Mesothelin-specific antibody and uses thereof

Abstract

The present invention relates to an antibody or an antigen-binding fragment thereof comprising a single domain antibody specifically binding to mesothelin, an antibody conjugate comprising same, a pharmaceutical composition for preventing or treating cancer or a tumor, and a composition for diagnosing or monitoring cancer or a tumor. When the antibody or composition comprising same is used, mesothelin-related cancer or tumors can be effectively prevented, treated, or diagnosed.

Description

Antibodies specific for mesothelin and their uses

[0001] The present invention relates to antibodies specific for mesothelin and uses thereof.

[0002]

[0003] Mesothelin (MSLN) is a 69-71 kDa precursor polypeptide and glycoprotein that is expressed on the cell surface and helps cells adhere to each other and transmit signals. Although it is expressed at a low level in general tissues, overexpression has been confirmed in several types of solid cancers, including mesothelioma, pancreatic cancer, and ovarian cancer. Various anticancer targeting studies are underway. Korean Patent Publication No. 10-2017-0036503 describes the development of antibody-based targeted therapies for lung, ovarian, and pancreatic cancers that express mesothelin (Korean Patent Publication No. 10-2017-0036503; Chang, K, et al., Int J Cancer, 50(3):373, 1992).

[0004]

[0005] Meanwhile, it is reported that there are more than 300,000 antibodies currently in commercial use, but they can only be observed mainly in fixed cells, and because of this, it was not possible to confirm protein folding or interactions between proteins in real time within the cell. In addition, existing antibodies were too large or chemically unstable to be usefully used in living cells. However, in 1993, Hamers-Casterman reported an antibody derived from a camelid animal that was composed of only a heavy chain, which is a different structure from existing antibodies (two heavy chains and two light chains), but was a single-domain antibody that functioned as a complete antibody (Hamers-Casterman, C. et al. 1993. Nature 363:446-448). Conventional antibodies are 150 kDa, and recombinant antibodies are 25-50 kDa, but single-domain antibodies derived from camels, llamas, and sharks are the smallest antibodies at 12-13 kDa, so they can easily move into cells (Cortez-Retamozo, V. et al. 2004. Cancer Res. 64:2853-2857), and they are easy to genetically manipulate, so they can be easily expressed in bacteria and yeast (Arbabi-Ghahroudii, M. et al. 1997. FEBS Lett. 414:521-526). In addition, single-domain antibodies have the characteristics of being highly water-soluble and stable even under extreme pH conditions and temperature conditions up to 90℃ (Dumoulin, M. et al. 2002. Protein Sciii.11:500-515, Dumoulin, M. et al. 2003. Nature 424:783-788), and various studies on single-domain antibodies have been conducted recently.

[0006]

[0007] In the present invention, in order to develop an antibody that binds more specifically to mesothelin, antibodies that bind to mesothelin were screened to establish a novel single-domain antibody, and the present invention was completed by confirming that the antibody specifically binds to the mesothelin antigen.

[0008]

[0009] One object of the present invention is to provide an antibody or antigen-binding fragment thereof comprising a single domain antibody that specifically binds to mesothelin.

[0010] Another object of the present invention is to provide an antibody conjugate comprising the antibody of claim 1 or an antigen-binding fragment thereof conjugated to an immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent, diagnostic agent, antiviral agent, antimicrobial agent or drug.

[0011] Another object of the present invention is to provide a nucleic acid molecule encoding the antibody or an antigen-binding fragment thereof.

[0012] Another object of the present invention is to provide an expression vector comprising the polynucleotide.

[0013] Another object of the present invention is to provide a host cell transformed with the expression vector.

[0014] Another object of the present invention is to provide a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to mesothelin using the host cell.

[0015] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer or tumor, comprising the antibody or an antigen-binding fragment thereof, or the antibody conjugate as an active ingredient.

[0016] Another object of the present invention is to provide a composition for diagnosing or monitoring cancer or tumor, comprising the antibody or fragment thereof.

[0017]

[0018] The antibody of the present invention can be usefully used for the prevention, treatment, and diagnosis of mesothelin-related cancer or tumor.

[0019]

[0020] Figure 1 is a diagram schematically illustrating an antigen immunization and blood collection schedule according to one embodiment of the present invention.

[0021] Figure 2 is a diagram showing the results of evaluating the antibody titer in blood of a single domain antibody according to one embodiment of the present invention.

[0022] Figure 3 is a diagram showing the biopanning results of a single domain antibody according to one embodiment of the present invention.

[0023] Figure 4 is a diagram showing the biopanning results of a single domain antibody according to one embodiment of the present invention.

[0024] Figure 5 is a diagram showing the cell binding ability of a single domain antibody according to one embodiment of the present invention.

[0025] Figure 6 is a diagram showing the antigen affinity of a single domain antibody according to one embodiment of the present invention.

[0026]

[0027] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

[0028] Furthermore, those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Furthermore, such equivalents are intended to be encompassed by the present invention.

[0029] Additionally, numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the present invention.

[0030]

[0031] One aspect of the present invention is an antibody or antigen-binding fragment thereof that specifically binds to mesothelin.

[0032] In the present invention, the term "antibody" 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.

[0033] The term "antibody" as used herein refers to antibodies in its broadest sense, encompassing a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antigen-binding fragments thereof, as long as they exhibit the desired antigen-binding activity. The term "antibody" also encompasses conventional four-chain antibodies, single-domain antibodies, and antigen-binding fragments thereof.

[0034] A conventional four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies are composed of five of the basic heterotetrameric units along with an additional polypeptide called the J chain, containing ten antigen-binding sites, whereas IgA antibodies contain two to five of the basic four-chain units that can polymerize to form multivalent aggregates in combination with the J chain. For IgG, a four-chain unit typically weighs about 150,000 daltons.

[0035] Each L chain is linked to an H chain by a single covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each H and L chain also has regularly spaced interchain disulfide bridges. Each H chain has, at its N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has, at its other end, a variable domain (VL) followed by a constant domain. The VL aligns with the VH, and the CL aligns with the first constant domain (CH1) of the heavy chain. The pairing of the VH and VL together forms a single antigen-binding site. L chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain (CH) of their heavy chains, immunoglobulins can be assigned to different classes, or isotypes. There are five classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. Of these, the γ and α classes are further divided into subclasses based on relatively minor differences in CH sequence and function; for example, humans express subclasses of IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0036]

[0037] In the present invention, the term "antigen-binding fragment" refers to any fragment of the antibody of the present invention that possesses antigen-binding activity, and is used interchangeably with antibody fragment and antibody part.

[0038] The above "antibody fragment" or "antigen-binding fragment" comprises a portion of a full-length antibody or an intact antibody, specifically the antigen-binding and / or variable region of an intact antibody. Examples of antibody fragments may include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody (scFv) molecules; single-domain antibodies (e.g., VHH), and multispecific antibodies formed from antibody fragments. An "Fv" is the smallest antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy chain and one light chain variable region domain in a compact, non-covalent association. A "single-chain Fv," also abbreviated "sFv" or "scFv," is an antibody fragment comprising VH and VL antibody domains linked in a single polypeptide chain. Specifically, the scFv polypeptide may further comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. "Diabodies" refer to small antibody fragments prepared by constructing sFv fragments with a short linker (about 5-10 residues) between the VH and VL domains to achieve interchain rather than intrachain pairing of the V domains, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossed" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains.

[0039] Fd refers to the heavy chain portion included in the Fab fragment. The Fab has a structure with a variable region of the light chain and heavy chain, a constant region (framework region (FR)) of the light chain, and the first constant region (CH1 domain) 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 antibodies are produced when cysteine ​​residues in the hinge region of Fab' form a disulfide bond. Fv (variable fragment) refers to the smallest antibody fragment that has only the heavy chain variable region and the light chain variable region. Double disulfide Fv (dsFv) has the heavy chain variable region and the light chain variable region linked by a disulfide bond, and single-chain Fv (scFv) has the heavy chain variable region and the light chain variable region covalently linked, generally via a peptide linker. These antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of whole antibodies with papain, and F(ab')2 fragments can be obtained by digestion with pepsin), or can be produced, for example, through genetic recombination technology.

[0040]

[0041] The term "single-domain antibody" as used herein refers to a single antigen-binding polypeptide having three complementarity determining regions (CDRs), and may be used interchangeably with "nanobody." Such single-domain antibodies can bind to an antigen without pairing with a corresponding CDR-containing polypeptide. Some of such single-domain antibodies may be engineered from camelid HCAb, and their heavy chain variable domains may be referred to as "VHH" (the variable domain of the heavy chain of a heavy chain antibody). A basic VHH has the following structure from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3.

[0042] Single domain antibodies according to the present invention may include, but are not limited to, heavy chain variable domains from heavy chain-only antibodies (e.g., VHH (variable domain of the heavy chain of a heavy chain antibody) in Camelidae), light chains derived from conventional four-chain antibodies, binding molecules naturally lacking a single domain (e.g., VH or VL), humanized heavy chain-only antibodies, human single domain antibodies produced by transgenic mice or rats expressing human heavy chain segments, and engineered domains and single domain scaffolds other than those derived from antibodies. The antibodies may be derived from any species including, but not limited to, mouse, rat, human, camel, llama, lamprey, fish, shark, goat, rabbit, and bovine. They may also include naturally occurring antibody molecules from species other than Camelidae.

[0043] Additionally, the antibody may be derived from a naturally occurring single-domain antigen-binding molecule known as a light chain-deficient heavy chain antibody (see, e.g., WO 94 / 04678 and Hamers-Casterman, et al., (1993) Nature 363:446-448). The variable domain derived from a heavy chain molecule naturally lacking a light chain is known herein as a VHH to distinguish it from the conventional VH of four-chain immunoglobulins. Such VHH molecules may be derived from antibodies produced by Camelidae species, such as camels, llamas, vicuñas, dromedaries, alpacas, and guanacos. Species other than Camelidae can produce heavy chain molecules naturally lacking light chains, and such VHHs are within the scope of the present disclosure.

[0044] Additionally, the antibodies may be recombinant, CDR-grafted, humanized, camelized, de-immunized, and / or produced in vitro (e.g., selected by phage display). In some embodiments, the amino acid sequence of the framework region may be altered by "camelization" of specific amino acid residues in the framework region. Camelization refers to the replacement or substitution of one or more amino acid residues in the amino acid sequence of a (naturally occurring) VH domain from a conventional four-chain antibody by one or more of the amino acid residues occurring at the corresponding position(s) in the VHH domain of a heavy chain antibody, and may be performed in a manner known in the art.

[0045] Additionally, the antibody may be a human antibody produced by a transgenic mouse or rat expressing a human heavy chain segment (see, e.g., patents US20090307787A1, US8,754,287, US20150289489A1, US20100122358A1, and WO 2004049794).

[0046] Additionally, naturally occurring VHH domains for a particular antigen or target may be obtained from a library (either naive or immunized) of Camelidae VHH sequences. Such methods may or may not involve screening such libraries using said antigen or target, or at least portions, fragments, antigenic determinants or epitopes thereof using one or more screening techniques known per se (e.g., libraries and techniques, see, e.g., patents WO 99 / 37681, WO 01 / 90190, WO 03 / 025020 and WO 03 / 035694).

[0047] Alternatively, improved synthetic or semi-synthetic libraries derived from (naive or immune) VHH libraries may be used, such as VHH libraries obtained from (naive or immune) VHH libraries by random mutagenesis and / or CDR shuffling, for example as described in patent WO 00 / 43507.

[0048] Additionally, the antibody can be generated from a conventional 4-chain antibody.

[0049]

[0050] The antibody of the present invention that specifically binds to mesothelin is a single domain antibody that can specifically bind to mesothelin and inhibit the activity of mesothelin, and has the characteristic of binding to mesothelin with high affinity.

[0051] The term "mesothelin (MSLN)" in the present invention refers to a 69-71 kDa precursor polypeptide, a glycoprotein, expressed on the cell surface that facilitates cell-to-cell adhesion and signal transmission. While low expression is observed in general tissues, overexpression has been confirmed in various solid tumors, including mesothelioma, pancreatic cancer, and ovarian cancer. Therefore, various anticancer targeting studies targeting mesothelin are currently underway.

[0052] The mesothelin described above is not particularly limited in type, but may specifically be human mesothelin. In addition, the mesothelin includes, but is not limited to, both natural and mutant mesothelin. The natural mesothelin generally refers to a polypeptide comprising the amino acid sequence of natural mesothelin, and the amino acid sequence of natural mesothelin generally refers to an amino acid sequence found in naturally occurring mesothelin. Information on the mesothelin can be obtained from known databases such as GenBank of the National Institutes of Health of the United States, and an example thereof may be, but is not limited to, mesothelin with NCBI Gene ID 10232 (https: / www.ncbi.nlm.nih.gov / gene / 10232).

[0053]

[0054] In the present invention, the term “specific” refers to selective recognition of an antigen binding protein for a specific epitope of an antigen.

[0055] The term "monospecific" above refers to an antigen binding protein having one or more binding sites each binding the same epitope of the same antigen, for example, natural antibodies are monospecific.

[0056] The term "epitope" refers to a protein determinant capable of specific antibody binding. Epitopes typically consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0057]

[0058] The present invention, as one specific example, may be characterized in that the single domain antibody or binding fragment thereof that specifically binds to the mesothelin comprises a CDR1 comprising an amino acid sequence represented by SEQ ID NO: 2; a CDR2 comprising an amino acid sequence represented by SEQ ID NO: 3; and a CDR3 comprising an amino acid sequence represented by SEQ ID NO: 4.

[0059] In addition, the present invention, as another specific example, may be characterized in that the single domain antibody or binding fragment thereof that specifically binds to the mesothelin is composed of an amino acid sequence having sequence ID No. 1 or at least 90% or more, specifically 95%, 96%, 97%, 98%, 99% or more sequence homology thereto.

[0060]

[0061] The term "CDR" is used interchangeably with "complementarity determining region" and is used to refer to hypervariable regions as defined by the Kabat system (see Kabat, Elvin A., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The Kabat complementarity determining region (CDR) is based on sequence variability and is the most commonly used.

[0062] The term "variable" refers to the fact that specific segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed across the entire range of the variable domain. Instead, it is concentrated in three segments, called complementarity determining regions (CDRs) or hypervariable regions (HVRs), in both heavy and light chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). The variable domains of native heavy and light chains each comprise four FR regions, which predominantly adopt a beta-sheet configuration, and in some cases form part of a beta-sheet structure, connected by three CDRs that form loop connections. In each chain, the CDRs are held together in close proximity by the FR regions, and the CDRs from the other chain contribute to the formation of the antigen-binding site of the antibody (see Kabat, Elvin A., Sequence of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0063] The term "variable domain", also referred to as "variable region," refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL," respectively. These domains are generally the most variable portions of an antibody compared to other antibodies of the same class and may contain the antigen-binding site. Heavy-chain-only antibodies from Camelidae species have a single heavy chain variable region, referred to as "VHH."

[0064] The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence than the variable domain, another portion of the immunoglobulin that contains the antigen-binding site. The constant domain contains the CH1, CH2, and CH3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.

[0065] The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. In some cases, an intact antibody may have more than one effector function.

[0066] The term "hypervariable region," "HVR," or "HV," as used herein, refers to the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops. Typically, single domain antibodies comprise three HVRs (or CDRs): HVR1 (or CDR1), HVR2 (or CDR2), and HVR3 (or CDR3). HVR3 (or CDR3) exhibits the greatest diversity of the three HVRs and is known to play a unique role in conferring fine specificity to antibodies (see Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996)).

[0067] The term "framework" or "FR" residues refers to variable-domain residues other than HVR residues, as defined herein.

[0068]

[0069] As used herein, the term "sequence homology" is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence, after sequence alignment and gap introduction, if necessary, and without considering any conservative substitutions as part of the sequence identity, to achieve the maximum percent sequence identity. The sequence homology may be used interchangeably with "percent (%) amino acid sequence identity" or "homology" with respect to peptide, polypeptide, or antibody sequences.

[0070] Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNATAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximal alignment over the full length of the sequences being compared.

[0071] Additionally, the expression “having” herein refers to an amino acid sequence represented by a specific sequence number may be understood to mean “comprising” the amino acid sequence, and in some embodiments, may be modified to “consisting essentially of” or “consisting of” the amino acid sequence.

[0072]

[0073] The present invention may be characterized by comprising at least one amino acid substitution in an antibody or binding fragment thereof that specifically binds to mesothelin according to any one of the above specific examples.

[0074] In addition, the present invention is characterized in that at least one amino acid substitution in an antibody or binding fragment thereof that specifically binds to mesothelin of any of the above specific examples is a conservative substitution.

[0075] In addition, the present invention is characterized in that in any one of the above specific examples, an antibody or binding fragment thereof that specifically binds to mesothelin, at least one amino acid substitution is a substitution of an amino acid with a non-genetically encoded amino acid or a synthetic amino acid.

[0076] Specifically, in the present invention, the antibody or antigen-binding fragment thereof that specifically binds to mesothelin may include an amino acid sequence variant.

[0077] Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleic acid sequence encoding the antibody, or by peptide synthesis. Such modifications can include, for example, deletions, insertions, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to achieve a final construct that retains the desired characteristics, such as antigen-binding. In some embodiments, substitutions, insertions, or deletions can occur within one or more hypervariable regions (HVRs), as long as such alterations do not substantially reduce the antibody's ability to bind antigen. For example, conservative alterations that do not substantially reduce binding affinity can be made in HVRs. Such alterations can be outside of HVR "hot spots" or CDRs.

[0078] Additionally, the amino acid substitution can be at least one (e.g., any one, two, three, four, five, six, seven, eight, nine, or ten) amino acid substitution. Additionally, the at least one amino acid substitution can be a conservative substitution, or a substitution with a non-genetically encoded amino acid or a synthetic amino acid. In some embodiments, the amino acid substitution can be in a CDR region and can comprise at least one (e.g., any one, two, three, or four) amino acid substitution in CDR1, CDR2, and / or CDR3. In some embodiments, the amino acid substitution can be in a FR region and can comprise at least one (e.g., any one, two, three, four, five, or six) amino acid substitution in FR1, FR2, FR3, and / or FR4.

[0079] Additionally, the amino acid sequence insertions may include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing more than 100 residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion may include an antibody having an N-terminal methionyl residue. Other insertional variants of the antibody molecule may include fusions to the N- or C-terminus of the antibody to a polypeptide or an enzyme (e.g., in the case of ADEPT) that increases the serum half-life of the antibody.

[0080] Additionally, one or more amino acid modifications can be introduced into the Fc region of an antibody or antigen-binding fragment thereof, including an antibody that specifically binds mesothelin provided herein, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0081]

[0082] In addition, the present invention may be characterized in that the antibody or binding fragment thereof that specifically binds to mesothelin in any one of the above specific examples is a heavy chain-only antibody fused to an Fc fragment.

[0083] In addition, the present invention may be characterized in that the heavy chain-only antibody in any one of the above specific examples or a binding fragment thereof is monomeric or multimeric.

[0084] In addition, the present invention may be characterized in that the Fc fragment of the antibody or binding fragment thereof that specifically binds to mesothelin of any one of the above specific examples is human IgG1, IgG2, IgG3 or IgG4.

[0085] In addition, the present invention may be characterized in that the single domain antibody or binding fragment thereof that specifically binds to mesothelin of any one of the above specific examples is fused to an Fc fragment via a peptide linker.

[0086] In addition, the present invention may be characterized in that the antibody or binding fragment thereof that specifically binds to mesothelin of any one of the above specific examples is composed of an amino acid sequence having sequence ID No. 5 or at least 90% or more, specifically 95%, 96%, 97%, 98%, 99% or more sequence homology thereto.

[0087] The antibody or fragment thereof may be characterized by comprising at least one amino acid substitution. In addition, the present invention may be characterized in that in any one of the above embodiments, the antibody or binding fragment thereof that specifically binds to mesothelin, at least one amino acid substitution is a conservative substitution. In addition, the present invention may be characterized in that in any one of the above embodiments, the antibody or binding fragment thereof that specifically binds to mesothelin, at least one amino acid substitution is a substitution of an amino acid with a non-genetically encoded amino acid or a synthetic amino acid. Specifically, the antibody or antigen-binding fragment thereof that specifically binds to mesothelin in the present invention may comprise an amino acid sequence variant.

[0088] The above terms, sequence homology, substitution, conservative substitution, variant, etc. are as explained above.

[0089]

[0090] As used herein, the term "heavy chain-only antibody" refers to a functional antibody that comprises a heavy chain but lacks the light chain commonly found in four-chain antibodies.

[0091] The antibodies or binding fragments thereof of the present invention may be monomeric or multimeric, and when multimeric, may be multispecific and multivalent (e.g., bispecific and bivalent), for example, comprising two or more different antibodies, or monospecific and multivalent (e.g., bivalent), comprising two or more copies of the same antibody. The antibodies may also be fused via a peptide linker. In some embodiments, the peptide linker may include flexible residues (e.g., glycine and serine) to allow the adjacent domains to move freely relative to one another. For example, a glycine-serine doublet may be a suitable peptide linker. The peptide linker may also be of any suitable length. In some embodiments, the peptide linker can be, but is not limited to, at least about any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids in length.

[0092] The term "Fc fragment," also referred to as "Fc region" or "fragment crystallizable region," is used to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Suitable native-sequence Fc regions for use in the antibodies described herein may include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, or IgG4.

[0093] The peptide linker may have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain-only antibody may be used as the linker (see patent WO 1996 / 34103). In some embodiments, the peptide linker may be an hIgG1 hinge, an hIgG2 hinge, an hIgG3 hinge, an hIgG4 hinge, or a variant thereof.

[0094]

[0095] In addition, the present invention may be characterized in that the antibody or binding fragment thereof that specifically binds to mesothelin of any one of the above specific examples comprises (a) a first antigen-binding portion comprising the single domain antibody; and (b) a second antigen-binding portion that specifically binds to a second epitope.

[0096] In addition, the present invention may be characterized in that the antibody or binding fragment thereof that specifically binds to mesothelin of any one of the above specific examples is a full-length antibody, Fab, Fab', (Fab')2, Fv, single-chain Fv (scFv), scFv-scFv, minibody, diabody or second single-domain antibody in the second antigen-binding portion.

[0097] In addition, the present invention may be characterized in that the first antigen-binding portion and the second antigen-binding portion of the antibody or binding fragment thereof that specifically binds to mesothelin of any one of the above specific examples are fused to each other via a peptide linker.

[0098]

[0099] The antibody or antigen-binding fragment thereof comprising an antibody specifically binding to the above mesothelin may be a multispecific antigen binding protein (MABP) or antigen-binding fragment thereof in which a single domain antibody of the present invention is fused to another mesothelin-specific antibody, a full-length 4-chain antibody or antigen-binding fragment thereof.

[0100] The multispecific antigen binding protein to which the single domain antibody is fused comprises (a) a first antigen binding portion comprising an antibody that specifically binds to mesothelin as described herein; and (b) a second antigen binding portion that specifically binds to a second epitope.

[0101] The second epitope may be an antigen other than mesothelin, or a second epitope in mesothelin.

[0102] The second antigen-binding moiety can be a full-length antibody, a Fab, a Fab', (Fab')2, an Fv, a single-chain Fv (scFv), an scFv-scFv, a minibody, a diabody, or a second single-domain antibody. Additionally, the second antigen-binding moiety can comprise a heavy chain comprising a VH and a light chain comprising a VL. In some embodiments, the first antigen-binding moiety can be fused to the second antigen-binding moiety at the N-terminus of the heavy chain, the N-terminus of the light chain, the N-terminus of the Fc region, the C-terminus of the heavy chain, or the C-terminus of the light chain. In some embodiments, the second antigen-binding moiety can comprise a Fab or a scFv. In some embodiments, the first antigen-binding moiety can be fused to the second antigen-binding moiety at the C-terminus of the Fab or scFv. In some embodiments, the second antigen-binding moiety can comprise a full-length 4-chain antibody. In some embodiments, the first antigen-binding moiety can be fused to a second antigen-binding moiety via a peptide linker. In some embodiments, the second antigen-binding moiety can comprise an Fc region, such as an IgG1 Fc, an IgG2 Fc, an IgG3 Fc, or an IgG4 Fc.

[0103] The multispecific antigen binding protein comprises at least two antigen-binding moieties that specifically bind at least two different epitopes. Some of the at least two antigen-binding moieties may be identical, as long as they have binding sites for two different epitopes. The multispecific antigen binding protein may also comprise any one of the different antigen-binding moieties of the antibodies described herein.

[0104] Additionally, the multispecific antibody can have any suitable number of valencies and any suitable number of specificities for the first and / or second epitopes of mesothelin. In some embodiments, the multispecific antibody can be divalent, trivalent, tetravalent, pentavalent, hexavalent, or higher valencies for mesothelin. In some embodiments, the multispecific antibody can be trispecific, tetraspecific, but is not limited thereto.

[0105] Techniques for producing multispecific antibodies according to the present invention include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (e.g., Milstein and Cuello, Nature 305: 537 (1983)); WO 93 / 08829; Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole" engineering (e.g., patent US5731168). Multispecific antibodies can also be produced by engineering electrostatic steering effects for producing antibody Fc-heterodimeric molecules (WO2009 / 089004A1); cross-linking of two or more antibodies or fragments (e.g., patent US4676980, and Brennan et al., Science, 229: 81 (1985)); The use of leucine zippers to produce bispecific antibodies (e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); the use of "diabody" technology to produce bispecific antibody fragments (e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and the use of single-chain Fv (sFv) dimers (e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and the production of trispecific antibodies, as described, for example, by Tutt et al., J. Immunol. 147: 60 (1991); and the creation of polypeptides comprising tandem single-domain antibodies (e.g., patent US20110028695; and Conrath et al., J. Biol. Chem., 2001; 276(10):7346-50).

[0106]

[0107] In the present invention, the term "a" indicates the presence of a specified number of binding sites in an antigen binding protein. For example, the terms "bivalent," "trivalent," "tetravalent," "pentavalent," and "hexavalent" indicate the presence of two binding sites, three binding sites, four binding sites, five binding sites, and six binding sites in an antigen binding protein.

[0108] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, full-length four-chain antibodies include those having heavy and light chains that include an Fc region. Full-length heavy-chain-only antibodies may comprise a heavy chain variable domain (e.g., VHH) and an Fc region.

[0109]

[0110] The antibody of the present invention may be a humanized antibody.

[0111] In the present invention, the term "humanized antibody" means an antibody in the form of a human antibody in which all or part of the CDR sequence of a mouse monoclonal antibody is grafted onto a human antibody. For example, a humanized variable region can be produced by recombining the CDRs of a mouse monoclonal antibody with FRs derived from a human antibody, and this can be produced by recombining it with the constant region of a specific human antibody.

[0112]

[0113] A single domain antibody according to the present invention may be a chimeric antibody. Particular chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In some embodiments, a chimeric antibody may comprise a non-human variable region (e.g., a variable region derived from a camelid species, such as a llama) and a human constant region. Additionally, a chimeric antibody may be humanized. Typically, a non-human antibody is humanized to reduce immunogenicity toward humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody will optionally also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody may be substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0114] The term "humanized antibody" is used as a subset of "chimeric antibody".

[0115] A "humanized" form of a non-human (e.g., llama or camelid) antibody is a chimeric antibody that contains minimal sequence derived from a non-human immunoglobulin. In some embodiments, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a CDR (as defined below) of the recipient are replaced with residues from a CDR of a non-human species (donor antibody) such as mouse, rat, rabbit, camel, llama, alpaca, or non-human primate having the desired specificity, affinity, and / or capacity.

[0116] In one example, framework ("FR") residues of a human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may include residues that are not found in either the recipient antibody or the donor antibody. These modifications may be made to further improve antibody performance, such as binding affinity.

[0117]

[0118] The term "antibody effector function" refers to those biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region), which vary with antibody isotype. Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation. "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component (Clq) of the complement system to antibodies (of the appropriate subclass) bound to their cognate antigen. "Antibody-dependent cell-mediated cytotoxicity" or ADCC refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) causes these cytotoxic effector cells to specifically bind antigen-bearing target cells and subsequently kill the target cells with cytotoxins.

[0119] As used herein, the term "binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair. Binding affinity may be expressed as Kd, Koff, Kon, or Ka. As used herein, the term equilibrium dissociation constant "KD" or "Kd" refers to the dissociation constant of a particular antibody-antigen interaction, describes the concentration of antigen required to occupy one-half of all antibody-binding domains present in solution of an antibody molecule at equilibrium, and is expressed in units of M. Measurements of KD assume that all binding agents are in solution. The dissociation constant (KD or Kd) is used as an indicator of the affinity of an antibody for its antigen. For example, simple analysis is possible using the Scatchard method, which utilizes antibodies labeled with various marker preparations, as well as over-the-counter drug products and assay kits, following the user's manual and experimental operating instructions attached to the kit. KD values ​​derived using these methods are expressed in units of M (Mols).

[0120]

[0121] Another aspect of the present invention provides an antibody conjugate comprising the antibody of claim 1 or an antigen-binding fragment thereof conjugated to an immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent, diagnostic agent, antiviral agent, antimicrobial agent or drug.

[0122] The above terms, “antibody specifically binding to mesothelin” and “antigen-binding fragment thereof” are as described above.

[0123] An antibody or antigen-binding fragment thereof comprising an antibody that specifically binds to the above mesothelin can be conjugated to an immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent, diagnostic agent, antiviral agent, antimicrobial agent or drug.

[0124] The term "conjugation" above refers to joining together, and in the present invention, the conjugate refers to a form in which an immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent, diagnostic agent, antiviral agent, antimicrobial agent, or drug is joined to a single-domain antibody of the present invention. The conjugation can be accomplished by any known method without limitation.

[0125]

[0126] Another aspect of the present invention provides a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof, comprising a single domain antibody that specifically binds to mesothelin, an expression vector comprising the nucleic acid molecule, and a host cell transformed with the expression vector.

[0127] In addition, another aspect of the present invention provides a method for producing an antibody or an antigen-binding fragment thereof, comprising the steps of (a) culturing the host cell under conditions that allow the antibody to be expressed; and (b) recovering the expressed antibody or antigen-binding fragment thereof.

[0128]

[0129] In the present invention, DNA encoding an antibody or antigen-binding fragment thereof, including an antibody that specifically binds to mesothelin as disclosed herein, can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding antibody heavy and light chains). Isolated and subcloned hybridoma cells (or phage- or yeast-derived colonies) can serve as a preferred source of such DNA. More particularly, the isolated DNA (which may be modified) can be used to clone constant and variable region sequences for the production of antibodies.

[0130] One exemplary method involves extracting RNA from selected cells, converting it to cDNA, and amplifying it by PCR using antibody-specific primers. Suitable primers are well known in the art and readily available from many commercial sources, as exemplified herein. To express a recombinant human or non-human antibody isolated by screening a combinatorial library, the DNA encoding the antibody is cloned into a recombinant expression vector and introduced into a host cell, including mammalian cells, insect cells, plant cells, yeast, and bacteria. In some embodiments, a modulator is introduced into and expressed by monkey COS cells, NS0 cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce the desired construct.

[0131] In the present invention, the nucleic acid molecule is present within a vector, where appropriate, together with a promoter that controls expression of the nucleic acid.

[0132] The term "vector" is used in its most general sense and includes any intermediate vehicle for nucleic acids, which allows the nucleic acid to be introduced into, for example, a prokaryotic and / or eukaryotic cell and, where appropriate, integrated into the genome. Vectors of this type are specifically replicated and / or expressed within the cell. The vector may comprise a plasmid, phagemid, bacteriophage, or viral genome. The plasmid generally refers to an extrachromosomal genetic material construct capable of replicating independently of chromosomal DNA, typically a circular DNA duplex.

[0133] Expression vectors containing antibody coding sequences and appropriate transcription and translation control signals can be constructed using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0134]

[0135] In the present invention, the transformant into which the expression vector has been introduced refers to a host cell or a recombinant host cell, meaning a cell into which the expression vector has been introduced. The recombinant host cell and host cell refer not only to a specific target cell but also to the progeny of such a cell. Since certain modifications may occur in subsequent generations due to mutation or environmental influences, such progeny may not be substantially identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Such cells may contain a vector as described above.

[0136] Additionally, substantial quantities of the antibodies disclosed herein can be produced using molecular biology techniques recognized in the art and current protein expression methodologies. More specifically, nucleic acid molecules encoding such antibodies can be incorporated into well-known and commercially available protein production systems, including various types of host cells, to provide preclinical, clinical, or commercial quantities of the desired pharmaceutical product. In some embodiments, nucleic acid molecules encoding the antibodies are engineered into vectors or expression vectors that provide efficient integration into selected host cells and subsequent high levels of antibody expression.

[0137]

[0138] Specifically, nucleic acid molecules encoding the antibodies disclosed herein and vectors comprising these nucleic acid molecules can be used to transfect suitable mammalian, plant, bacterial, or yeast host cells, although prokaryotic systems may also be used. Transfection can be accomplished by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of DNA into the nucleus. Nucleic acid molecules can also be introduced into mammalian cells via viral vectors. Methods for transforming mammalian cells are well known in the art. Methods for transforming plant cells are also well known in the art and include, for example, Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

[0139] Many commercially available host-expression vector systems can be used to express the antibodies disclosed herein. Such host-expression systems not only represent vehicles in which a coding sequence of interest can be expressed and subsequently purified, but also represent cells capable of in situ expressing a molecule of the invention when transformed or transfected with the appropriate nucleotide coding sequence. Such systems include microorganisms, such as bacteria (e.g., E. coli, B. subtilis, Streptomyces), transformed with a recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vector containing a regulator coding sequence; yeast (e.g., Saccharomyces, Pichia) transfected with a recombinant yeast expression vector containing a regulator coding sequence; insect cell systems infected with a recombinant virus expression vector (e.g., baculovirus) containing a regulator coding sequence; Plant cell systems (e.g., Nicotiana, Arabidopsis, duckweed, maize, wheat, potato, etc.) infected with a recombinant virus expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transfected with a recombinant plasmid expression vector containing a regulator coding sequence (e.g., Ti plasmid); or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0140] If the antibodies disclosed herein are produced by recombinant expression or any of the other techniques disclosed herein, they may be purified by any method known in the art for purifying immunoglobulins, or more generally by any other standard technique for purifying proteins.

[0141]

[0142] One aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer or a tumor, comprising an antibody or an antigen-binding fragment thereof, or an antibody conjugate that specifically binds to mesothelin, as an active ingredient.

[0143] In addition, the present invention provides a composition comprising an antibody or an antigen-binding fragment thereof, or an antibody conjugate that specifically binds to mesothelin, as an active ingredient, for the prevention or treatment of cancer or tumor.

[0144] The present invention also provides a method for preventing or treating cancer, comprising administering to a subject a pharmaceutical composition comprising a pharmaceutically effective amount of an antibody or antigen-binding fragment thereof comprising a single domain antibody disclosed herein, or an antibody conjugate comprising the antibody or antigen-binding fragment thereof.

[0145] The present invention also provides the use of an antibody or antigen-binding fragment thereof, or an antibody conjugate comprising the antibody or antigen-binding fragment thereof, comprising a single domain antibody disclosed herein for use in the prevention or treatment of cancer.

[0146] The terms “antibody”, “antigen-binding fragment”, “antibody conjugate”, “mesothelin”, and “specifically” are as described above.

[0147]

[0148] In the present invention, the cancer or tumor is a cancer caused by mesothelin overexpression, and specifically, may be selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, mesothelial cancer, peritoneal cancer, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer, but is not limited thereto.

[0149] A pharmaceutical composition according to the present invention may comprise an antibody or antigen-binding fragment thereof comprising one or more (e.g., two or three) single domain antibodies described herein, or a drug conjugate comprising said antibody or antigen-binding fragment thereof.

[0150] By administering the pharmaceutical composition according to the present invention to a subject, specifically a cancer patient, cancer can be prevented or treated.

[0151] Furthermore, the pharmaceutical compositions according to the present invention can be formulated as desired using techniques recognized in the relevant art, depending on the form of the antibody described herein, the intended mode of delivery, and numerous other variables. Furthermore, they can be formulated to contain suitable pharmaceutically acceptable carriers, including excipients and auxiliaries, which are relatively inert substances well known in the relevant art and which facilitate administration or aid in processing the active compounds into pharmaceutically optimized formulations for delivery. For example, various pharmaceutically acceptable carriers, including vehicles, auxiliaries, and diluents, are readily available from numerous commercial sources. Furthermore, classes of pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, tonicity adjusters, stabilizers, wetting agents, and the like, are also available. Specific, non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0152]

[0153] In another embodiment, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.

[0154] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier in addition to the antibody of the present invention or a binding fragment thereof, and the carrier may comprise a non-naturally occurring carrier.

[0155] The term "pharmaceutically acceptable carrier" of the present invention refers to a carrier or diluent that does not stimulate a living organism and does not inhibit the biological activity and properties of the administered compound. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

[0156] The above pharmaceutical composition may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories, and may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations are prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

[0157]

[0158] Additionally, the pharmaceutical composition of the present invention can be administered to a subject in a pharmaceutically effective amount.

[0159] In the present invention, the term "subject" may be used interchangeably with "subject" to refer to a subject to whom a pharmaceutical composition is to be administered. The subject refers to a mammal, including, but not limited to, a human, a bovine, a horse, a cat, a dog, a rodent, or a primate. In some embodiments, the subject is a human.

[0160] As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined according to factors including the type and severity of the individual, age, sex, type of disease, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered singly or in multiple doses. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects by taking all of the above factors into consideration, and it can be easily determined by those skilled in the art.

[0161] Additionally, the pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) to a subject, depending on the intended method. For example, parenteral administration may be used, but is not limited to, in vivo administration may be achieved via various routes. Appropriate formulations and routes of administration may be selected depending on the intended use and treatment regimen.

[0162] Dosage and frequency vary depending on the half-life of the antibody in the patient. Generally, human antibodies have the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. Dosage and frequency may vary depending on whether the treatment is prophylactic or therapeutic.

[0163] The duration of treatment depends on the disease being treated, the patient's age and condition, the stage and type of the patient's disease, and how the patient responds to treatment. The clinician will closely monitor the effectiveness of the therapy and may make any adjustments as needed. When agents are used in combination, two or more therapeutic agents may be administered simultaneously or sequentially in any order. That is, the antibodies disclosed herein may be administered prior to, concurrently with, or subsequent to the administration of the second therapeutic agent.

[0164]

[0165] The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent, or may be administered together with another active ingredient that exhibits a preventive or therapeutic effect against mesothelin-related diseases. When the pharmaceutical composition of the present invention is administered together with another active ingredient, this may be referred to as "combined administration" or "combined administration." In this case, the antibody or antigen-binding fragment thereof that specifically binds to mesothelin of the present invention and the other active ingredient may be administered as a single mixture or in separate forms.

[0166] In the present invention, "combined administration," "combined administration," or "combining" should be understood to refer to simultaneous, separate, sequential, or reverse administration, with no limit to the order. That is, combination is not limited to simultaneous administration, but can also be a dosage form in which the two substances act together on an individual, enabling each substance to perform its respective function at a level equal to or greater than its original function. Therefore, when the term "combined administration" is used herein, whether the administration is sequential, reverse, or separate, the order of administration is not particularly limited, and the interval between administrations of the secondary components can be such that the beneficial effects of the combination are not lost.

[0167] For example, the pharmaceutical composition of the present invention may be administered together with a substance selected from the group consisting of 5-fluorouracil (5-FU), oxaliplatin, doxorubicin, sorafenib, and cetuximab.

[0168]

[0169] As used herein, the term "prevention" may refer to any action that inhibits or delays the onset of a disease by administering the composition of the present invention. Furthermore, as used herein, the term "treatment" may refer to any action that improves or beneficially alters the symptoms of a disease by administering the composition of the present invention.

[0170]

[0171] Another aspect of the present invention is a composition for diagnosing or monitoring a mesothelin-overexpressing cancer or tumor, comprising an antibody or antigen-binding fragment thereof that binds to mesothelin.

[0172] Additionally, the present invention relates to a composition comprising an antibody or antigen-binding fragment thereof that binds to mesothelin for use in diagnosing or monitoring cancer or tumors.

[0173]

[0174] Another aspect of the present invention is a method for providing information for diagnosing a mesothelin-overexpressing cancer or tumor, comprising a step of detecting mesothelin in a biological sample through an antigen-antibody reaction using an antibody or an antigen-binding fragment thereof that binds to mesothelin.

[0175]

[0176] In the present invention, the antibody or antigen-binding fragment thereof comprising an antibody that specifically binds to mesothelin may be linked to, fused to, conjugated to (e.g., covalently or non-covalently) or otherwise associated with a diagnostic moiety or a biocompatibility modifier. For example, a peptide or polypeptide (e.g., a biotoxin, a biomarker, a purification tag, etc.), a protein, a polymer, a nucleic acid molecule, a small molecule, a mimetic, a synthetic drug, an inorganic molecule, an organic molecule, or a radioisotope may be conjugated or associated therewith.

[0177] Additionally, the antibody or antigen-binding fragment thereof, including the antibody that specifically binds to mesothelin, may be conjugated or associated with a diagnostic or detectable agent, marker, or reporter, which may be a biological molecule (e.g., a peptide or nucleotide), a small molecule, a fluorophore, or a radioisotope. The labeled modulator may be useful for monitoring the development or progression of a disease associated with mesothelin, such as cancer, or for determining the efficacy of a particular therapy comprising the antibodies disclosed herein (i.e., theragnosis), or as part of a clinical trial procedure to determine a future course of treatment. Such markers or reporters may also be useful for purifying the antibodies disclosed herein.

[0178]

[0179] The method for providing information for the above diagnosis can detect mesothelin by reacting an antibody specific to mesothelin of the present invention with a separated biological sample of an individual suspected of having cancer or a tumor and detecting the formation of an antigen-antibody complex, thereby providing information for diagnosing cancer or a tumor.

[0180] Specifically, it may be a method for providing information for diagnosing cancer or a tumor, or a method for diagnosing cancer or a tumor, comprising the steps of: (a) treating a separated biological sample of an individual suspected of having a mesothelin-related disease with the antibody to detect mesothelin through an antigen-antibody reaction; and (b) comparing the level of mesothelin detected in (a) with a control group, and determining that the individual is suffering from mesothelin-related cancer or a tumor if the level of mesothelin is higher than that of the control group.

[0181] In the present invention, the term "biological sample" may be a cell sample, i.e., a sample containing cells such as cancer cells.

[0182]

[0183] The term "antigen-antibody complex" of the present invention refers to a combination of a mesothelin protein antigen in a sample and an antibody of the present invention that recognizes the same, and the formation of such an antigen-antibody complex can be detected by any method, such as a colorimetric method, an electrochemical method, a fluorimetric method, a luminometric method, a particle counting method, a visual assessment, or a scintillation counting method. However, it is not necessarily limited to these and various applications and applications are possible.

[0184] Various labels can be used to detect the above antigen-antibody complex. Specific examples include, but are not limited to, enzymes, fluorescent substances, ligands, luminescent substances, microparticles, or radioactive isotopes. At this time, enzymes used as detection labels include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase, β-latamase, etc.; fluorescent substances include fluorescein, Eu3+, Eu3+ chelate, or cryptate, etc.; ligands include biotin derivatives, etc.; luminescent substances include acridinium ester, isoluminol derivatives, etc.; microparticles include colloidal gold, colored latex, etc.; and radioactive isotopes include 57Co, 3H, 125I, 125I-Bonton Hunter reagent, etc.

[0185] For example, antigen-antibody complexes can be detected using an enzyme-linked immunosorbent assay (ELISA). ELISA includes various ELISA methods, including a direct ELISA that uses a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA that uses a labeled secondary antibody that recognizes a capture antibody in a complex of antibodies that recognize the antigen attached to the solid support, a direct sandwich ELISA that uses another labeled antibody that recognizes the antigen in a complex of antibodies and antigen attached to the solid support, and an indirect sandwich ELISA that uses a labeled secondary antibody that recognizes the antibody after reacting it with another antibody that recognizes the antigen in a complex of antibodies and antigen attached to the solid support.

[0186] The above antibodies may have a detection label, and if they do not have a detection label, these antibodies can be captured and identified by treating them with another antibody having a detection label.

[0187]

[0188] The antibody or antigen-binding fragment of the present invention can be used to detect mesothelin or mesothelin-expressing cells or to determine the amount of mesothelin or mesothelin-expressing cells. By detecting or determining the amount of a complex between mesothelin and the antibody or antigen-binding fragment of the present invention, mesothelin or mesothelin-expressing cells are detected or the amount of mesothelin or mesothelin-expressing cells is determined.

[0189] The formation of a complex indicates the presence of mesothelin or mesothelin-expressing cells. Detection or determination of such amounts can be accomplished by a variety of methods, including but not limited to immunodetection using an antibody or antigen-binding fragment of the present invention. Methods utilizing antibodies to detect peptides or proteins are well known and include ELISA, competitive binding assays, and similar methods. Typically, such assays utilize an antibody or antibody fragment that specifically binds to a target peptide or protein, which is then conjugated directly or indirectly to a label providing detection, such as an indicator enzyme, radiolabel, fluorophore, or paramagnetic particle. The methods of the present invention enable quantitative and / or qualitative evaluations, such as absolute and / or relative evaluations, of mesothelin levels or the levels of mesothelin-expressing cells.

[0190]

[0191] Hereinafter, the present invention will be described in more detail through examples. These examples are intended to explain the present invention more specifically, and the scope of the present invention is not limited by these examples.

[0192]

[0193] Example 1: Confirmation of alpaca immunization and antibody formation against MSLN antigen.

[0194] Alpacas were immunized three times by intramuscular injection with human MSLN immune antigen (100 µg) mixed with GERBU immune adjuvant. To confirm antibody formation in the blood, 10 ml of blood was collected before immunization, on days 2 to 3, and 14 days (Day 42) after the third immunization. Expi-CHO cells expressing MSLN were seeded at 2 x 10 per well. 5 After dispensing the cells individually, they were washed three times with DPBS. Afterwards, they were blocked with FACS buffer (2% FBS, 1% EDTA, 0.02% sodium azide in DPBS) at 4°C for 1 hour. After washing three times with DPBS, the serum diluted with FACS buffer was treated with the cells and reacted for 1 hour at 4°C. After washing the cells three times with DPBS, they were treated with MonoRab Anti-camelid VHH cocktail-alexa 647 antibody and reacted for 30 minutes at 4°C while blocking from light. Afterwards, the cells were washed three times with DPBS and analyzed using an Accuri C6 (BD) instrument.

[0195]

[0196] Example 2: Library construction and evaluation

[0197] To amplify the gene of a single-domain antibody against the immune antigen of Example 1, peripheral blood mononuclear cells (PBMCs) were isolated from blood using Ficoll. Total RNA extracted from the isolated PBMCs was amplified using specific primers to produce a gene fragment encoding a single-domain antibody, which was then cloned into the pComb3x vector.

[0198]

[0199] Example 3: Library amplification

[0200] The immune library produced in Example 2 was transformed into the XL1-blue strain. The transformed XL1-blue was inoculated into 10 ml of 2xYT medium containing 2% glucose and 100 ug / ml ampicillin and cultured in a shaking shaker at 37°C. The culture was incubated until the absorbance reached 0.5 at OD600, and 1x10 M13K07 phage (Invitrogen) was added. 11 pfu / ml. After that, the culture was stationary cultured at 37°C for 30 minutes and then further cultured for 30 minutes at 200 rpm in a shaking incubator. The culture was centrifuged at room temperature and 4,000 rpm for 15 minutes and the supernatant was removed. Next, 10 ml of 2xYT medium containing 100 ug / ml ampicillin and 50 ug / ml kanamycin was added to resuspend the culture pellet and cultured overnight at 250 rpm in a shaking incubator at 30°C. After that, the supernatant was recovered by centrifugation at 4,000 rpm for 30 minutes at 4°C. The supernatant was precipitated using the PEG precipitation method and centrifuged at 12,000 rpm at 4°C for 30 minutes. After resuspending the pellet in PBS, centrifugation was performed at 12,000 rpm at 4°C for 5 minutes, and the supernatant was stored at 4°C until use.

[0201]

[0202] Example 4: Biopanning

[0203] To select single-domain antibodies specific for immune antigens, microplates were coated with immune antigens at a concentration of 5 ug / ml overnight at 4°C. The library to be used in the experiment to select single-domain antibodies was dispensed into a 96-well microplate and incubated at room temperature for 30 minutes. The library was then transferred to a new well and incubated at room temperature for 30 minutes. This process was repeated four times to reduce nonspecific binding of the library. The library was transferred to a 1.7 ml tube and stored at 4°C until use. The microplate coated with immune antigens was then washed five times with PBST and blocked with 5% skimmed milk for 2 hours at room temperature. After washing five times with PBST, the library with reduced nonspecific binding was dispensed at 5 x 10^12 virions / well with binding solution (2.5% skimmed milk, 0.5% tween20) and incubated at room temperature for 30 minutes. In addition, the plate was washed 10 times with washing solution (PBS, 0.5% Tween 20). Single-domain antibodies specifically bound to the immune antigen were selectively eluted by adding 5 μg of the immune antigen per well and incubating at 500 rpm at room temperature for 30 minutes. The eluted phage was infected with XL1-blue cells in logarithmic growth and then plated on 2xYT agar. The panning for the second selection was repeated under the same conditions as above. Each single phage clone generated on the agar plate was amplified and screened using FACS.

[0204]

[0205] Example 5: Phage screening

[0206] Expi-CHO cells overexpressing immune antigens were washed with DPBS and centrifuged at 1,200 rpm for 3 minutes at room temperature. The supernatant was removed, and the cells were resuspended in 2% skim milk and blocked at 4°C for 30 minutes. The cells were centrifuged at 1,200 rpm for 3 minutes at room temperature, the supernatant was removed, and the cells were washed three times with DPBS and inoculated into 3 × 10 5 Cells were dispensed into a 96-well microplate at a density of 100 μl / well. Polyclonal phage obtained by biopanning or monoclonal phage amplified from a single colony was added to each well, and the cells were incubated at 4°C for 1 hour and washed twice with DPBS. Afterwards, M13 major coat protein Alexa Flour 647 (Santacruz) was dispensed onto the cells, and the cells were incubated at 4°C for 30 minutes while protected from light. The cells were washed twice with DPBS, resuspended in DPBS, and then subjected to FACS analysis using an Accuri C6 (BD) instrument. Clones selected using the FACS system were subjected to base sequence analysis.

[0207]

[0208] Example 6: Expression and purification of single-domain antibodies

[0209] The clones selected in Example 5 above were cloned into the TGEX-Fc expression vector. For the expression of single-domain antibodies, Expi-CHO cells with a viability of 95-99% were counted and 7x10 6Cells were added to 25 ml of Expi-CHO expression medium (Gibco) and cultured overnight at 125 rpm in a 37°C shaking incubator maintained at 8% CO2. Then, 20 µg of plasmid DNA and 1 ml of OptiPRO medium were added to a mixture of 80 µl of ExpiFectamine CHO reagent (Gibco) and 920 µl of OptiPRO medium, reacted at room temperature for 5 minutes, and then added to the cultured cells. The cells were cultured for 20 hours at 125 rpm in a shaking incubator maintained at 8% CO2. Afterwards, 150 µl of ExpiFectamine CHO enhancer (Gibco) and 6 ml of ExpiCHO Feed (Gibco) were added and cultured for 5 days at 125 rpm in a 32°C shaking incubator maintained at 5% CO2. The cultured cells were centrifuged at 4,000 rpm for 30 minutes at 4°C, and the supernatant was filtered using a 0.2 μm syringe filter. The supernatant was then loaded onto a HiTrap protein G HP column (GE Healthcare), washed with PBS, and single-domain antibodies were separated from the column using IgG elution buffer (Thermo). The eluted sample was neutralized with 1 M Tris-HCl (pH 9.0) and stored at 4°C until use.

[0210]

[0211] Example 7: Evaluation of binding of single-domain antibodies to immune antigens using ELISA

[0212] The binding of the purified single-domain antibody in Example 6 to the immune antigen was confirmed through ELISA. Specifically, the immune antigen was dispensed at a concentration of 0.3 ug / ml into a 96-well microplate and coated overnight at 4°C. The microplate coated with the immune antigen was washed three times with PBST and blocked with 2% skim milk for 2 hours at room temperature. After washing three times with PBST, the purified single-domain antibody was serially diluted from 10 nM and reacted for 1 hour at room temperature. The microplate was washed three times with PBST and reacted with Peroxidase Affinipure Goat anti-human IgG for 1 hour at room temperature. Afterwards, the microplate was washed three times with PBST, treated with TMB One Component HRP microwell substrate (Surmodics), and developed for 5 minutes at room temperature. The reaction was then stopped with 1 M sulfuric acid, and the absorbance was measured to evaluate the binding of the single-domain antibody to the immune antigen.

[0213]

[0214] Example 8: Evaluation of binding ability of single domain antibodies to immune antigens using FACS

[0215] The binding of the purified single-domain antibody and the immune antigen in Example 6 was confirmed through FACS. Specifically, Expi-CHO cells overexpressing the immune antigen (MSLN) were washed with DPBS and centrifuged at 1,200 rpm for 3 minutes at room temperature. The supernatant was removed, the cells were resuspended in 2% skim milk, and blocked at 4°C for 30 minutes. The cells were centrifuged at 1,200 rpm for 3 minutes at room temperature, the supernatant was removed, and the cells were washed twice with DPBS. Afterwards, 3x10 5Cells were seeded at a density of 100 μl / well and treated with single-domain antibodies at various concentrations. An isotype control antibody was used as a negative control. The cells were incubated at 4°C for 1 hour and then washed twice with DPBS. Afterwards, anti-human IgG Fc APC antibody (Biolegend) was treated and incubated for 30 minutes at 4°C, protected from light. The cells were washed twice with DPBS, resuspended in 100 μl of DPBS, and subjected to FACS analysis using an Accuri C6 (BD) instrument.

[0216]

[0217] Example 9 Affinity evaluation of single domain antibodies with immune antigens

[0218] The affinity (Kd) of the purified single-domain antibody and the immune antigen protein in Example 6 was evaluated using an Octet RED 96e (ForteBio) instrument. Specifically, the anti-human Fc-coated biosensor tip (ForteBio) was saturated to 1.5 nm in a 96-well microplate (Greiner) dispensed with 5 ug / ml of the single-domain antibody. The MSLN protein was diluted two-fold in 1X kinetic buffer (ForteBio) to a concentration of 10 to 400 nM, and reacted at 30°C with stirring at 1,000 rpm. The binding and dissociation reactions of the samples were analyzed for 200 and 400 ch, respectively. The resulting data were analyzed using a 1:1 interaction model (global fitting) method.

[0219]

[0220] Experimental Example 1: Confirmation of antibody production by MSLN immunization and biopanning

[0221] To select single-domain antibodies that specifically bind to the MSLN protein, alpacas were immunized three times with the MSLN antigen (see Fig. 1), and single-domain antibodies that specifically bind to MSLN-expressing cell lines in the blood were evaluated.

[0222] As a result, as can be confirmed in Fig. 2, it was confirmed that the amount of single-domain antibodies specifically binding to the MSLN-expressing cell line in the blood after immunization increased compared to before immunization. Phages binding to the MSLN antigen were selected through biopanning from an immune library constructed from blood whose titer was confirmed, and it was confirmed that the phages selected in the second round were amplified (Fig. 3).

[0223]

[0224] Experimental Example 2: Production of MSLN antigen-specific single-domain antibodies

[0225] A single-domain antibody clone that specifically binds to the human MSLN protein, an immune antigen, was selected using the method described in Example 5 above, and its base sequence was analyzed. The amino acid sequences of the selected clones are as follows: [Table 1 - Single-domain Antibody Sequence] and [Table 2 - CDR Sequence of Single-domain Antibody].

[0226] Single domain antibody Single domain antibody sequence Sequence number MSLN Nb#A2QVQLVESGGGLVQPGGSQRLSCVASRNISSIGVMGWYRQAPGKERELVAALGSRGATELGDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTARYGMYVYWGQGTQVTVSS1

[0227] Single domain antibody CDR1 SEQ ID NO CDR2 SEQ ID NO CDR3 SEQ ID NO MSLN Nb#A2RNISSIGV2LGSRGAT3TARYGMYVY4

[0228] In addition, using the selected clone, a single domain antibody specifically binding to MSLN containing human IgG Fc was expressed and purified by the method described in Example 6, and the amino acid sequence of the single domain antibody containing human IgG Fc is shown in [Table 3 - Sequence of human IgG1 fusion single domain antibody].

[0229] Single domain antibody Single domain antibody sequence + Human IgG1 Fc sequence Sequence number MSLN Nb#A2-hIgG1QVQLVESGGGLVQPGGSQRLSCVASRNISSIGVMGWYRQAPGKERELVAALGSRGATELGDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTARYGMYVYWGQGTQVTVSSGPGGPEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK5

[0230] Experimental Example 3: Antigen binding capacity of MSLN-specific single-domain antibodies

[0231] The antigen binding ability of the MSLN-specific single-domain antibody produced in Experimental Example 2 was evaluated using ELISA and FACS according to the methods described in Examples 7 and 8 above.

[0232] As a result, as can be confirmed in Fig. 4, it was confirmed that the single domain antibody produced in Experimental Example 2 binds to the MSLN protein in a concentration-dependent manner.

[0233] In addition, as can be confirmed in Fig. 5, it was confirmed that the single domain antibody produced in Experimental Example 2 had a binding capacity of 2.349 nM (EC50 value) in Expi-CHO cells expressing the MSLN protein.

[0234]

[0235] Experimental Example 4: Antigen Affinity Evaluation of MSLN-Specific Single-Domain Antibodies

[0236] The MSLN-specific single-domain antibody produced in Example 1 was evaluated for affinity with the MSLN protein in the same manner as described in Example 9.

[0237] As a result, as can be confirmed in Fig. 6, it was confirmed that the single domain antibody produced in Experimental Example 2 had an antigen affinity of 0.577 nM between the MSLN protein and the MSLN protein.

[0238]

[0239] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. An antibody or antigen-binding fragment thereof comprising a single domain antibody that specifically binds to mesothelin.

2. In paragraph 1, An antibody or antigen-binding fragment thereof, wherein the single domain antibody comprises a CDR1 comprising an amino acid sequence represented by SEQ ID NO: 2; a CDR2 comprising an amino acid sequence represented by SEQ ID NO: 3; and a CDR3 comprising an amino acid sequence represented by SEQ ID NO:

4.

3. An antibody or an antigen-binding fragment thereof, wherein the single domain antibody in claim 1 comprises an amino acid sequence having sequence number 1 or at least 95% sequence homology thereto.

4. An antibody or antigen-binding fragment thereof, comprising at least one amino acid substitution according to any one of claims 1 to 3.

5. An antibody or antigen-binding fragment thereof, wherein at least one amino acid substitution in paragraph 4 is a conservative substitution.

6. An antibody or antigen-binding fragment thereof, wherein in paragraph 5, at least one amino acid substitution is a substitution of an amino acid with a non-genetically encoded amino acid or a synthetic amino acid.

7. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the single domain antibody is a heavy chain-only antibody fused to an Fc fragment.

8. An antibody or antigen-binding fragment thereof, wherein the heavy chain-only antibody in paragraph 7 is monomeric or multimeric.

9. An antibody or an antigen-binding fragment thereof, wherein the Fc fragment in paragraph 7 is human IgG1, IgG2, IgG3 or IgG4.

10. An antibody or antigen-binding fragment thereof, wherein the single domain antibody in claim 7 is fused to an Fc fragment via a peptide linker.

11. An antibody or antigen-binding fragment thereof, wherein the heavy chain-only antibody in claim 7 comprises an amino acid sequence having sequence ID NO: 5 or at least 95% sequence homology thereto.

12. An antibody or antigen-binding fragment thereof comprising at least one amino acid substitution in claim 11.

13. An antibody or antigen-binding fragment thereof, wherein at least one amino acid substitution in claim 12 is a conservative substitution.

14. An antibody or antigen-binding fragment thereof, wherein at least one amino acid substitution is a substitution of an amino acid with a non-genetically encoded amino acid or a synthetic amino acid.

15. An antibody or antigen-binding fragment thereof, comprising (a) a first antigen-binding portion comprising the single domain antibody; and (b) a second antigen-binding portion that specifically binds to a second epitope.

16. An antibody or an antigen-binding fragment thereof, in claim 15, wherein the second antigen-binding portion is a full-length antibody, Fab, Fab', (Fab')2, Fv, single-chain Fv (scFv), scFv-scFv, minibody, diabody or a second single-domain antibody.

17. An antibody or an antigen-binding fragment thereof, in claim 15, wherein the first antigen-binding portion and the second antigen-binding portion are fused to each other via a peptide linker.

18. An antibody conjugate comprising the antibody of claim 1 or an antigen-binding fragment thereof conjugated to an immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent, diagnostic agent, antiviral agent, antimicrobial agent or drug.

19. A nucleic acid molecule encoding the antibody of paragraph 1 or an antigen-binding fragment thereof.

20. An expression vector comprising the nucleic acid molecule of claim 19.

21. A host cell transformed with the expression vector of clause 20. 22.(a) a step of culturing the host cell of claim 21 under conditions that allow the antibody to be expressed; and (b) a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to mesothelin, comprising the step of recovering the expressed antibody or antigen-binding fragment thereof.

23. A pharmaceutical composition for preventing or treating cancer or a tumor, comprising the antibody of paragraph 1 or an antigen-binding fragment thereof, or the antibody conjugate of paragraph 18, as an active ingredient.

24. A pharmaceutical composition according to claim 23, wherein the cancer or tumor is selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, mesothelial cancer, peritoneal cancer, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

25. A pharmaceutical composition according to claim 23, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

26. A composition for diagnosing or monitoring cancer or a tumor, comprising an antibody or fragment thereof that specifically binds to mesothelin according to any one of claims 1 to 3.

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