structure having a variable region of the light chain and heavy chain, a constant region of the light chain, and a first constant region (CH1) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 is generated when cysteine ​​residues in the hinge regions of two Fab's form disulfide bonds. Fv refers to a minimal antibody fragment having only a heavy chain variable region and a light chain variable region. In two-chain Fv, the variable region of the heavy chain and the variable region of the light chain are connected by non-covalent bonds, and in single-chain Fv (scFv), the variable region of the heavy chain and the variable region of the light chain are generally connected by covalent bonds through peptide linkers or directly at the C-terminus, so they can form a structure similar to a dimer, like two-chain Fv.

[0049] An antigen-binding fragment or antibody fragment according to one aspect of the present invention can be obtained using a proteolytic enzyme and can also be produced through genetic recombination technology.

[0050] An antibody according to one aspect of the present invention is in the Fv form (e.g., scFv) or in the complete antibody form. Additionally, the heavy chain constant region may be any one of the isotypes gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε). For example, the constant region is gamma 1 (IgG1), gamma 3 (IgG3), or gamma 4 (IgG4). The light chain constant region may be in the kappa or lambda form.

[0051] An antibody according to one aspect of the present invention consists of a complete human antibody sequence. The term "human antibody" refers to a molecule derived from human immunoglobulin, wherein the entire amino acid sequence constituting the antibody, including a complementation determining region and a structural region, is composed of human immunoglobulin. If necessary, the antibody according to one aspect of the present invention may be modified into various forms, such as humanized antibodies and chimeric antibodies, according to methods known in the art.

[0052] According to one aspect of the present invention, an "antibody variable domain" refers to a light chain and heavy chain portion of an antibody molecule comprising the amino acid sequences of a Complementarity Determining Region (CDR) and a Framework Region (FR). H refers to the mutable domain of the heavy chain. V L ... refers to the variable domain of the light chain.

[0053] An anti-MSLN antibody or an antigen-binding fragment thereof according to one aspect of the present invention may comprise a complementary determining region (CDRs) consisting of a heavy chain CDR1 with an amino acid sequence of SEQ ID NO. 2 or 16, a heavy chain CDR2 with an amino acid sequence of SEQ ID NO. 4 or 18, a heavy chain CDR3 with an amino acid sequence of SEQ ID NO. 6 or 20, a light chain CDR1 with an amino acid sequence of SEQ ID NO. 9 or 23, a light chain CDR2 with an amino acid sequence of SEQ ID NO. 11 or 25, and a light chain CDR3 with an amino acid sequence of SEQ ID NO. 13 or 27.

[0054] According to one aspect of the present invention, "complementary determining regions" (CDRs; i.e., CDR1, CDR2, and CDR3) refer to amino acid residues of an antibody variable domain that are present for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2, and CDR3 on the heavy chain and light chain, respectively.

[0055] An antibody according to one aspect of the present invention comprises a heavy chain FR1 having 90% or more homology with the amino acid sequence of SEQ ID NO. 1 or 15, a heavy chain FR2 having 90% or more homology with the amino acid sequence of SEQ ID NO. 3 or 17, a heavy chain FR3 having 90% or more homology with the amino acid sequence of SEQ ID NO. 5 or 19, a heavy chain FR4 having 90% or more homology with the amino acid sequence of SEQ ID NO. 7 or 21, a light chain FR1 having 90% or more homology with the amino acid sequence of SEQ ID NO. 8 or 22, a light chain FR2 having 90% or more homology with the amino acid sequence of SEQ ID NO. 10 or 24, a light chain FR3 having 90% or more homology with the amino acid sequence of SEQ ID NO. 12 or 26, and a sequence having 90% or more homology with the amino acid sequence of SEQ ID NO. 14 or 28. It may include framework regions (FRs) of light chain FR4.

[0056] According to one aspect of the present invention, a “framework region” (FR) is a variable domain residue other than a CDR residue, and each variable domain typically has four FRs identified as FR1, FR2, FR3, and FR4 in each of the heavy chain and light chain.

[0057] An antibody according to one aspect of the present invention may comprise the following sets of CDRs (i) or (ii): (i) heavy chain CDR1 of SEQ ID NO. 2, heavy chain CDR2 of SEQ ID NO. 4, heavy chain CDR3 of SEQ ID NO. 6, light chain CDR1 of SEQ ID NO. 9, light chain CDR2 of SEQ ID NO. 11, and light chain CDR3 of SEQ ID NO. 13; and (ii) heavy chain CDR1 of SEQ ID NO. 16, heavy chain CDR2 of SEQ ID NO. 18, heavy chain CDR3 of SEQ ID NO. 20, light chain CDR1 of SEQ ID NO. 23, light chain CDR2 of SEQ ID NO. 25, and light chain CDR3 of SEQ ID NO. 27.

[0058] Specifically, an antibody according to one aspect of the present invention may comprise a combination of CDR and FR of any one of (a) and (b) below: (a) heavy chain CDR1 of SEQ ID NO. 2, heavy chain CDR2 of SEQ ID NO. 4, heavy chain CDR3 of SEQ ID NO. 6, light chain CDR1 of SEQ ID NO. 9, light chain CDR2 of SEQ ID NO. 11, and light chain CDR3 of SEQ ID NO. 13; and a heavy chain FR1 consisting of a sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO. 1, a heavy chain FR2 consisting of a sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO. 3, a heavy chain FR3 consisting of a sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO. 5, a heavy chain FR4 consisting of a sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO. 7, a light chain FR1 consisting of a sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO. 8, a light chain FR2 consisting of a sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO. 10, a light chain FR3 consisting of a sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO. 12, and a light chain FR4 consisting of a sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO. 14; and (b) heavy chain CDR1 of SEQ ID NO. 16, heavy chain CDR2 of SEQ ID NO. 18, heavy chain CDR3 of SEQ ID NO. 20, light chain CDR1 of SEQ ID NO. 12, light chain CDR2 of SEQ ID NO. 25, and light chain CDR3 of SEQ ID NO. 27;and a heavy chain FR1 consisting of a sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO. 15, a heavy chain FR2 consisting of a sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO. 17, a heavy chain FR3 consisting of a sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO. 19, a heavy chain FR4 consisting of a sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO. 21, a light chain FR1 consisting of a sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO. 22, a light chain FR2 consisting of a sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO. 24, a light chain FR3 consisting of a sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO. 26, and a light chain FR4 consisting of a sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO. 28.;

[0059] An antibody according to one aspect of the present invention may include a heavy chain variable region composed of the amino acid sequence of SEQ ID NO. 29 or 31, and a light chain variable region composed of the amino acid sequence of SEQ ID NO. 30 or 32.

[0060] An anti-MSLN antibody or its antigen-binding fragment according to one aspect of the present invention may comprise an antibody or its antigen-binding fragment in which a portion of the amino acid sequence is substituted through a conservative substitution in the anti-MSLN antibody or its antigen-binding fragment according to the present invention.

[0061] The term "conservative substitution" above refers to a modification of a polypeptide comprising substituting one or more amino acids with amino acids having similar biochemical properties that do not cause a loss of the biological or biochemical function of the polypeptide. "Conservative amino acid substitution" is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain. A class of amino acid residues having a similar side chain is defined in the relevant art and is well known. These classes are amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0062] An antibody according to one aspect of the present invention can still retain activity even if it has a conservative amino acid substitution as described above.

[0063]

[0064] In another aspect, the present invention provides a nucleic acid encoding the anti-MSLN antibody or its antigen-binding fragment. Descriptions of the MSLN, antibody, antigen-binding fragment, and anti-MSLN antibody in the anti-MSLN antibody or its antigen-binding fragment may be applied to the nucleic acid.

[0065] The nucleic acid according to one aspect of the present invention may be present in cells or cell lysates, or in a partially purified or substantially pure form. The nucleic acid is “isolated” or “substantially purified” when purified from other cellular components or other contaminants, e.g., nucleic acids or proteins of other cells, by standard techniques including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other methods well known in the art. The nucleic acid according to one aspect of the present invention may be, for example, DNA or RNA, and may or may not contain intron sequences. The nucleotides, which are the basic building blocks of the nucleic acid, may include natural nucleotides as well as analogues in which sugar or base sites are modified. The sequence of the nucleic acid encoding the heavy chain and light chain variable regions according to one aspect of the present invention may be modified. Such modification may include the addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0066] A nucleic acid encoding an anti-MSLN antibody according to one aspect of the present invention may include a nucleic acid encoding a heavy chain variable region consisting of a nucleic acid sequence represented by either SEQ ID NO. 33 or 34, and a nucleic acid encoding a light chain variable region consisting of a nucleic acid sequence represented by either SEQ ID NO. 35 or 36.

[0067] Considering the modifications having the aforementioned biological equivalent activity, the antibody or nucleic acid molecule encoding it according to one aspect of the present invention is interpreted to include a sequence that exhibits substantial identity with the sequence described in SEQ ID NO. The said substantial identity may include a sequence that exhibits 90% or more homology, specifically 95% or more homology, more specifically 96% or more, 97% or more, 98% or more, or 99% or more homology when any other sequence is arranged to correspond as much as possible with the sequence according to one aspect of the present invention and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0068] The above homology may be determined by sequence comparison and / or alignment by methods known in the art. For example, the sequence homology of the nucleic acid or protein of the present invention may be determined using a sequence comparison algorithm (e.g., NCBI Basic Local Alignment Search Tool; BLAST), manual alignment, visual inspection, etc.

[0069]

[0070] In another aspect, the present invention provides a recombinant expression vector comprising a nucleic acid encoding the anti-MSLN antibody or its antigen-binding fragment. Descriptions of the MSLN, antibody, antigen-binding fragment, and anti-MSLN antibody in the anti-MSLN antibody or its antigen-binding fragment, and descriptions of the nucleic acid in the nucleic acid may be applied to the recombinant expression vector.

[0071] For the expression of an anti-MSLN antibody or an antigen-binding fragment thereof according to one aspect of the present invention, DNA encoding a partial or full-length light chain and a heavy chain may be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma expressing a target antibody), and the DNA may be “linked to operate” to transcription and translation control sequences and inserted into an expression vector. The vector components may include, but are not limited to, one or more of the following: a signal sequence, a replication origin, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0072] A “vector” according to one aspect of the present invention may include a plasmid vector; a cosmid vector; and viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors, as a means for expressing a target gene in a host cell. In the vector, a nucleic acid encoding an antibody or an antigen-binding fragment thereof may be operatively linked to a promoter.

[0073] According to one aspect of the present invention, "operationally linked" means that a gene encoding an antibody or its antigen-binding fragment is ligated into a vector so that a transcription and translation control sequence within the vector performs the intended function of regulating the transcription and translation of the antibody gene. The expression vector and the expression control sequence may be selected to be compatible with the expression cells used. According to one aspect of the present invention, the light chain gene and the heavy chain gene of the antibody may be inserted into separate vectors, or both genes may be inserted into the same expression vector. The antibody gene may be inserted into the expression vector by standard methods (e.g., ligation of the antibody gene fragment and the complementary restriction enzyme site on the vector, or blunt end ligation if no restriction enzyme site is present).

[0074] Alternatively, a recombinant expression vector according to one aspect of the present invention may include a sequence encoding a signal peptide that facilitates the secretion of an antibody chain from a transformed cell. The antibody chain gene and the signal peptide-coding sequence may be cloned into a vector in a frame such that the signal peptide binds to the amino terminus of the antibody chain and is expressed. The signal peptide may be an immunoglobulin signal peptide or a heterogeneous signal peptide (i.e., a signal peptide derived from a protein other than immunoglobulin). Additionally, the recombinant expression vector may include a regulatory sequence that controls the expression of the antibody chain gene in the transformed cell. The "regulatory sequence" may include a promoter, enhancer, and other expression control elements (e.g., a polyadenylation signal) that control the transcription or translation of the antibody chain gene. A person skilled in the art may recognize that the design of the expression vector may vary by selecting a different regulatory sequence depending on factors such as the selection of the cell to be transformed and the expression level of the protein.

[0075] A recombinant expression vector according to one aspect of the present invention may also include other sequences to be fused to an antibody gene to facilitate the purification of the antibody expressed from the vector. These sequences may be, for example, genes such as glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), 6x His(hexahistidine; Quiagen, USA). The vector may include antibiotic resistance genes commonly used in the art as selection markers, such as genes for resistance to ampicillin, gentamicin, cabbageillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0076]

[0077] In another aspect, the present invention provides cells transformed with the recombinant expression vector. The description of the anti-MSLN antibody or its antigen-binding fragment, the nucleic acid, MSLN in the recombinant expression vector, the antibody, the antigen-binding fragment, the anti-MSLN antibody, the nucleic acid, and the recombinant expression vector may be applied to the cells.

[0078] Cells according to one aspect of the present invention may include animal cells, plant cells, yeast, E. coli, and insect cells, but are not limited thereto.

[0079] Specifically, according to one aspect of the present invention, the cell may be a prokaryotic cell including Escherichia coli, Bacillus subtilis, Streptomyces p., Pseudomonas sp., Proteus mirabilis, and Staphylococcus sp. Alternatively, according to one aspect of the present invention, the cell may be a fungus such as Aspergillus sp., a lower eukaryotic cell including Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces p., and Neurospora crassa, or a eukaryotic cell such as a cell of a higher eukaryote (e.g., an insect).

[0080] Alternatively, cells according to one aspect of the present invention may be derived from plants or mammals. For example, COS-7 (monkey kidney cells-7), BHK (baby hamster kidney), CHO (Chinese hamster ovary), CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS0, U20S, or HT1080 may be used, but are not limited thereto. Specifically, the cells may be human HEK293F cells, but are not limited thereto.

[0081] Various cell / vector combinations may be used to express an anti-MSLN antibody according to one aspect of the present invention. Specifically, expression vectors suitable for eukaryotic cells include, but are not limited to, expression vectors derived from SV40, bovine papillomavirus, anenovirus, adeno-associated virus, cytomegalovirus, and retrovirus. Expression vectors suitable for use in bacterial cells may include E. coli-derived bacterial plasmids such as pET, pRSET, pBluescript, pGEX2T, pUC, col E1, pCR1, pBR322, pMB9, and derivatives thereof; plasmids having a broader host range such as RP4; phage DNA such as various phage lambda derivatives such as λgt10, λgt11, and NM989; and other DNA phages such as M13 and filamentous single-stranded DNA phages. Expression vectors useful for yeast cells may include the YEp plasmid and its derivatives. Vectors useful for insect cells may include pVL941.

[0082] The above vector can be transfected or transfected into cells. Various techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic cells for "transfection" or "transfection," such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection, may be used.

[0083]

[0084] In another aspect, the present invention provides a method for producing an anti-MSLN antibody or an antigen-binding fragment thereof, comprising the steps of: culturing the transformed cells; and recovering an antibody that specifically binds to MSLN or an antigen-binding fragment thereof from the cultured cells. Descriptions of the anti-MSLN antibody or the antigen-binding fragment thereof, the nucleic acid, the recombinant expression vector, MSLN in the transformed cells, the antibody, the antigen-binding fragment, the anti-MSLN antibody, the nucleic acid, the recombinant expression vector, the transformation, and the cell may be applied to the method of production.

[0085] When a recombinant expression vector capable of expressing an anti-MSLN antibody or an antigen-binding fragment thereof according to one aspect of the present invention is introduced into a mammalian cell, said antibody or its antigen-binding fragment can be produced by culturing the cell for a period sufficient to cause the antibody to be expressed in the cell, or specifically for a period sufficient to cause the antibody to be secreted into a culture medium in which the cell is cultured.

[0086] The above cells may be cultured in various media, and commercially available media may be used as culture media without limitation. All other essential supplements known to those skilled in the art may also be included in appropriate concentrations. Suitable culture conditions for protein expression in selected host cells, such as temperature and pH, are already in use and will be obvious to those skilled in the art.

[0087] Alternatively, depending on the case, the expressed antibody can be separated from the cell culture medium and uniformly purified. The separation or purification of the antibody may be performed by a conventional protein separation and purification method, for example, by chromatography. The chromatography may include, for example, affinity chromatography using a protein A column or protein G column, ion exchange chromatography, hydrophobic chromatography, or hydroxyapatite chromatography. In addition to the chromatography, the antibody may be separated and purified by combining filtration, ultrafiltration, salting out, dialysis, etc.

[0088]

[0089] In another aspect, the present invention provides an antibody-drug conjugate (ADC) comprising an anti-MSLN antibody or an antigen-binding fragment thereof, and a drug. The description of the MSLN, antibody, antigen-binding fragment, and anti-MSLN antibody in the anti-MSLN antibody or its antigen-binding fragment thereof may be applied to the antibody-drug conjugate.

[0090] In an antibody-drug conjugate according to one aspect of the present invention, the anticancer drug must remain stably bound to the antibody until the anticancer drug is delivered to target cancer cells. The drug delivered to the target must be released from the antibody to induce apoptosis of the target cells. To this end, the drug must be stably bound to the antibody and, at the same time, possess sufficient cytotoxicity to induce apoptosis of the target cells when released from them.

[0091] The above-mentioned drug refers to a compound that exhibits a therapeutic effect on target cells, which is a preparation exhibiting a pharmacological effect and may be bound to an antibody or its antigen-binding fragment according to one aspect of the present invention, and may be separated from said antibody or its antigen-binding fragment by acidic conditions. The above-mentioned drug may be one or more selected from the group consisting of immunomodulators, cytotoxic agents, chemotherapy agents, proteolytic agents (TPDs), and antibody-oligonucleotide conjugates (AOCs), or the above-mentioned drug may be a cytotoxin, a radioisotope, an antiproliferative agent, a pro-apoptotic agent, a chemotherapy agent, and a therapeutic nucleic acid, but is not limited thereto.

[0092] An antibody-drug conjugate according to one aspect of the present invention can be internalized into a cell and can mediate antibody-dependent cytotoxicity.

[0093] In one aspect of the present invention, "cytotoxic activity" refers to the effect of an antibody-drug conjugate or an intracellular metabolite of an antibody-drug conjugate on cell death, inhibition of cell proliferation, or inhibition of growth. Cytotoxic activity is defined as the IC50, which is the concentration (molar or mass) per unit volume at which half of the cells survive. 5O It can be expressed as a value.

[0094] In one aspect of the present invention, the term “cytotoxic” generally refers to a preparation that inhibits or prevents the function of a cell and / or destroys a cell. Representative cytotoxics may include antibiotics, tubulin polymerization inhibitors, alkylating agents that bind to and destroy DNA, and preparations that disrupt the function or protein synthesis of essential cellular proteins such as protein kinases, phosphatases, topoisomerases, enzymes, and cyclins. Examples of cytotoxins include Taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, and procaine. Tetracaine, lidocaine, propranolol, and puromycin and their analogs or homologues are included, but not limited to, tetracaine, lidocaine, propranolol, and puromycin.

[0095] For radiotherapy applications, an antibody according to one aspect of the present invention may comprise a high-energy radioisotope. The radioisotope may bind directly to the antibody, for example, at a cysteine ​​residue present within the antibody, or may mediate the binding of the antibody to the radioisotope using a chelate. Radioisotopes suitable for radiotherapy include, but are not limited to, α-emitted, β-emitted, and Auger electrons. Radioisotopes useful for diagnostic applications may include positron emitters and γ-emitted.

[0096] The above antiproliferative agents and apoptosis promoters include PPAR-gamma (e.g., cyclopenthenone prostaglandins (cyPGs)), retinoids, triterpenoids (e.g., cycloartan, lupan, uric acid, oleanan, friedelan, dammaran, cucurbitacin, and limonoid triterpenoids), inhibitors of EGF receptors (e.g., HER4), rapamycin, calcitriol (1,25-dihydroxycholecalciferol (vitamin D)), aromatase inhibitors (FEMARA® (Retrozone)), telomerase inhibitors, iron chelating agents (e.g., 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (Triaffin)), apoptin (viral protein 3 - VP3 from chicken anemia virus), inhibitors of Bcl-2 and Bcl-X(L), TNF-alpha, FAS ligands, TNF-related apoptosis-inducing ligands (TRAIL / Apo2L), activators of TNF-alpha / FAS ligand / TNF-related apoptosis-inducing ligand (TRAIL / Apo2L) signaling, and inhibitors of PI3K-Akt survival pathway signaling (e.g., UCN-01 and geldanamycin) may be included.

[0097] A “chemotherapeutic agent” according to one aspect of the present invention is a chemical compound useful for the treatment of cancer, regardless of its mechanism of action. The class of chemotherapeutic agents may include, but is not limited to, alkylating agents, metabolic antagonists, spindle-toxic plant alkaloids, cytotoxic / antitumor antibiotics, focisome isomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents may include compounds used in “targeted therapy” and traditional chemotherapy.

[0098] An antibody-drug conjugate according to one aspect of the present invention can be prepared by a known method by conjugating a drug to an antibody or its antigen-binding fragment. The antibody and the drug may be directly bound through their own linkers, or indirectly bound through a linker or other substance. Major mechanisms that cause the drug to be cleaved from the antibody may include hydrolysis of lysosomes (hydrazone, acetal, and cis-aconitate-like amides) at an acidic pH, peptide cleavage by lysosomal enzymes (cathepsin and other lysosomal enzymes), and reduction of disulfides. As a result of these various cleavage mechanisms, the mechanisms for linking the drug to the antibody are very diverse, and any suitable linker may be used.

[0099] Suitable linkers for binding an antibody and a drug according to one aspect of the present invention are well known in the art and may include, for example, a disulfide group, a thioether group, an acid-degradable group, a photodegradable group, a peptidase-degradable group, and an esterase-degradable group.

[0100] In one aspect of the present invention, when a drug is directly bound, the linking group may be, for example, a disulfide bond utilizing an SH group or a maleimide-mediated linkage. For example, the intramolecular disulfide bond of the antibody Fc region and the disulfide bond of the drug are reduced to connect the two via a disulfide bond. Additionally, there are methods involving maleimide and methods involving the genetic engineering introduction of cysteine ​​into the antibody.

[0101] In one aspect of the present invention, an antibody and a drug may be indirectly combined through another substance (linker). The linker may have one or more types of functional groups that react with the antibody, the drug, or both. Examples of functional groups include amino groups, carboxyl groups, mercapto groups, maleimide groups, pyridinyl groups, etc.

[0102] In another aspect, the present invention provides a bispecific antibody comprising an anti-MSLN antibody or an antigen-binding fragment thereof and a drug. The descriptions of the MSLN, antibody, antigen-binding fragment, and anti-MSLN antibody in the anti-MSLN antibody or its antigen-binding fragment may be applied to the bispecific antibody. A bispecific antibody is an antibody capable of simultaneously recognizing two different antigens and is characterized by having two different antigen-binding sites, unlike conventional monoclonal antibodies. For example, a bispecific antibody may bind to an MSLN through the first antigen-binding site to target cancer cells, and bind to other immune cells (e.g., CD3 on T cells) or cancer cell surface antigens through the second antigen-binding site.

[0103] In another aspect, the present invention provides a composition for the prevention or treatment of cancer comprising the anti-MSLN antibody or its antigen-binding fragment, the antibody-drug conjugate, or the bispecific antibody. Descriptions of MSLN, antibody, antigen-binding fragment, anti-MSLN antibody, drug, antibody-drug conjugate, bispecific antibody, and chimeric antigen receptor in the anti-MSLN antibody or its antigen-binding fragment, the antibody-drug conjugate, and the bispecific antibody may be applied to the composition for the prevention or treatment of cancer.

[0104] A cancer or tumor according to one aspect of the present invention may be associated with the expression or overexpression of MSLN, and specifically, may be pancreatic cancer, ovarian cancer, mesothelioma, biliary tract cancer, lung cancer, gastric cancer, colorectal cancer, cervical cancer, esophageal cancer, breast cancer, kidney cancer, or metastatic cancer derived from any of the above. The cancer may be a primary cancer or a metastatic cancer.

[0105] In one aspect of the present invention, "cancer" and "tumor" are used interchangeably and refer to or mean a physiological condition of mammals characterized by uncontrolled cell growth and proliferation.

[0106] In one aspect of the present invention, "prevention" means any act of inhibiting the occurrence of cancer or tumor or delaying its progression by administering a composition according to one aspect of the present invention, and "treatment" means inhibiting the development of cancer or tumor, or alleviating or eliminating cancer or tumor.

[0107] A composition for cancer prevention or treatment according to one aspect of the present invention may be a pharmaceutical composition, and said pharmaceutical composition may be characterized by comprising a therapeutically effective amount of an anti-MSLN antibody or an antigen-binding fragment thereof, an antibody-drug conjugate or a bispecific antibody, and a pharmaceutically acceptable additive or carrier.

[0108] The above "pharmaceuticalally acceptable carrier" is a substance that may be added to the active ingredient to help formulate or stabilize the preparation and does not cause significant toxic effects to the patient.

[0109] The above additives refer to carriers or diluents, etc., that do not irritate the patient and do not impair the biological activity and properties of the administered compound. As pharmaceutical carriers acceptable for compositions formulated as liquid solutions, sterile and biocompatible saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof may be used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Additionally, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets.

[0110] In one aspect of the present invention, a pharmaceutically acceptable carrier may comprise a sterile aqueous solution or dispersion and a sterile powder for preparing a sterile injectable solution or dispersion for extemporaneous administration. The composition may specifically be formulated for parenteral injection. The composition may be formulated as a solution, a microemulsion, a liposomal formulation, or other ordered formulations suitable for high drug concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. In some cases, an isotonic agent, for example, sugar, a polyalcohol, such as mannitol, sorbitol, or sodium chloride, may be included in the composition. Each formulation may be prepared using methods well known in the pharmaceutical field.

[0111] The dosage of a composition for preventing or treating cancer according to one aspect of the present invention is not particularly limited but may be varied depending on various factors including the patient's health condition and body weight, the severity of the disease, the type of drug, the route of administration, and the time of administration. A composition for preventing or treating cancer according to one aspect of the present invention may be administered once or multiple times a day in a single dose through various routes of oral or parenteral routes typically permitted in mammals, including humans, rats, mice, and livestock. Specifically, it may be administered in a conventional manner through oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intra-arterial, transdermal, intranasal, inhalation, intraocular, intrapulmonary, or intradermal routes, but is not limited thereto.

[0112] A composition for preventing or treating cancer according to one aspect of the present invention may be administered to a patient as a bolus or by continuous infusion as needed. For example, a bolus administration of an antigen-binding fragment of the anti-MSLN antibody of the present invention, represented by a Fab fragment, may be in an amount of 0.0025 to 100 mg / kg body weight, 0.025 to 0.25 mg / kg, 0.010 to 0.10 mg / kg, or 0.10 to 0.50 mg / kg. In the case of continuous infusion, the antigen-binding fragment of the anti-MSLN antibody of the present invention, represented by the Fab fragment, may be administered at a dose of 0.001 to 100 mg / kg body weight / min, 0.0125 to 1.25 mg / kg / min, 0.010 to 0.75 mg / kg / min, 0.010 to 1.0 mg / kg / min, or 0.10 to 0.50 mg / kg / min for a period of 1 hour to 24 hours, 1 hour to 12 hours, 2 hours to 12 hours, 6 hours to 12 hours, 2 hours to 8 hours, or 1 hour to 2 hours. When administering the anti-MSLN antibody or its antigen-binding fragment, antibody-drug conjugate, or bispecific antibody according to one aspect of the present invention, the dose may be about 1 to 10 mg / kg body weight, 2 to 8 mg / kg, or 5 to 6 mg / kg. Full-length anti-MSLN antibodies may typically be administered via an infusion lasting for a period of 30 to 35 minutes, but are not limited thereto. The frequency of administration depends on the severity of the condition. The frequency may range from 3 times per week to once every 1 or 2 weeks.

[0113] In some cases, cancer can be prevented or treated by effectively targeting tumor cells expressing or overexpressing MSLN by using an antibody or its antigen-binding fragment, antibody-drug conjugate, or bispecific antibody according to one aspect of the present invention in combination with other conventional anticancer agents. The antibody or its antigen-binding fragment, antibody-drug conjugate, or bispecific antibody may be used with other anti-neoplastic agents or immunogenic agents, for example, cells transfected with genes encoding attenuated cancer cells, tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), or antigen-transmitting cells (for example, dendritic cells pulsed with tumor-derived antigens or nucleic acids); standard cancer therapies, for example, chemotherapy, radiation therapy, or surgery; or other antibodies.

[0114] An anti-MSLN antibody or its antigen-binding fragment, antibody-drug conjugate or bispecific antibody, or a pharmaceutical composition containing the same according to one aspect of the present invention may be administered simultaneously with or sequentially with a conventional anticancer agent.

[0115]

[0116] In another aspect, the present invention provides a composition for cancer prediction or diagnosis comprising an anti-MSLN antibody or an antigen-binding fragment thereof. The descriptions of the anti-MSLN antibody or the antigen-binding fragment thereof, MSLN, antibody, antigen-binding fragment, anti-MSLN antibody, and cancer in the composition for cancer prevention or treatment may be applied to the composition for cancer prediction or diagnosis.

[0117] In one aspect of the present invention, "cancer prediction" may mean predicting or diagnosing whether there is a possibility of cancer developing in a subject, whether there is a relatively high probability of cancer developing, what the causative factors of cancer are, or whether cancer has already developed. Additionally, in one aspect of the present invention, "cancer diagnosis" means confirming the existence or characteristics of a pathological state in a subject, and for the purposes according to one aspect of the present invention, diagnosis may mean confirming whether cancer has developed. A composition, kit, or method according to one aspect of the present invention may be used to delay the onset of cancer or prevent it from developing in any specific subject, specifically an animal subject with a high risk of developing cancer, through special and appropriate management. Additionally, a composition, kit, or method according to one aspect of the present invention may be used clinically to determine treatment by diagnosing cancer early and selecting the most appropriate treatment method.

[0118] Cancer or tumors can be predicted or diagnosed by measuring the level of MSLN expression in a subject's sample using the anti-MSLN antibody or its antigen-binding fragment according to one aspect of the present invention. The expression level can be measured according to conventional immunoassay methods, and can be measured through radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, ELISA (enzyme-linked immunosorbent assay), capture-ELISA, inhibition or competition analysis, sandwich analysis, flow cytometry, immunofluorescence staining, and immunoaffinity purification using the antibody against MSLN, but is not limited thereto.

[0119] A composition for cancer prediction or diagnosis according to one aspect of the present invention may further include a label that enables quantitative or qualitative measurement of the formation of an antigen-antibody complex, a conventional tool used in immunological analysis, a reagent, etc.

[0120] In one aspect of the present invention, labels that enable qualitative or quantitative measurement of the formation of the antigen-antibody complex include, but are not limited to, enzymes, fluorescent agents, ligands, luminescent agents, microparticles, redox molecules, and radioisotopes. Enzymes available for use as detection labels include, but are not limited to, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphphenolpyruvate decarboxylase, β-latamase, etc. Fluorescent materials include, but are not limited to, fluorescein, isothiocyanates, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehydrides, fluorescarmine, etc. Ligands include, but are not limited to, biotin derivatives, etc. Luminescent materials include, but are not limited to, acridinium esters, luciferin, luciferase, etc. Microparticles include, but are not limited to, colloidal gold, colored latex, etc. Redox molecules include ferrocene, ruthenium complexes, viologen, quinone, Ti ions, Cs ions, diimides, 1,4-benzoquinone, hydroquinone, K4W(CN) 8 , [Os(bpy)3] 2+ , [RU(bpy)3] 2+ , [MO(CN)8] 4- These include, but are not limited to. Radioisotopes include 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, 186There are Re, etc., and are not limited to this.

[0121] In one aspect of the present invention, examples of the tool or reagent include, but are not limited to, suitable carriers, solvents, cleaning agents, buffers, stabilizers, etc. If the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction stopping agent. The carrier may be a soluble carrier or an insoluble carrier, and an example of a soluble carrier is a physiologically acceptable buffer known in the art, e.g., PBS, and an example of an insoluble carrier may be polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluoropolymer, cross-linked dextran, polysaccharide, other paper, glass, metal, agarose, and combinations thereof.

[0122] A cancer or tumor according to one aspect of the present invention may be associated with the expression or overexpression of MSLN, and specifically, may be pancreatic cancer, ovarian cancer, mesothelioma, biliary tract cancer, lung cancer, gastric cancer, colorectal cancer, cervical cancer, esophageal cancer, breast cancer, kidney cancer, or metastatic cancer derived from any of the above. The cancer may be a primary cancer or a metastatic cancer.

[0123] A composition for predicting or diagnosing cancer according to one aspect of the present invention can predict or diagnose cancer if the MSLN protein level is higher than that of a normal control group.

[0124] In another aspect, the present invention provides a cancer prediction or diagnosis kit comprising the cancer prediction or diagnosis composition. The description of the cancer prediction or diagnosis composition may be applied to the kit.

[0125] A kit according to one aspect of the present invention may include an anti-MSLN antibody or an antigen-binding fragment thereof according to one aspect of the present invention, and a label that generates a detectable signal. The label may include, but is not limited to, a chemical substance bound to the antibody (e.g., biotin), an enzyme (alkaline phosphatase, β-galactosidase, horseradish peroxidase, luciferase, or cytochrome P450), a radioactive substance (e.g., C14, I125, P32, and S35), a fluorescent substance (e.g., fluorescein), a luminescent substance, a chemiluminescent substance, and FRET (fluorescence resonance energy transfer). In this case, regarding the substrate for the enzyme, when alkaline phosphatase is used as the enzyme, chromogenic reaction substrates such as bromochloroindoleyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate, and ECF (enhanced chemifluorescence) are used as substrates; and when horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resolupin benzyl ether, luminol, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), and TMB (tetramethylbenzidine) are used as substrates. Substrates such as ABTS (2,2'-Azinedi[3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate) may be used, but are not limited thereto.

[0126] A kit according to one aspect of the present invention can predict or diagnose cancer by analyzing the intensity of a signal produced by the reaction between a sample and an antibody. The measurement of enzyme activity or signal used for diagnosis can be performed according to various methods known in the art, thereby enabling qualitative or quantitative analysis of MSLN expression.

[0127]

[0128] In another aspect, the present invention provides a method for providing information for cancer prediction or diagnosis, comprising the step of treating a sample of a subject with the anti-MSLN antibody or an antigen-binding fragment thereof. Descriptions of the anti-MSLN antibody or an antigen-binding fragment thereof, MSLN in the composition for cancer prediction or diagnosis, antibody, antigen-binding fragment, anti-MSLN antibody, cancer, prediction or diagnosis may be applied to the method for providing information.

[0129] A method for providing information according to one aspect of the present invention may further include the step of measuring MSLN protein levels.

[0130] According to one aspect of the present invention, the protein level measurement is a process of confirming the presence and expression level of the MSLN protein in a biological sample isolated from a subject for the prediction or diagnosis of cancer, and may be performed by western blotting, ELISA (enzyme-linked immunosorbent assay), radioimmuno assay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining (IHC staining), immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, fluorescence-activated cell sorter analysis (FACS), or protein chip technology, but is not limited thereto.

[0131] A method for providing information according to one aspect of the present invention may further include the step of comparing the MSLN protein level with a normal control group. The normal control group refers to a control group that has not developed cancer.

[0132] In addition, a method for providing information according to one aspect of the present invention may further include a step of predicting or diagnosing that the measured MSLN protein level is higher than that of a normal control group as cancer.

[0133] The technology according to one aspect of the present invention relates to a chimeric antigen receptor (hereinafter CAR) comprising an anti-MSLN antibody or an antigen-binding fragment thereof. More specifically, the technology according to one aspect relates to designing a CAR that recognizes and eliminates cancer cells using an antibody or antigen-binding fragment targeting mesothelin (MSLN). MSLN is overexpressed in various cancer cells, such as mesothelioma, pancreatic cancer, ovarian cancer, and some lung cancers; accordingly, anti-MSLN CARs are used to maximize selectivity and efficacy against these cancer cells.

[0134] A CAR based on one aspect contains an anti-MSLN antibody or an antigen-binding fragment as an antigen-binding domain, thereby selectively binding to MSLN-expressing cells. The antigen-binding site of the antibody may consist of a single-chain variable fragment (scFv), which is stably expressed on the surface of T cells to recognize target antigens. Additionally, a CAR based on one aspect includes co-stimulatory signaling domains such as CD3ζ and 4-1BB to enhance T cell activation and proliferation and induce apoptosis of target cancer cells.

[0135] In particular, the technology according to one aspect includes a configuration in which a drug is combined with or co-administered to a CAR. The drug may be an anticancer agent, an immunomodulator, or a cytotoxic drug that assists in the activation of T cells or acts directly on cancer cells to promote apoptosis. This configuration expands the therapeutic scope of CAR-T cell therapy and maximizes therapeutic efficiency by suppressing the immune evasion mechanisms of cancer cells. Consequently, the anti-MSLN CAR and drug combination therapy according to one aspect can provide an effective treatment option for relapsed or advanced cancer.

[0136] The structure and effects of the present invention will be explained in more detail below through examples and experimental examples. However, the following examples and experimental examples are provided for illustrative purposes only to aid in understanding the present invention, and the scope and range of the present invention are not limited by them.

[0137]

[0138] [Example 1] Preparation of Mesothelin (MSLN) Antigen and Cell Line

[0139]

[0140] [Example 1-1] Preparation of Antigen (MSLN)

[0141] To clone antigens for the extracellular domain (ECD) of mesothelin (MSLN), polymerase chain reaction (hereinafter 'PCR') was performed using a primer set (Table 1) containing restriction enzyme Sfi° sites at the 5' and 3' ends for human (hMSLN, Uniprot accession No. Q13421-3), mouse (mMSLN, Uniprot accession No. Q61468-1), and monkey (rhMSLN, Uniprot accession No. F6Q1U7) MSLNs. Using the obtained PCR products and the N293F vector, expression vectors were constructed to express proteins in which 6xHis is fused to the amino terminus of the antigen's extracellular domain.

[0142] [Table 1]

[0143]

[0144] PCR primers for MSLN cloning

[0145]

[0146] Transfection was performed using PEI (polyethylenimine, 23966, Polysciences) under optimized conditions. Human HEK293F cells were 5 x 10⁶ per ml. 5 Inoculate the cell count into the medium (#Freestyle 293 AGT type; AG100009P1, Thermo.) and 1x10 6 The cells were cultured until the cell / mL concentration was reached. Each expression vector was mixed with PEI to form a polyplex, which was then added to the cells for transformation. Subsequently, 5 g / L of Soytone (#212488, DIFCO) was added, and the cells were cultured for an additional 6 days. To purify the His-tagged MSLNs, 3 mL of Ni-NTA resin (#30230, QIAGEN) was placed in an empty column, and the resin was packed with 20 mL of binding buffer (10 mM imidazole). The filtered culture medium was flowed through the packed resin at a gravity flow rate of 0.2 mL / min to bind to the resin. After washing with 100 mL of wash buffer (20 mM imidazole), the cells were eluted with elution buffer (250 mM imidazole). The eluent obtained from the elution buffer was buffer-exchanged with DPBS (Dulbecco's phosphate-buffered saline) via dialysis (1 L, 3 times). Protein concentration was measured using a nano-drop. Each protein was purified and its purity was confirmed using SDS-PAGE (Fig. 1A) and size exclusion chromatography (#TSK-GEL G-3000 SWXL Size Exclusion Chromatography (SEC), Tosoh) (Fig. 1B).

[0147]

[0148] [Examples 1-2] Establishment of MSLN overexpressing cell line and temporary expression

[0149] Cell lines for human (hMSLN) and monkey (rhMSLN) MSLNs were constructed using MIA PaCa-2 cell lines that lack MSLN expression. Each synthesized gene was cloned into a pcDNA3.1 / neo expression vector using the Nhe1 / HindIII restriction enzyme site. The constructed human MSLN (pcDNA3.1 / neo-hMSLN) and monkey MSLN (pcDNA3.1 / neo-rhMSLN) expression vectors were prepared into linear plasmids using ScaI and PvuI, respectively, and then transformed into MIA PaCa-2 cells. After transformation, the cells were cultured for 2 weeks in DMEM medium treated with G-418 (1 mg / ml). Subsequently, single cells were selected and cultured using a 96-well plate. For human or monkey MSLNs, amatuximab, which cross-links to monkey MSLN, was used as the primary antibody to confirm MSLN expression on the surface of the respective cell lines prepared as follows. Each cell was cultured in 2 x 10⁶ 5 Cells were prepared and incubated with the primary antibody (2 µg / ml) at 4°C for 30 minutes. Afterward, the cells were washed with cold PBS and incubated under dark conditions at 4°C for 30 minutes using an anti-human IgG secondary antibody (Vector, USA) conjugated with FITC (fluorescein isothiocyanate; Vector, USA). Fluorescence-stained cells were suspended in 100 µl of PBS containing 2% FBS (fetal bovine serum), and MSLN expression was confirmed using a flow cytometer, Cytoflex (Beckman Coulter, USA). Finally, MIA PaCa-2 / hMSLN and MIA PaCa-2 / rhMSLN cell lines were established (Fig. 1C, right and left).

[0150] In addition, temporary expression of mouse MSLN was confirmed by transforming MIA PaCa-2 cells with a mouse MSLN (pcDNA3.1 / zeo-mMSLN) expression vector constructed using the synthetic gene's NheI / EcoRI restriction enzyme site, and mouse MSLN expression was confirmed by flow cytometry using anti-Mouse MSLN (LSBio, #LS-C179484) as the primary antibody and FITC-fluorescence-conjugated anti-red IgG (Invitrogen, #11-4811-85) as the secondary antibody (Fig. 1C, middle).

[0151]

[0152] [Example 2] MSLN Human Monoclonal Antibody Screening

[0153]

[0154] [Example 2-1] Panning via phage display

[0155] Monoclonal antibody screening against human MSLN utilized antigens constructed by fusing hFc to the amino terminus of the extracellular domain of MSLN. After adding 2 mL of DPBS containing 50 µg of recombinant MSLN-hFc to an immunosorb tube, the recombinant MSLN was coated onto the tube by rotating at 4°C for 8 hours. Subsequently, blocking was induced for 1 hour using DPBS containing 4% skim milk. 1 x 10⁶ 11 After infecting Escherichia coli with a fully human single-chain variable fragment (scFv) library phage (Y-BioLogics Co., Ltd.) having diversity, the obtained Escherichia coli were cultured at 30°C for 16 hours. The culture medium was centrifuged to concentrate the supernatant with PEG (polyethylene glycol), and then dissolved in PBS (phosphate buffered saline) buffer to prepare a human antibody library phage.

[0156] Antibody library phages were placed in immunosorbent tubes and incubated at room temperature for 2 hours. After washing with 1x PBST and 1x PBS, 100 mM TAE and Tris-HCl (pH 7.5) solutions were sequentially treated to elute only scFv-phages specifically bound to the antigen. A pool of positive phages was obtained through a panning process in which the eluted phages were reinfected into E. coli to amplify them. Using the phages amplified in the first round of panning, the second and third rounds of panning were performed by increasing the number of PBST (PBS + tween-20) washes while maintaining the same procedure for the remainder. After the third round of panning, it was confirmed that the number of phage colonies against the antigen had been enriched by more than 200-fold compared to the results of the second round of panning, as shown in Table 2.

[0157] [Table 2]

[0158]

[0159] Comparison of antibody titers according to panning rounds

[0160]

[0161] [Example 2-2] Screening of Positive Phages

[0162] Thousands of single clones were selected from the positive phage pool of the third round of panning, and after infecting them with helper phages and culturing them in 96-deep-well plates, single scFv-phages present in the supernatant were transferred to an immuno-plate coated with recombinant MSLN to perform enzyme-linked immunosorbent assay (ELISA). At this time, a single phage ELISA for MSLN-hFc and an ELISA for the non-specific antigen control protein IGA6-Fc were performed simultaneously to confirm whether the obtained positive phage clones were specific to MSLN. As a result, single scFv-phage clones with strong binding ability to MSLN proteins were selected, and Figure 2 is a schematic diagram of a representative clone among the various single clones.

[0163] For the selected monoclones above, phagemid DNA was isolated using a DNA purification kit (Qiagen, Germany) and DNA sequencing was performed. As a result of analyzing the sequences of the CDR3 regions of the heavy and light chains using the Ig BLAST program on the NCBI webpage, the two types of single scFv-phages (CSA0682, CSA1092) were identified as different clones (Table 3), and the amino acid sequences of the heavy and light chain variable regions for each clone are as listed in Table 4.

[0164] [Table 3]

[0165]

[0166] Characteristics of MSLN-specific single clones

[0167]

[0168] [Table 4]

[0169]

[0170] Amino acid sequences of the heavy and light chain variable regions of the anti-MSLN monoclone

[0171]

[0172] [Examples 2-3] Conversion of scFv form to IgG form

[0173] To convert selected monoclonal phage antibodies from scFv form to IgG form, the nucleotide sequence of the heavy chain variable region was cloned into the pNATVH vector (Y-Biologics) using the restriction enzyme SfiI / NheI site, and the N293F HC vector was prepared. The nucleotide sequence of the light chain variable region was cloned into the pNATVL vector (Y-Biologics) using the restriction enzyme SfiI / Bgl± site, and the N293F LC vector was prepared.

[0174] HEK293F cells were co-transfected with N293F HC and N293F LC vectors. On day 7 of culture, the culture medium was centrifuged at 8,000 rpm for 30 minutes to remove cell debris, and the solution was filtered using a bottle top filter with a pore size of 0.22 μm (Steritop-GP Filter Unit, #SCGPS01RE, Millipore). Meanwhile, 4 ml of Protein A Sepharose resin slurry (KANEKA KanCapA) was placed in an empty column (#BR731-1550, Bio-rad), and the resin was packed and washed with 100 ml of DPBS. The filtered medium was loaded onto the packed resin and flowed at a rate of 1 ml per minute (#EP-1 Econo pump, Bio-Rad). After washing with 150 ml of DPBS, the solution was eluted with 10 ml of 0.1 M glycine-HCl (pH 3.3). 10% of 1 M Tris-HCl (pH 9.0) was added to the eluent to neutralize the pH, and the buffer was changed to DPBS using an Amicon Ultra-10 (#UFC901096, Millipore). This process was repeated approximately three times, after which the solution was concentrated to about 1 ml, and the concentration of the purified antibody was measured using a Nano-drop. Subsequently, the purity of the three purified anti-MSLN monoclonal antibodies was confirmed through SDS-PAGE (Fig. 3A) and SEC-HPLC (size exclusion-high performance liquid chromatography) analysis (Fig. 3B).

[0175]

[0176] [Example 3] Confirmation of the binding specificity of anti-MSLN monoclonal antibody to antigen

[0177]

[0178] The specific binding affinity of the selected anti-MSLN monoclonal antibodies to the antigen was analyzed using a flow cytometer with MSLN-non-expressing (MIA PaCa-2) or overexpressing human pancreatic cancer cell lines (MIA PaCa-2 / hMSLN). Each cell was 2 x 10⁶ 5 The cells were prepared and reacted with anti-MSLN monoclonal antibodies at a concentration of 2 µg / ml as the primary antibody at 4°C for 30 minutes. After washing the cells with cold PBS, they were cultured in the cancer at 4°C for 30 minutes using an anti-human IgG secondary antibody (Vector, USA) conjugated with FITC (fluorescein isothiocyanate; Vector, USA). Fluorescence-stained cells were suspended in 100 µl of PBS containing 2% FBS (fetal bovine serum) and analyzed using a flow cytometer, Cytoflex (Beckman Coulter, USA) (Fig. 4).

[0179] As a result, it was confirmed that the two selected anti-MSLN monoclonal antibodies (CSA0682, CSA1092) did not bind to MIA PaCa-2 cell lines that do not express MSLN (Fig. 4A), whereas they specifically bound to MIA PaCa-2 / hMSLN cell lines that overexpress human MSLN (Fig. 4B).

[0180]

[0181] [Example 4] Confirmation of binding affinity of anti-MSLN monoclonal antibody to antigen

[0182]

[0183] The binding affinity of two selected anti-MSLN monoclonal antibodies (CSA0682 and CSA1092) to human MSLN was confirmed via ELISA analysis. Antigen proteins were dispensed at 100 ng per well into 96-well plates and fixed overnight at 4°C. Subsequently, non-specific protein binding was blocked by adding 200 µl of PBS-T containing 4% skim milk to all wells and incubating at 37°C for 1 hour. Anti-MSLN monoclonal antibodies were serially diluted in 1 / 5 increments from 100 nM to 0.00000205 nM, and 100 µl of each was sequentially added to the column of each well and incubated at 37°C for 1 hour. After the reaction, the samples were washed three times with PBS-T. Goat anti-Human IgG (H+L) Secondary Antibody conjugated with HRP (horseradish peroxidase) and HRP (Invitrogen) were diluted 1:5,000 and incubated at 37°C for 1 hour. After washing three more times with PBS-T, 100 µl of TMB substrate was added to each well, and color development was allowed for approximately 2–3 minutes to prevent saturation. The reaction was stopped with 50 µl of 2.5 M sulfuric acid (H2SO4), and absorbance was measured at 450 nm using a spectrophotometer (SpectraMax spectrophotometer, Molecular Devices, USA). The results were analyzed using GraphPad Prism 9 software.

[0184] As a result, the affinity dissociation constants (K) of the anti-MSLN monoclonal antibodies CSA0682 and CSA1092 against the human MSLN antigen D The affinities were 0.021 nM and 1.211 nM, respectively (Fig. 5). Compared to the control antibody, CSA0682 showed similar affinity, while CSA1092 showed lower affinity compared to the control antibody (Fig. 5).

[0185]

[0186] [Example 5] Confirmation of cross-reactivity of anti-MSLN monoclonal antibody

[0187]

[0188] The cross-reactivity of the two selected anti-MSLN monoclonal antibodies (CSA0682, CSA1092) was confirmed by flow cytometry using MIA PaCa-2 cell lines in which human (hMSLN) and monkey (rhMSLN) MSLN were stably overexpressed, or in which mouse (mMSLN) MSLN was transiently overexpressed.

[0189] The experimental method was carried out in the same manner as in Example 3 above, and the number of cells used was 3 x 10 5 The antibody concentration was serially diluted from 666.67 nM to 0.0085 nM in 1 / 5 increments.

[0190] As a result, it was confirmed that the anti-MSLN monoclonal antibodies CSA0682 and CSA1092 cross-linked to mouse and monkey MSLN (Fig. 6, AC). In addition, the cellular binding affinity of the two anti-MSLN monoclonal antibodies (CSA0682, CSA1092) to antigens in human, mouse, and monkey MSLN overexpressing cell lines is as described in Table 5.

[0191] [Table 5]

[0192]

[0193] Interspecies cross-reactivity and cell binding affinity of anti-MSLN monoclonal antibodies

[0194]

[0195] [Example 6] Verification of Cell Internalization of Anti-MSLN Monoclonal Antibody

[0196]

[0197] The intracellular uptake of anti-MSLN monoclonal antibodies (CSA0682, CSA1092) was measured using real-time image analysis equipment with human MSLN-overexpressing cell lines (MIA PaCa-2 / hMSLN). 1 x 10⁶ antibodies were placed per well in a 96-well plate. 4 10 MIA PaCa-2 / hMSLN cells were attached for 24 hours, and each 4 µg / ml antibody was mixed with an equal amount of Incucyte® FabFluor red antibody labeling reagent (Sartorius, Germany) and incubated in the dark at 37°C for 15 minutes, after which the cells were treated. The amount of antibody intracellular influx was quantified by checking the amount of antibody accumulating in the cell lysosomes over time at 15-minute intervals for 48 hours using the IncuCyte ZOOM HD / 2CLR System (Essen Biosciences, USA).

[0198] As a result, the antibody showing the fastest cellular internalization was CSA1092, followed by CSA0682 and the control antibody anetumab (Fig. 7A). In addition, the degree of cellular internalization of the antibodies after 24 hours (RCU x μm 2 As a result of quantitatively analyzing / well), CSA1092 (17.79 x 10 4 ), CSA0682 (13.04 x 10 4 ), anetumab (5.81 X 10 4 ...and in particular, CSA1092 was about 3.1 times better than the control antibody, and CSA0682 was about 2.2 times better (Fig. 7B).

[0199] Through this, it was found that the anti-MSLN antibody of the present invention or its antigen-binding fragment can be utilized as a therapeutic agent capable of delivering drugs through cell internalization.

[0200]

[0201] [Example 7] Cancer cell death effect of toxin-conjugated anti-MSLN monoclonal antibody

[0202]

[0203] To confirm the potential for developing the two anti-MSLN monoclonal antibodies (CSA0682, CSA1092) of the present invention as antibody-drug conjugates, cancer cell apoptosis experiments were performed against the two anti-MSLN monoclonal antibodies using a ZAP antibody internalization kit (Advance Targeting System, USA) on MSLN-nonexpressing (MIA PaCa-2) or MSLN-overexpressing (MIA PaCa-2 / hMSLN) human pancreatic cancer cell lines. 2.5 x 10⁶ cells per well were placed in a 96-well plate (Nunc, USA). 3 After seeding cells and allowing them to adhere for 24 hours, the cell culture medium was treated with CSA0682, CSA0682-Fab ZAP, CSA1092, and CSA1092-Fab ZAP at concentrations ranging from 1 fM to 10 nM, and the positive control drug Saporin at concentrations ranging from 1 fM to 1 μM, as shown in Fig. 8. Subsequently, the cell lines were cultured in a 37°C 5% CO2 incubator for 72 hours, after which 50 μl of XTT / PMS solution was added and the cells were cultured for an additional 2 hours. Absorbance was measured at 450 nm using an ELISA reader (SpectraMax ABS Plus spectrophotometer, Molecular Devices, USA). In addition, to compare the apoptotic effects of each antibody, the amount of sample required to reach 50% of the maximum apoptotic effect (IC10) was calculated. 50 ) was measured.

[0204] As a result, when treated with two anti-MSLN monoclonal antibodies (CSA0682, CSA1092), no cancer cell death occurred in MIA PaCa-2 cells that were under- or over-expressed with human MSLN; whereas, for the two toxin-conjugated anti-MSLN monoclonal antibodies (CSA0682-Fab ZAP, CSA1092-Fab ZAP), the cancer cell death effect increased with increasing antibody concentration in MIA PaCa-2 cells over-expressing human MSLN, and at this time, the EC of CSA0682-Fab ZAP and CSA1092-Fab ZAP 50 The values ​​were 2.205 pM and 3.888 pM, respectively (Fig. 8). On the other hand, the positive control drug saporin caused apoptosis due to nonspecific cell influx at high concentrations of 100 nM or higher.

[0205] Through this, it was found that the anti-MSLN antibody of the present invention or its antigen-binding fragment can be developed as an anti-MSLN antibody-drug conjugate capable of killing cancer cells by targeting MSLN expressed on the surface of cancer cells.

Claims

1. An anti-MSLN (mesothelin) antibody or its antigen-binding fragment comprising the following complementarity determining regions (CDRs): Heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO. 2 or 16, Heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO. 4 or 18, Heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO. 6 or 20, Light chain CDR1 consisting of the amino acid sequence of SEQ ID NO. 9 or 23, A light chain CDR2 consisting of the amino acid sequence of SEQ ID NO. 11 or 25, and Light chain CDR3 consisting of the amino acid sequence of SEQ ID NO. 13 or 27.

2. In claim 1, the antibody is an anti-MSLN antibody or an antigen-binding fragment thereof comprising the following framework regions (FRs): A heavy chain FR1 consisting of a sequence having 90% or more homology with the amino acid sequence of SEQ ID NO. 1 or 15, A heavy chain FR2 consisting of a sequence having 90% or more homology with the amino acid sequence of SEQ ID NO. 3 or 17, Heavy chain FR3 consisting of a sequence having 90% or more homology with the amino acid sequence of SEQ ID NO. 5 or 19, A heavy chain FR4 consisting of a sequence having 90% or more homology with the amino acid sequence of SEQ ID NO. 7 or 21, Light chain FR1, consisting of a sequence having 90% or more homology with the amino acid sequence of SEQ ID NO. 8 or 22, Light chain FR2, consisting of a sequence having 90% or more homology with the amino acid sequence of SEQ ID NO. 10 or 24, A light chain FR3 consisting of a sequence having 90% or more homology with the amino acid sequence of SEQ ID NO. 12 or 26, and Light chain FR4 consisting of a sequence having 90% or more homology with the amino acid sequence of SEQ ID NO. 14 or 28.

3. In claim 1, the antibody comprises an anti-MSLN antibody or an antigen-binding fragment thereof, wherein the antibody comprises a set of CDRs of any one of (i) and (ii) below: (i) heavy chain CDR1 of SEQ ID NO. 2, heavy chain CDR2 of SEQ ID NO. 4, heavy chain CDR3 of SEQ ID NO. 6, light chain CDR1 of SEQ ID NO. 9, light chain CDR2 of SEQ ID NO. 11, and light chain CDR3 of SEQ ID NO. 13; and (ii) heavy chain CDR1 of SEQ ID NO. 16, heavy chain CDR2 of SEQ ID NO. 18, heavy chain CDR3 of SEQ ID NO. 20, light chain CDR1 of SEQ ID NO. 23, light chain CDR2 of SEQ ID NO. 25, and light chain CDR3 of SEQ ID NO.

27.

4. The anti-MSLN antibody or its antigen-binding fragment, wherein the antibody comprises a heavy chain variable region formed of the amino acid sequence of SEQ ID NO. 29 or 31 and a light chain variable region formed of the amino acid sequence of SEQ ID NO. 30 or 32.

5. In Paragraph 1, The above antigen-binding fragment is an anti-MSLN antibody or its antigen-binding fragment, which is scFv, (scFv)2, Fab, Fab', or F(ab')2.

6. A nucleic acid encoding an anti-MSLN antibody of any one of claims 1 to 5 or an antigen-binding fragment thereof.

7. In Paragraph 6, The nucleic acid comprises a nucleic acid encoding a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO. 29 or 31, and a nucleic acid encoding a light chain variable region consisting of the amino acid sequence of SEQ ID NO. 30 or 32.

8. A recombinant expression vector comprising nucleic acid according to paragraph 6.

9. Cells transformed with a recombinant expression vector according to paragraph 8.

10. A step of culturing cells according to paragraph 9; and A method for preparing an anti-MSLN antibody or an antigen-binding fragment thereof, comprising the step of recovering an antibody that specifically binds to mesothelin (MSLN) or an antigen-binding fragment thereof from the cultured cells.

11. An antibody-drug conjugate (ADC) comprising the anti-MSLN antibody of claim 1 or an antigen-binding fragment thereof, and a drug.

12. In Paragraph 11, The above drug is an antibody-drug conjugate selected from the group consisting of immunomodulators, cytotoxic agents, chemotherapy agents, proteolytic agents (TPDs), and antibody-oligonucleotide conjugates (AOCs).

13. A bispecific antibody comprising the anti-MSLN antibody of claim 1 or an antigen-binding fragment thereof.

14. A composition for the prevention or treatment of cancer, comprising the anti-MSLN antibody of claim 1 or an antigen-binding fragment thereof, the antibody-drug conjugate of claim 11 or the bispecific antibody of claim 13.

15. In Paragraph 14, A composition for preventing or treating cancer, wherein the above cancer is pancreatic cancer, ovarian cancer, mesothelioma, biliary tract cancer, lung cancer, stomach cancer, colorectal cancer, cervical cancer, esophageal cancer, breast cancer, kidney cancer, or metastatic cancer derived from any of the above.

16. A composition for cancer prediction or diagnosis comprising an anti-MSLN antibody of any one of claims 1 to 5 or an antigen-binding fragment thereof.

17. In Paragraph 16, A composition for predicting or diagnosing cancer, wherein the above cancer is pancreatic cancer, ovarian cancer, mesothelioma, biliary tract cancer, lung cancer, stomach cancer, colorectal cancer, cervical cancer, esophageal cancer, breast cancer, kidney cancer, or metastatic cancer derived from any of the above.

18. In Paragraph 16, The above composition is a composition for predicting or diagnosing cancer, which predicts or diagnoses cancer if the MSLN protein level is higher than that of a normal control group.

19. A kit for cancer prediction or diagnosis comprising a composition according to paragraph 16.

20. A method for providing information for cancer prediction or diagnosis, comprising the step of treating a sample of a subject with an anti-MSLN antibody of any one of claims 1 to 5 or an antigen-binding fragment thereof.

21. In Paragraph 20, The above information provision method further comprises the step of measuring MSLN protein levels.

22. In Paragraph 21, The above information provision method includes the step of comparing the MSLN protein level with a normal control group; and A method for providing information, further comprising the step of predicting or diagnosing cancer if the measured MSLN protein level is higher than that of a normal control group.

23. A chimeric antigen receptor (CAR) comprising the anti-MSLN antibody of claim 1 or an antigen-binding fragment thereof.

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