Antibodies or antigen binding fragments thereof

Abstract

Provided is an antibody or an antigen-binding fragment thereof that is useful for the examination of cancer, in particular for early cancer. An antibody or an antigen-binding fragment thereof, comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence represented by SAS, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6.

Description

Technical Field

[0001] The present disclosure relates to an antibody or an antigen-binding fragment thereof, etc. Background Art

[0002] Cancer is the leading cause of death in Japan, and the number of deaths is increasing year by year as the average life expectancy increases. Early diagnosis is considered to be the most important factor in reducing the number of cancer deaths, but there are often no characteristic symptoms in the early stages, and by the time the cancer is diagnosed, it has already progressed, making treatment difficult. In addition, in cancers such as pancreatic cancer, even if the cancerous tissue can be removed surgically, the recurrence rate after surgery is high, and postoperative monitoring is essential. Therefore, simple cancer screening methods such as blood tests play an important role because they can achieve reliable results through appropriate implementation.

[0003] So far, many antibodies against cancer antigens have been produced, especially monoclonal antibodies, which are commonly used because of their high sensitivity and specificity and can be used semi-permanently. In the case of pancreatic cancer, which is the most difficult to diagnose at an early stage, a test method using CA19-9 monoclonal antibodies has been reported (Non-Patent Document 1). In addition, it has been shown that extracellular vesicles secreted by cancer cells are involved in the generation, progression, drug resistance, and metastasis of cancer (Non-Patent Document 2). Prior art literature Non-patent literature

[0004] Non-patent literature 1: Erxi W. Shuang Z. Kruttika B. Qingyong M.: CA19-9 and pancreatic cancer. Clin Adv Hematol Oncol. 201311(1):53-55. Non-patent document 2: Sunami Y. Haubler J. Zourelidis A. Kleef J.: Cancer-associated fibroblasts and tumor cells in pancreatic cancer microenvironment and metastasis: Paracrine regulators, reciprocation and exosome. Cancers. 202214(3):744. Summary of the invention Problems to be solved by the invention

[0005] An object of the present disclosure is to provide an antibody or an antigen-binding fragment thereof that is useful for cancer, particularly for early cancer detection. Technical solutions to the problem

[0006] In view of the above-mentioned problems, the present inventors conducted in-depth research and found that if it is an antibody or an antigen-binding fragment thereof, it contains: a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence represented by SAS, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6, then the above-mentioned problems can be solved. The present inventors further conducted research based on the above-mentioned insights and completed the present invention. That is, the present invention includes the following aspects.

[0007] Item 1. An antibody or antigen-binding fragment thereof comprising: A heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, A heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, A heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, A light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence represented by SAS, and A light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:6.

[0008] Item 2. The antibody or antigen-binding fragment thereof according to Item 1, wherein the antibody is a monoclonal antibody.

[0009] Item 3. The antibody or antigen-binding fragment thereof according to Item 1, wherein the antibody is IgG.

[0010] Item 4. The antibody or antigen-binding fragment thereof according to Item 1, wherein the antigen-binding fragment is Fab, F(ab')2, miniantibody, scFv-Fc, Fv, scFv, diabody, triabody or tetrabody.

[0011] Item 5. A polynucleotide comprising a coding sequence for the antibody or antigen-binding fragment thereof according to any one of Items 1 to 4.

[0012] Item 6. A cell comprising the polynucleotide described in Item 5.

[0013] Item 7. A cancer testing agent comprising the antibody or antigen-binding fragment thereof according to any one of Items 1 to 4.

[0014] Item 8. The test agent according to Item 7, wherein the cancer is metastatic cancer.

[0015] Item 9. The test agent according to Item 7, wherein the cancer is pancreatic cancer.

[0016] Item 10. The test agent according to Item 7, wherein the cancer is early pancreatic cancer.

[0017] Item 11. A pharmaceutical composition for diagnosing cancer, comprising the antibody or antigen-binding fragment thereof according to any one of Items 1 to 4 labeled with a diagnostic agent.

[0018] Item 12. The pharmaceutical composition according to Item 11, wherein the diagnostic agent is a radioactive substance.

[0019] Item 13. The pharmaceutical composition according to Item 11, wherein the cancer is pancreatic cancer.

[0020] Item 14. A pharmaceutical composition for treating cancer, comprising the antibody or antigen-binding fragment thereof described in any one of Items 1 to 4.

[0021] Item 15. The pharmaceutical composition for treating cancer according to Item 14, wherein the cancer is pancreatic cancer.

[0022] Item 16. A pharmaceutical composition for inhibiting cancer metastasis, comprising the antibody or antigen-binding fragment thereof described in any one of Items 1 to 4.

[0023] Item 17. The pharmaceutical composition for treating cancer according to Item 16, wherein the cancer is pancreatic cancer. Effects of the Invention

[0024] According to the present disclosure, an antibody or an antigen-binding fragment thereof that is useful for cancer, particularly for early cancer detection, can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The figure shows the clone PEMb14, which was produced in Example 1 to produce an antibody that reacts with extracellular vesicles. The left figure shows the presence of one lymphocyte observed by transmitted light. The right figure shows that the lymphocyte-derived antibody reacts with extracellular vesicles observed by immunofluorescence staining. DETAILED DESCRIPTION

[0026] In the present specification, the expressions “comprise” and “include” include the concepts of “comprises”, “comprising”, “consisting essentially of” and “consisting only of”.

[0027] In the present specification, amino acid sequences are represented by single letter symbols.

[0028] 1. Antibodies or antigen-binding fragments thereof The present disclosure, in one embodiment, relates to an antibody or an antigen-binding fragment thereof (sometimes referred to as "the antibody or fragment thereof of the present disclosure" in the present specification), comprising: a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence represented by SAS, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6. This is described below.

[0029] The antibody or fragment thereof disclosed herein contains a heavy chain variable region and a light chain variable region. The heavy chain variable region contains: a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3. The light chain variable region contains: a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence represented by SAS, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.

[0030] "Heavy chain" refers to a polypeptide chain of about 50 to 70 kDa, wherein the amino terminal portion contains a variable region of about 120 to 130 or more amino acids, and the carboxyl terminal portion contains a constant region. The constant region can be one of five different types, called alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), depending on the amino acid sequence of the heavy chain constant region. Different heavy chains have different sizes, with α, δ, and γ containing approximately 450 amino acids, and μ and ε containing approximately 550 amino acids. When these different types of heavy chains are combined with light chains, five known classes of antibodies are produced, including the four subclasses of IgG (IgG1, IgG2, IgG3, and IgG4), namely IgA, IgD, IgE, IgG, and IgM.

[0031] "Light chain" refers to a polypeptide chain of about 25 kDa, wherein the amino terminal portion contains a variable region of about 100 to about 110 or more amino acids, and the carboxyl terminal portion contains a constant region. The approximate length of a light chain is 211 to 217 amino acids. Depending on the amino acid sequence of the constant domain, there are two different types, called kappa (κ) or lambda (λ).

[0032] "Variable region" refers to a part of the light chain or heavy chain of an antibody, which is generally located at the amino terminus of the light chain or heavy chain, with a length of about 120 to 130 residues in the heavy chain and about 100 to 110 residues in the light chain, and is involved in binding and specificity to a specific antigen. The variable region has a wide range of sequence differences between different antibodies, and the variability of the sequence is concentrated in the CDR (complementary determining region) present in the variable region. The part with low variability in the variable region is called the framework region. The CDR of the light chain and heavy chain is mainly involved in the interaction between the antibody and the antigen.

[0033] "CDR" refers to one of the three hypervariable regions (H1, H2 or H3) in the non-framework region of the β-sheet framework of the antibody heavy chain (H) variable region, or one of the three hypervariable regions (L1, L2 or L3) in the non-framework region of the β-sheet framework of the antibody light chain (L) variable region. CDR is a hypervariable region sequence dispersed within the framework region sequence.

[0034] The antibodies or fragments thereof disclosed herein may preferably contain a heavy chain variable region and / or a light chain variable region, The heavy chain variable region comprises: (a) the amino acid sequence shown in SEQ ID NO: 13, or (b) an amino acid sequence having an identity of 85% or more to the amino acid sequence shown in SEQ ID NO: 13, The light chain variable region comprises: (c) the amino acid sequence shown in SEQ ID NO: 14, or (d) an amino acid sequence having 85% or more identity with the amino acid sequence shown in SEQ ID NO:14.

[0035] The amino acid sequence (b) has an amino acid mutation relative to the amino acid sequence (a). In addition, the amino acid sequence (d) has an amino acid mutation relative to the amino acid sequence (c).

[0036] Examples of amino acid mutations include substitution, deletion, addition, insertion, etc. From the viewpoint of being less likely to impair the antigen-binding ability of the antibody or fragment thereof disclosed herein, the amino acid mutation is preferably a substitution, and more preferably a conservative substitution.

[0037] "Conservative substitution" means that an amino acid residue is replaced with an amino acid residue having a similar side chain. For example, substitutions between amino acid residues having basic side chains such as lysine, arginine, and histidine are conservative substitutions. In addition, substitutions between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having non-polar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine are also conservative substitutions.

[0038] The "consistency" of an amino acid sequence refers to the degree of consistency of the amino acid sequences of two or more amino acid sequences that can be compared. Therefore, the higher the consistency of two amino acid sequences, the higher the consistency or similarity of their sequences. The level of consistency of the amino acid sequence is determined, for example, using the sequence analysis tool FASTA and using default parameters. Alternatively, the algorithm BLAST based on Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc Natl Acad Sci USA. 87: 2264-2268 (1990), Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc Natl Acad Sci USA. 90: 5873-7 (1993)) can be used to determine. A program called BLASTX based on this BLAST algorithm has been developed. The specific techniques of these analysis methods are well known, and the website of the National Center of Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov / ) may be referred to. In addition, the "identity" of the base sequence is also defined based on the above.

[0039] In each of the amino acid sequences (b) and (d), the identity may be preferably 90% or more, more preferably 95% or more, further preferably 98% or more, and further preferably 99% or more.

[0040] An example of the amino acid sequence (b) includes: (b') An amino acid sequence in which one or more amino acids are substituted, deleted, added, or inserted (preferably substitution, more preferably conservative substitution) relative to the amino acid sequence shown in SEQ ID NO: 13.

[0041] An example of the amino acid sequence (d) includes: (d') An amino acid sequence in which one or more amino acids are substituted, deleted, added, or inserted (preferably substitution, more preferably conservative substitution) relative to the amino acid sequence shown in SEQ ID NO: 14.

[0042] In each of the amino acid sequences (b') and (d'), the plurality of moieties is, for example, 2 to 20, preferably 2 to 10, more preferably 2 to 5, and even more preferably 2 or 3.

[0043] Antibodies are used to include monoclonal antibodies and polyclonal antibodies. Antibodies can be of any isotype, such as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, IgM, etc. Antibodies can be human antibodies or non-human antibodies. Examples of non-human antibodies include, but are not limited to, mouse antibodies, rat antibodies, rabbit antibodies, monkey antibodies, chimpanzee antibodies, etc. Antibodies can also be chimeric antibodies such as mouse-human chimeric antibodies. Antibodies can be partially or fully humanized antibodies. The antibodies disclosed herein are preferably monoclonal antibodies.

[0044] In the present specification, the antigen-binding fragment of an antibody is not particularly limited as long as it contains heavy chain CDR1 to 3 and light chain CDR1 to 3, and examples thereof include Fab, F(ab')2, minibody, scFv-Fc, Fv, scFv, diabody, triabody, tetrabody, etc.

[0045] Fab includes a heavy chain fragment including a heavy chain variable region and CH1 in a heavy chain constant region and a light chain including a light chain variable region and a light chain constant region (CL), and has a structure in which the heavy chain variable region and the light chain variable region are associated through the above-mentioned non-covalent intermolecular interaction or bonded through a disulfide bond. In Fab, CH1 and CL may also form a disulfide bond with each other through the thiol groups of the cysteine ​​residues respectively present thereon.

[0046] F(ab')2 has a structure comprising two pairs of the above-mentioned Fab, wherein CH1s form a disulfide bond with each other via thiol groups of cysteine ​​residues contained therein.

[0047] Minibodies have a structure in which two fragments of the heavy chain variable region constituting the following scFV are bound to CH3 and the CH3 fragments are associated with each other through non-covalent intermolecular interactions.

[0048] scFv-Fc has a structure in which two antibody fragments including the following scFv, CH2 and CH3 are associated with each other through non-covalent intermolecular interactions between CH3 similarly to the above-mentioned miniantibodies, and disulfide bonds are formed between thiol groups of cysteine ​​residues contained in each CH3.

[0049] Fv is also called the smallest structural unit of an antibody, and is a structure in which the heavy chain variable region and the light chain variable region are associated through non-covalent intermolecular interactions. In Fv, the thiol groups of cysteine ​​residues present in the heavy chain variable region and the light chain variable region can also form disulfide bonds with each other.

[0050] scFv is a structure in which the C-terminus of the heavy chain variable region and the N-terminus of the light chain variable region are connected by a linker, or a structure in which the N-terminus of the heavy chain variable region and the C-terminus of the light chain variable region are connected by a linker, and is also called a single-chain antibody.

[0051] Diabodies, triabodies and tetrabodies are structures in which the scFv described above forms dimers, trimers and tetramers, respectively, and similarly to Fv, etc., they are associated in a structurally stable state through non-covalent intermolecular interactions between variable regions.

[0052] The antibodies or fragments thereof disclosed in the present invention have binding properties (preferably specific binding properties) to extracellular vesicles. The International Society for Extracellular Vesicles (ISEV) defines extracellular vesicles as "particles that do not have a nucleus released from the cell (cannot replicate) and are surrounded by a lipid bilayer membrane." Extracellular vesicles are divided into (1) exosomes (diameter 50-150nm), (2) microvesicles (diameter 100-1000nm), and (3) apoptotic bodies (diameter 5μm) according to their production mechanisms. As constituent protein components of extracellular vesicles, known are CD9, CD63, CD81 tetraspanins, ALIX, Tsg101, Hsp70, Hsp90, various integrins, various selectins, CD40, Annexin V, etc. The extracellular vesicles to which the antibodies or fragments thereof of the present disclosure bind may preferably be extracellular vesicles released from cancer cells, more preferably extracellular vesicles released from pancreatic cancer cells, and even more preferably extracellular vesicles released from early pancreatic cancer cells.

[0053] In the present specification, early pancreatic cancer refers to pancreatic cancer of stage 0 or stage 1. In the present specification, the staging of cancer is based on the UICC 8th edition classification.

[0054] The antibodies or fragments thereof disclosed herein may also be bound or fused to other peptides, oligopeptides or proteins. Examples of other peptides, oligopeptides or proteins include albumin (e.g., serum albumin), protein tags (e.g., biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag), fluorescent proteins (e.g., GFP, Azami-Green, ZsGreen, GFP2, HyPer, Sirius, BFP, CFP, Turquoise, Cyan, TFP1, YFP, Venus, ZsYellow, Banana, KusabiraOrange, RFP, DsRed, AsRed, Strawberry, Jred, KillerRed, Cherry, HcRed, mPlum), luminescent proteins (e.g., luciferase, β-galactosidase, chloramphenicol acetyltransferase, β-glucuronidase), secretory signal sequences (e.g., Igκ signal sequences), protease recognition sequences (e.g., TEV protease recognition sequences), expression enhancing sequences, soluble sequences, multimerization domains (e.g., cartilage oligomer matrix protein domains, leucine zipper domains, collagen-like domains, cholera toxin B subunit domains, Tetrabrachion coiled core domains, reovirus σ1 protein domains, hepatitis D antigen domains), etc. In the case where the antibody or fragment thereof disclosed in the present invention is combined or fused with a multimerization domain, an antibody or antigen-binding fragment thereof homologous or heterologous to the antibody or fragment thereof disclosed in the present invention may be further combined or fused to the multimerization domain to form a multimer (homomultimer or heteromultimer).

[0055] The antibodies or fragments thereof of the present disclosure may also be chemically modified as long as the binding ability to the antigen is not significantly impaired.

[0056] The C-terminus of the antibody or fragment thereof disclosed herein may be a carboxyl group (-COOH), a carboxylate group (-CO - ), amide group (-CONH2), ester group (-COOQ). Here, as Q in the ester group, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. can be used. 1-6 Alkyl; for example, cyclopentyl, cyclohexyl, etc. 3-8 Cycloalkyl; for example, phenyl, α-naphthyl, etc. C 6-12 Aryl; for example, benzyl, phenethyl, etc. phenyl-C 1-2 Alkyl; α-naphthylmethyl etc. α-naphthyl-C 1-2 Alkyl etc.C 7-14 Arylalkyl; pivaloyloxymethyl, etc.

[0057] The carboxyl group (or carboxylate group) other than the C-terminus of the antibody or fragment thereof disclosed herein may also be amidated or esterified. As the ester in this case, for example, the ester at the C-terminus described above is used.

[0058] The antibodies or fragments thereof disclosed herein may also include: the amino group of the amino acid residue at the N-terminus is protected by a protecting group (e.g., a C-terminus such as a formyl group or an acetyl group); 1-6 Alkanoyl etc. 1-6 acyl, etc.), a pyroglutamic acid-containing product in which the N-terminal glutamine residue that can be cleaved in vivo is converted to glutamic acid, a product in which the substituents on the side chains of the amino acids in the molecule (e.g., -OH, -SH, amino, imidazolyl, indolyl, guanidinyl, etc.) are protected by appropriate protecting groups (e.g., C-terminal groups such as formyl and acetyl) 1-6 Alkanoyl etc. 1-6 Acyl groups, etc.), or conjugated proteins called glycoproteins etc. to which sugar chains are bound.

[0059] The antibodies or fragments thereof of the present disclosure can be produced, for example, by a method comprising the steps of culturing a host transformed with a polynucleotide comprising a coding sequence for the antibodies or fragments thereof of the present disclosure (polynucleotide of the present disclosure), and collecting fractions containing the antibodies of the present disclosure.

[0060] "Polynucleotide" can be a nucleotide in the form of a polymer of any length of any one of deoxyribonucleotides or ribonucleotides or their analogs. The sequence of a polynucleotide is composed of 4 nucleotide bases: adenine (A), cytosine (C), guanine (G) and thymine (T). When the polynucleotide is RNA, uracil (U) is used instead of thymine. Polynucleotides can include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, etc. In addition, polynucleotides refer to both double-stranded molecules and single-stranded molecules. In addition, polynucleotides can also be subjected to chemical modifications as exemplified below. In order to prevent decomposition by hydrolases such as nucleases, the phosphate residues (phosphate esters) of each nucleotide can be replaced with chemically modified phosphate residues such as thiophosphates (PS), methyl phosphates, dithiophosphates, etc. In addition, the hydroxyl group at position 2 of the sugar (ribose) of each ribonucleotide may be replaced by OR (R represents, for example, -CH3, -CH2CH2OCH3, -CH2CH2NHC(NH)NH2, -CH2CONHCH3, -CH2CH2CN, etc.). Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl or cationic functional group at position 5 of the pyrimidine base or replacing the carbonyl group at position 2 with a thiocarbonyl group, etc. Furthermore, the phosphate moiety or the hydroxyl moiety may be modified by, for example, biotin, amino group, lower alkylamine group, acetyl group, etc., but is not limited to these.

[0061] The polynucleotides of the present invention are not particularly limited as long as they contain the coding sequence of the antibody or its fragment disclosed herein. Preferably, the polynucleotides of the present invention contain the above-mentioned coding sequence in a state capable of expressing the antibody or its fragment disclosed herein. In addition to the above-mentioned coding sequence, the polynucleotides of the present invention may also contain other sequences. As other sequences, there can be mentioned: secretion signal peptide coding sequences, promoter sequences, enhancer sequences, repressor sequences, insulator sequences, replication origins, drug resistance gene coding sequences, etc., which are adjacent to the antibody coding sequence disclosed herein. In addition, the polynucleotides of the present invention may be linear polynucleotides or circular polynucleotides (vectors, etc.).

[0062] Specific examples of the polynucleotides disclosed herein include: (I) a polynucleotide comprising a base sequence encoding at least one selected from the group consisting of a heavy chain, a heavy chain variable region, and heavy chain CDRs1-3 of the antibody or a fragment thereof disclosed herein, (II) a polynucleotide comprising a base sequence encoding at least one selected from the group consisting of a light chain, a light chain variable region, and light chain CDRs1-3 of the antibody or a fragment thereof disclosed herein, (III) a combination of a polynucleotide comprising a base sequence encoding at least one selected from the group consisting of a heavy chain, a heavy chain variable region, and heavy chain CDRs1-3 of the antibody or a fragment thereof disclosed herein and a polynucleotide comprising a base sequence encoding at least one selected from the group consisting of a light chain, a light chain variable region, and light chain CDRs1-3 of the antibody or a fragment thereof disclosed herein, and the like.

[0063] The polynucleotides disclosed herein can also be obtained by chemical synthesis or by isolation from an appropriate source (e.g., cDNA obtained from cells expressing antibodies such as hybridoma cells selected for antibody expression) by PCR amplification using synthetic primers that can hybridize to the 3' and 5' ends of the sequence or by cloning using oligonucleotide probes specific for a specific nucleic acid sequence. Subsequently, the amplified nucleic acid generated by PCR can be cloned into a replicable cloning vector using any method known in the art.

[0064] When the polynucleotides disclosed in the present invention are obtained, vectors for producing antibodies or fragments thereof can be prepared by using recombinant DNA techniques known in the art, and expression vectors containing coding sequences of antibodies or fragments thereof and appropriate transcription and translation control signals can be constructed using known methods. In addition, constant regions vary greatly between species such as humans and rabbits, but constant regions between antibodies of the same species are very similar, so antibodies of humans, rabbits, etc. can be prepared by cloning into vectors containing nucleotide sequences encoding constant regions of each species.

[0065] The expression vector can be introduced into the host cell by conventional methods, and the transfected cells can then be cultured by conventional methods to produce the antibodies or fragments thereof disclosed herein. In order to express the entire immunoglobulin molecule, vectors encoding both the heavy chain and the light chain can be simultaneously expressed in the host cell.

[0066] To express the antibodies or fragments thereof of the present disclosure, a variety of host-expression vector systems may be utilized. For example, bacteria (e.g., Escherichia coli, Bacillus subtilis) transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing an antibody coding sequence, yeast (e.g., Saccharomyces Pichia) transformed with a recombinant yeast expression vector containing an antibody coding sequence, insect cell lines infected with a recombinant virus expression vector containing an antibody coding sequence (e.g., baculovirus), plant cell lines infected with a recombinant virus expression vector (e.g., Cauliflower Mosaic Virus, Tobacco Mosaic Virus (TMV)) or transformed with a recombinant plasmid expression vector containing an antibody coding sequence (e.g., Ti plasmid), or mammalian cell lines (e.g., COS, CHO, HEK293, 3T3 cells) having a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter).

[0067] When the antibodies or fragments thereof of the present disclosure are produced by recombinant expression, they can be purified by methods known in the art for purifying immunoglobulin molecules, such as chromatography (e.g., ion exchange chromatography, protein A chromatography, gel permeation column chromatography), centrifugation, salting out, etc. Further, the antibodies or fragments thereof of the present disclosure can be fused with known heterologous polypeptide sequences to facilitate purification. For example, the antibodies or fragments thereof of the present disclosure can be purified by recombinantly adding a polyhistidine tag (His tag), a FLAG tag, a hemagglutinin tag (HA tag) or a myc tag.

[0068] 2. Cells In one aspect, the present disclosure relates to a cell comprising a polynucleotide of the present disclosure.

[0069] The above-mentioned cells include, for example, cells transformed with the polynucleotide of the present disclosure.

[0070] The above-mentioned cells are preferably isolated cells. The cells may be cultured cells, primary cultured cells, or secondary cultured cells. The cells may also be cell line cells. The cells may also be iPS cells.

[0071] Examples of the above cells include Escherichia coli K12 and other Escherichia coli, Bacillus subtilis MI114 and other Bacillus bacteria, Saccharomyces cerevisiae AH22 and other yeasts, Sf cell lines derived from Spodoptera frugiperda or HighFive cell lines derived from Trichoplusia ni, insect cells such as olfactory nerve cells, animal cells, etc. As animal cells, preferably cultured cells derived from mammals, specifically COS7 cells, CHO cells, HEK293 cells, HEK293FT cells, Hela cells, PC12 cells, N1E-115 cells, SH-SY5Y cells, etc. can be mentioned.

[0072] 3. Disease examination medicine The disease testing agent disclosed in the present invention is characterized by comprising the antibody disclosed in the present invention or a fragment thereof. The testing agent disclosed in the present invention is a drug for testing the possibility of having cancer, particularly early stage cancer.

[0073] The disease testing agent disclosed in the present invention can be used in the following method: a method for testing (or determining) the possibility of having cancer, especially early stage cancer, comprising the step of measuring the amount or concentration of an antigen bound to the antibody or fragment thereof disclosed in the biological sample collected from the subject (sometimes referred to as "the testing method disclosed in the present invention" in this specification). The method is described below.

[0074] The cancer to be detected is not particularly limited, and includes cancers of all types and stages. As the type of cancer, pancreatic cancer is preferred, and early pancreatic cancer (stage 0 or stage 1) is particularly preferred. As the type of pancreatic cancer, for example, pancreatic ductal carcinoma, pancreatic endocrine tumor, intraductal papillary mucinous neoplasm of the pancreas, mucinous cystic neoplasm, acinar cell carcinoma, etc., preferably pancreatic ductal carcinoma. In addition, the cancer to be detected may be metastatic cancer. As the metastatic site, when the primary cancer is pancreatic cancer, for example, the liver, peritoneum, lung, lymph node, bone, etc. may be mentioned.

[0075] The subject is an organism to be examined in the present disclosure, and its species is not particularly limited. Examples of the subject's species include various mammals such as humans, monkeys, rabbits, mice, rats, dogs, and cats, and preferably humans.

[0076] The subject's cancer status is not particularly limited. Examples of the subject include: a sample whose cancer status is unknown, a sample with no history of cancer, a sample with a history of cancer and having completed cancer treatment, and a sample that has been determined to have cancer (or not have cancer) by other determination methods.

[0077] Biological samples are not particularly limited as long as they are samples that can contain antigens that bind to the antibodies or fragments thereof disclosed herein. Examples of biological samples include body fluids such as whole blood, serum, plasma, saliva, spinal fluid, joint fluid, urine, tissue fluid, sweat, tears, saliva, and samples derived from these body fluids. As samples derived from body fluids, there are no particular limitations as long as they are samples prepared from body fluids, and examples include samples obtained by concentrating and purifying antigens bound to the antibodies or fragments thereof disclosed herein from body fluids, etc. As body fluids, preferably whole blood, serum, plasma, etc. can be mentioned. The biological sample can be used alone or in combination of two or more.

[0078] Biological samples can be collected from subjects by methods known to those skilled in the art. For example, whole blood can be collected by using a syringe or the like to collect blood. It should be noted that it is desirable that blood collection be performed by medical personnel such as doctors and nurses. Serum is a portion of blood from which blood cells and specific blood coagulation factors have been removed, and can be obtained, for example, as a supernatant after blood coagulation. Plasma is a portion of blood from which blood cells have been removed, and can be obtained, for example, as a supernatant when centrifugation is performed without coagulating blood.

[0079] The inspection method disclosed herein includes a step of determining the amount or concentration of an antigen bound to an antibody or a fragment thereof disclosed herein, and the method is not particularly limited. As this method, for example, immunoassay can be cited. Immunoassay can be widely used whether it is a direct method, an indirect method, a uniform method, an inhomogeneous method, a competitive method, a non-competitive method, etc. As an immunoassay, more specifically, for example, ELISA (for example, a direct method, an indirect method, a sandwich method, a competitive method, etc.), an immunoradiometric assay (RIA), an immunoradiometric assay (IRMA), an enzyme immunoassay (EIA), a sandwich EIA, immunochromatography, Western blotting, immunoprecipitation, a slit or dot blot assay, immunohistostaining, fluorescent immunoassay, an immunoassay using avidin-biotin or streptavidin-biotin system, an immunoassay using a surface plasmon resonance (SPR) method, etc.

[0080] Since the antibody or fragment thereof of the present disclosure binds to extracellular vesicles, the amount or concentration of extracellular vesicles constituting the antigen of the antibody of the present disclosure can be measured by combining with an antibody or fragment thereof against a constituent component of the extracellular vesicle. As an example of its inspection method, there can be cited: sandwich method, ExoCounter, exosome sensor chip, etc. In addition, the detection method can be used alone or in combination of two or more.

[0081] In the step of determining the amount or concentration of the antigen bound to the antibody or fragment thereof disclosed herein, the type of label (e.g., labeled antibody, etc.) used when detecting the antigen is not particularly limited. As labels, for example, fluorescent substances, luminescent substances, pigments, enzymes, colloidal gold, radioactive isotopes, etc. can be cited. Among these, enzyme labels such as peroxidase and alkaline phosphatase are more preferred from the viewpoints of safety, economy, detection sensitivity, etc.

[0082] The details of the method for measuring and calculating the amount or concentration of the antigen bound to the antibody or fragment thereof of the present disclosure are described.

[0083] As one embodiment thereof, there can be exemplified the steps of contacting the antibody or fragment thereof disclosed herein with a biological sample collected from a subject, and measuring the amount of antigen bound to the antibody or fragment thereof disclosed herein.

[0084] The contact method with the biological sample collected from the subject is not particularly limited, and an appropriate method can be selected according to the type of method for measuring the amount or concentration of the antigen bound to the antibody or fragment thereof of the present disclosure (eg, various immunoassays, etc.).

[0085] Examples of the contact method include a method in which only one of the antibody or fragment thereof disclosed herein and the biological sample is fixed to a solid phase, a method in which both of them are contacted without being fixed to a solid phase, etc. Among these, from the viewpoint of efficiency, etc., a method in which only the antibody or fragment thereof disclosed herein is fixed to a solid phase is preferably mentioned.

[0086] When only one of the antibody or fragment thereof of the present disclosure and the biological sample is immobilized on a solid phase, after immobilization, the solid phase is preferably washed with a solution containing tris(hydroxymethylaminomethane) (Tris) and / or an ether-type nonionic surfactant.

[0087] As a solid phase, as long as the antibody or its fragment and the biological sample disclosed in the present invention can be fixed, there is no particular limitation. As the solid phase, for example, culture plates, slides, membranes, etc. containing polyethylene, glass, nitrocellulose, etc. as the main components can be cited. The solid phase can be coated with a component for more easily fixing the antibody or its fragment and the biological sample disclosed in the present invention, such as a reactive compound (for example, a compound with a reactive group, colloidal gold, etc.). As a compound with a reactive group, a group that can form a covalent bond with a sugar chain or a sugar chain derivative, such as (1H-imidazole-1-yl) carbonyl, succinimidyloxycarbonyl, epoxy, aldehyde, amino, thiol, carboxyl, azido, cyano, active ester (1H-benzotriazole-1-yloxycarbonyl, pentafluorophenyloxycarbonyl, p-nitrophenyloxycarbonyl, etc.) or halogenated carbonyl (chlorinated carbonyl, fluorinated carbonyl, brominated carbonyl, iodinated carbonyl) and the like can be cited.

[0088] As a compound which has an easily reactive group, epoxysilane, polylysine, etc. are mentioned, for example.

[0089] When a solid phase coated with a reactive compound is used, the reactive compound is preferably blocked with a buffer containing bovine serum albumin (BSA) or the like, and the blocking time is preferably 60 minutes or longer. In addition, the blocking solution preferably contains tris (hydroxymethylaminomethane) (Tris) and / or an ether-type nonionic surfactant.

[0090] There is no particular limitation on the method for determining the amount of antigen bound to the antibody or fragment thereof disclosed herein, and an appropriate method can be selected according to the type of the above-mentioned method for determining the amount or concentration of the antigen bound to the antibody or fragment thereof disclosed herein (e.g., various immunoassays, etc.). The determination can be performed, for example, by quantifying the signal derived from the label of the marker used. As a more specific method, for example, it can be performed in the following manner: contacting a labeled antibody or fragment thereof disclosed herein or a labeled antibody against an antigen with an antigen bound to the antibody or fragment thereof disclosed herein, and quantifying the signal derived from the label of the bound labeled antibody.

[0091] Based on the obtained signal amount, the amount of antigen bound to the antibody or fragment thereof of the present invention can be calculated. For example, in the case of a non-competitive method, the obtained signal amount can be directly used as the amount of antigen bound to the antibody or fragment thereof of the present invention. In addition, as another example, in the case of a competitive method, since the obtained signal amount is inversely proportional to the amount of antigen bound to the antibody or fragment thereof of the present invention, the amount of antigen bound to the antibody or fragment thereof of the present invention can be calculated from the signal amount obtained based on the relationship.

[0092] According to the inspection method of the present disclosure, the antigen value bound to the antibody or fragment thereof of the present disclosure can be provided as an indicator for detecting the presence of cancer such as pancreatic cancer, thereby assisting in determining the possibility of the presence of cancer such as pancreatic cancer.

[0093] As one embodiment, the inspection method of the present invention preferably further includes the following step: when the measured value of the amount or concentration of the antigen bound to the antibody or fragment thereof of the present invention is above a predetermined critical value, the step of determining that the subject has a high possibility of suffering from cancer such as pancreatic cancer. Here, "the possibility of suffering from cancer such as pancreatic cancer" means "the possibility of suffering from cancer such as pancreatic cancer at the time of biological sample collection".

[0094] According to the inspection method disclosed in the present invention, the possibility of having cancer can be determined. In addition, since the inspection method disclosed in the present invention can determine the possibility of having cancer with higher sensitivity, the inspection method disclosed in the present invention can more accurately determine that a subject who actually has cancer is "suffering from cancer" (that is, the possibility of misjudging as "not suffering from cancer" can be further reduced).

[0095] From the perspective of sensitivity, specificity, positive predictive value, negative predictive value, etc., the critical value can be appropriately set by a person skilled in the art, for example, it can be set to the average, percentile or maximum value of the value of the amount or concentration of the antigen bound to the antibody or fragment thereof of the present invention in a biological sample collected from a non-cancer subject. More specifically, for example, the amount or concentration of the antigen bound to the antibody or fragment thereof of the present invention in a biological sample collected from a non-cancer subject and a subject suffering from cancer such as pancreatic cancer is measured, and the measured value is used to perform a statistical analysis such as an analysis based on a receiver operating characteristic (ROC) curve, so that the critical value can be set (more specifically, a method using the Youden index can be exemplified).

[0096] In addition, the amount or concentration of the antigen bound to the antibody or fragment thereof of the present invention in a biological sample collected from the same subject before a certain period of time may also be used as a critical value. The "certain period of time" is not particularly limited as long as the amount or concentration of the antigen bound to the antibody or fragment thereof of the present invention can change within the same subject. For example, the period of about 1 month to 10 years, 2 months to 5 years, 3 months to 2 years, and 4 months to 1 year can be cited.

[0097] As an improved method of the test method disclosed in the present invention, there can be cited a method comprising the following steps: when the amount or concentration of the antigen bound to the antibody or fragment thereof disclosed in the present invention is higher than the amount or concentration of the antigen bound to the antibody or fragment thereof in a biological sample collected from the same subject a certain period of time ago, the step of judging that the subject is likely to have a cancer such as pancreatic cancer in the future is high. By using a test method including this step, the risk of developing cancer in the future is judged.

[0098] The "certain period" is not particularly limited as long as the amount or concentration of the antigen bound to the antibody or fragment thereof of the present disclosure can vary within the same subject, and examples thereof include periods of about 1 month to 10 years, 2 months to 5 years, 3 months to 2 years, and 4 months to 1 year.

[0099] The degree of "high" is not particularly limited, and the amount or concentration of the antigen bound to the antibody or fragment thereof of the present invention can be exemplified as 2 times or more, 4 times or more, 8 times or more, or 20 times or more of the amount or concentration of the antigen bound to the antibody or fragment thereof of the present invention in a biological sample collected from the same subject a certain period of time ago.

[0100] In the case where the test method disclosed herein determines that the subject is likely to suffer from a cancer such as pancreatic cancer, by further combining other diagnostic methods in the test method disclosed herein, it is possible to diagnose the subject with a cancer such as pancreatic cancer with higher accuracy. As other diagnostic methods, there are no particular restrictions, and various known diagnostic methods can be used. As such diagnostic methods, for example, biopsy, PET examination, CT examination, ultrasound examination, tumor marker examination, etc. can be cited. Among these, from the viewpoint of being able to diagnose pancreatic cancer with higher accuracy, preferably biopsy, PET examination, CT examination, ultrasound examination, etc. can be cited. The diagnostic method can be used alone or in combination of two or more.

[0101] The test agent disclosed herein may be in the form of a composition comprising the antibody disclosed herein or a fragment thereof. The composition may contain other components as required. Examples of other components include: a matrix, a carrier, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant, a thickener, a humectant, a colorant, a fragrance, a chelating agent, and the like.

[0102] The test agent of the present disclosure may also be in the form of a kit comprising the antibody or fragment thereof of the present disclosure. The kit may include instruments, reagents, etc. for implementing the test method of the present disclosure.

[0103] Examples of the instrument include test tubes, microtiter plates, agarose particles, latex particles, purification columns, epoxy-coated slides, colloidal gold-coated slides, and the like.

[0104] Examples of the reagent include antibodies against antigens of the antibodies of the present disclosure or fragments thereof, labeled antibodies thereof, and standard samples (positive control, negative control).

[0105] As an antibody against an antigen of the antibody of the present disclosure or a fragment thereof, an antibody against a molecule (eg, protein) to which the antibody of the present disclosure binds can be used.

[0106] The type of label used to make the labeled antibody is not particularly limited. As the label, for example, fluorescent substances, luminescent substances, pigments, enzymes, colloidal gold, radioisotopes, etc. can be enumerated. Among these, from the viewpoints of safety, economy, detection sensitivity, etc., enzyme labels such as peroxidase and alkaline phosphatase are preferred.

[0107] As a standard sample, an antigen of the antibody or fragment thereof of the present disclosure is used. The antigen of the antibody or fragment thereof of the present disclosure can be separated and purified from a biological sample such as an extracellular vesicle secreted by cancer cells by immunoprecipitation, for example, using the antibody or fragment thereof of the present disclosure.

[0108] In addition, the present invention also provides a pharmaceutical composition for diagnosing cancers such as pancreatic cancer, comprising the antibody or fragment thereof of the present invention labeled with a diagnostic agent. When it is determined by the test method of the present invention that the subject is highly likely to suffer from cancers such as pancreatic cancer, the subject can be diagnosed with cancers such as pancreatic cancer with higher accuracy by administering the pharmaceutical composition containing the antibody or fragment thereof of the present invention labeled with a diagnostic agent, for example, intravascularly, intramuscularly, subcutaneously, or intraperitoneally and performing a systemic examination.

[0109] The pharmaceutical composition for diagnosing a disease disclosed in the present invention may also contain a pharmaceutically acceptable carrier. As pharmaceutically acceptable carriers that can be used, there can be cited: phosphate buffered saline solution, water, emulsions such as oil / water emulsions, various types of wetting agents, and other standard pharmaceutical carriers known in the art. In addition, other ingredients, such as pharmaceutical grade stabilizers, buffers, preservatives, excipients, etc., may also be used in the pharmaceutical composition for diagnosing a disease disclosed in the present invention. The preparation of the pharmaceutical composition taking into account pH, isotonicity, stability, etc. can be implemented by known methods.

[0110] Examples of diagnostic reagents for labeling the antibodies or fragments thereof disclosed herein include radioactive substances, fluorescent materials, luminescent materials, bioluminescent materials, photoacoustic imaging materials, and the like. Examples of radioactive substances include zirconium ( 89 Zr), iodine ( 131 I. 125 I. 124 I. 123 I and 121 I), indium ( 115 In, 113In, 112 In and 111 In), Technetium ( 99 Tc), thallium ( 201 Ti、Ga( 68 Ga, 67 As fluorescent materials, there are umbelliferone, fluorescein, fluorescein isothiocyanate, etc., as luminescent materials, there are luminol, etc., as bioluminescent materials, there are luciferase, luciferin, aequorin, etc., as photoacoustic imaging materials, there are gold nanoparticles, single-layer carbon nanotubes, indocyanine green, methylene blue, etc.

[0111] As methods for labeling the antibodies or fragments thereof disclosed herein, there can be mentioned: methods of indirectly binding the compound using a linker such as polyethylene glycol, and methods of directly binding the compound by forming a disulfide bond at a cysteine ​​residue of the antibody or fragment thereof. In addition, the compound can be directly bound to the antibody or fragment thereof using a heterobifunctional crosslinker such as N-succinyl-3-(2-pyridyldithio) propionate. Further, the compound can be bound by oxidizing the sugar chain of the antibody Fc region.

[0112] The dosage of the antibody or fragment thereof labeled with a diagnostic agent contained in the pharmaceutical composition for diagnosing cancers such as pancreatic cancer disclosed herein is not particularly limited as long as it is a pharmacologically effective amount, and can be appropriately determined according to race, sex, age, type of cancer, etc., and is generally 0.01 to 1,000 mg / kg, preferably 0.1 to 100 mg / kg. The time for preferentially concentrating the antibody or fragment thereof labeled with a diagnostic agent disclosed herein at the binding site in the subject and for removing the antibody or fragment thereof that is not bound to the binding site to the background level can be appropriately determined according to the type of diagnostic agent used, the method of administration, etc., for example, about 6 to 48 hours after administration. In addition, in the case of monitoring a disease, for example, the time interval after administration is 5 to 20 days, and it is repeated from the initial diagnosis to 1 to 12 months.

[0113] The presence of an antibody or fragment thereof labeled with a diagnostic agent contained in the pharmaceutical composition for diagnosing a disease disclosed herein can be detected by examining the whole body of the subject using a known method. The detection method depends on the type of diagnostic agent used, and methods that can be used in the present invention include: computed tomography, positron emission tomography, magnetic resonance imaging, ultrasonography, photoacoustic imaging, and the like.

[0114] Diseases that can be diagnosed using the pharmaceutical composition of the present disclosure include cancers such as pancreatic cancer, and the pharmaceutical composition of the present disclosure is particularly useful for diagnosing cancers that highly express antigens of the antibodies or fragments thereof of the present disclosure. That is, when it is determined by the test method of the present disclosure that the subject is likely to have a cancer such as pancreatic cancer, the administration of the pharmaceutical composition of the present disclosure can diagnose the subject's pancreatic cancer with higher accuracy.

[0115] 4. Cancer treatment In the case where the test method disclosed herein determines that the subject is highly likely to have a cancer such as pancreatic cancer, or in the case where the subject is diagnosed with a cancer such as pancreatic cancer in combination with other diagnostic methods, the subject's cancer can be treated by treating the subject with cancer. In addition, the test method disclosed herein can determine the possibility of having a cancer such as pancreatic cancer with higher sensitivity, so the test method disclosed herein or in combination with other diagnostic methods can more accurately treat a subject who actually has cancer (i.e., the possibility of excluding a subject who actually has cancer from the treatment target can be further reduced).

[0116] There are no particular restrictions on the method of cancer treatment, and various known treatment methods can be used. As treatment methods, for example, chemotherapy, surgical treatment, radiotherapy, immunotherapy, etc. can be mentioned. These can be implemented according to known methods.

[0117] As the therapeutic drug used in chemotherapy, there is no particular limitation, and various anticancer drugs can be used. As anticancer drugs, for example, alkylating agents, metabolic antagonists, microtubule inhibitors, antibiotic anticancer drugs, topoisomerase inhibitors, platinum preparations, molecular targeted drugs, hormone agents, biological agents, etc. As alkylating agents, for example, cyclophosphamide, ifosfamide, nitrosourea, dacarbazine, temozolomide, nimustine, busulfan, melphalan, procarbazine, ranimustine, etc. can be mentioned. Examples of the metabolic antagonist include enocitabine, carmofur, capecitabine, tegafur, uracil tegafur, tegafur (Tegafur-Gimeracil-Oteracil Potassium), gemcitabine, cytarabine, cytarabine alkylphosphite (Cytarabine), and cytarabine alkylphosphite (Cytarabine). Ocfosfate, Nelarabine, Fluorouracil, Fludarabine, Pemetrexed, Pentostatin, Methotrexate, Cladribine, Doxifluridine, Hydroxycarbamide, Mercaptopurine, etc. Examples of microtubule inhibitors include alkaloid anticancer drugs such as Vincristine, and taxane anticancer drugs such as Docetaxel and Paclitaxel.Examples of antibiotic anticancer drugs include Mitomycin C, Doxorubicin, Epirubicin, Daunorubicin, Bleomycin, Actinomycin D, Aclarubicin, Idarubicin, Pirarubicin, Peplomycin, Mitoxantron, Amrubicin, Zinostatin Stimalamer, etc. Examples of topoisomerase inhibitors include CPT-11, Irinotecan, and Nogitecan, which have a topoisomerase I inhibitory effect; and Etoposide and Sobuzoxane, which have a topoisomerase II inhibitory effect. Examples of platinum preparations include cisplatin, nedaplatin, oxaliplatin, and carboplatin. Examples of the hormone agent include dexamethasone, finasteride, tamoxifen, anastrozole, exemestane, ethinylestradiol, chlormadinone, goserelin, bicalutamide, flutamide, prednisolone, leuprorelin, letrozole, estramustine, toremifene, fosfestrol, mitotane, methyltestosterone, medroxyprogesterone, and mepitiostane. Examples of biological agents include immune checkpoint inhibitors such as anti-PD-1 antibodies and anti-PD-L1 antibodies, interferon α, β, and γ, interleukin 2, Ubenimex, and dried BCG.Examples of molecular targeted drugs include rituximab, alemtuzumab, trastuzumab, cetuximab, panitumumab, imatinib, dasatinib, nilotinib, gefitinib, erlotinib, temsirolimus, bevacizumab, VEGF trap, sunitinib, sorafenib, tocilizumab, bortezomi, gemtuzumab-ozogamicin, ibritumomab-ozogamicin, ibritumomab-ozogamicin, and vegetative control. In addition to the molecular target drugs specified here, the following molecular target drugs may also be included: human epidermal growth factor receptor 2 inhibitors, epidermal growth factor receptor inhibitors, Bcr-Abl tyrosine kinase inhibitors, epidermal growth factor tyrosine kinase inhibitors, mTOR inhibitors, vascular endothelial growth factor receptor 2 inhibitors (α-VEGFR-2 antibodies) and other inhibitors targeting angiogenesis, various tyrosine kinase inhibitors such as MAP kinase inhibitors, inhibitors targeting cytokines, proteasome inhibitors, antibody-anticancer drug combination preparations and other molecular targeted drugs, etc. These inhibitors also include antibodies.

[0118] Representative pancreatic cancer therapeutic drugs include gemcitabine, TS-1, erlotinib, a combination of gemcitabine and erlotinib, a combination of gemcitabine and nab-paclitaxel, and a combination of four drugs (oxaliplatin, levofolinate, irinotecan, and fluorouracil).

[0119] In addition, the present invention also provides a pharmaceutical composition for treating a disease comprising an antibody or a fragment thereof disclosed herein. Specifically, for example, a pharmaceutical composition for treating a disease comprising an antibody or a fragment thereof disclosed herein labeled with a radioactive substance such as various anticancer drugs, lysine and other toxin compounds, radioactive substances such as radioactive metal ions (e.g., compounds with radioactivity such as α emitters), etc., is also provided. As its labeling method, there can be cited: a method of indirectly binding a compound using a linker such as polyethylene glycol, and a method of directly binding a compound by forming a disulfide bond on a cysteine ​​residue of an antibody or a fragment thereof. In addition, a heterologous bifunctional cross-linking agent such as N-succinyl-3-(2-pyridyldithio) propionate can also be used to directly bind the compound to an antibody or a fragment thereof. Further, the compound can also be bound by oxidizing the sugar chain in the Fc region of the antibody.

[0120] The pharmaceutical composition for treating diseases disclosed herein may also contain a pharmaceutically acceptable carrier. As pharmaceutically acceptable carriers that can be used, there can be cited: phosphate buffered saline solution, water, emulsions such as oil / water emulsions, various types of wetting agents, and other standard pharmaceutical carriers known in the art. In addition, other ingredients, such as pharmaceutical grade stabilizers, buffers, preservatives, excipients, etc., may also be used in the pharmaceutical composition for treating diseases disclosed herein. The preparation of the pharmaceutical composition taking into account pH, isotonicity, stability, etc. can be implemented by known methods.

[0121] The dosage of the antibody or fragment thereof and the antibody or fragment thereof labeled with a therapeutic agent contained in the pharmaceutical composition for treating a disease disclosed herein is not particularly limited as long as it is a pharmacologically effective amount, and can be appropriately determined according to race, sex, age, type of cancer, etc., and can generally be 0.01 to 1,000 mg / kg, preferably 0.1 to 100 mg / kg, once within 1 to 180 days, or twice or three times a day or more.

[0122] Diseases that can be treated using the pharmaceutical composition of the present disclosure include cancers such as pancreatic cancer, and the pharmaceutical composition of the present disclosure is useful for treating cancers that highly express a molecule to which the antibody or fragment thereof of the present disclosure binds. That is, when it is determined by the test method of the present disclosure that the subject is likely to have a cancer such as pancreatic cancer, the subject can be treated for a cancer such as pancreatic cancer by administering the pharmaceutical composition of the present disclosure.

[0123] In addition, the present disclosure also provides a pharmaceutical composition for inhibiting cancer metastasis comprising the antibody or fragment thereof of the present disclosure. The composition and use of the pharmaceutical composition are the same as those of the above-mentioned pharmaceutical composition. Example

[0124] Hereinafter, the present invention will be described in detail based on Examples, but the present invention is not limited to these Examples and the like.

[0125] Example 1. Preparation of rabbit monoclonal antibodies that bind to extracellular vesicles from pancreatic cancer cells (a) Lymph 1. Cell Preparation Frozen human early pancreatic cancer tissue (produced by Proteogenex, stage 1, tumor proportion 85%, 55 years old, female) was minced and suspended in PBS at 0.06 mg / mL as an immune antigen. JW / CSK rabbits (13 weeks old, female) were immunized with a mixture of immune antigen and FCA antigen solution, and hypertrophic lymph nodes were collected after 3 weeks. The lymphocytes obtained by homogenization and hemolytic treatment were frozen in cryoprotective medium (produced by Japan Zenyaku Industry, Cellbanker1).

[0126] (b) Preparation of extracellular vesicles At the Osaka International Cancer Center, tumor tissue was surgically removed from early pancreatic cancer patients with tumors less than 2 cm. The tumor tissue was washed with physiological saline containing antibiotics, chopped into pieces of about 1 mm, and a DMEM medium containing 1 mg / mL collagenase (manufactured by Sigma-Aldrich, catalog number: 044-29765) was added thereto for shaking treatment (200 rpm, 37°C, 15 minutes), and then tumor cells derived from the tumor tissue were collected through a 200 μm mesh. The tumor cells were cultured in a DMEM medium containing 10 ng / mL basic FGF and 2 ng / mL TGF-β, and the culture supernatant was collected every 2 to 4 days and stored at -80°C. On the other hand, fresh culture medium was added to the tumor cells and culture was continued.

[0127] After thawing the culture supernatant stored at -80°C, centrifuge (300×g, 4°C, 5 minutes; 1,200×g, 4°C, 20 minutes; 10,000×g, 4°C, 30 minutes) and collect the supernatant. Extracellular vesicles were isolated from the collected supernatant using MagCapture Exosome Isolation Kit PS (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., catalog number: 290-84103) according to the operating procedures. The protein concentration of extracellular vesicles was quantified using the BCA protein assay kit (manufactured by ThermoFisher, catalog number: 23225) using BSA solution as the standard.

[0128] (c) Screening of lymphocytes producing antibodies that bind to extracellular vesicles In a 196,000-well microchamber with a diameter of 20 μm per well, the extracellular vesicles of (b) were immobilized and washed, and then blocked with 1% BSA solution at room temperature for 30 minutes. After removing the blocking solution, the lymphocytes of (a) (2×10 5After culturing for 45 minutes, the microchamber was set in the ASONE Cell Picking System (manufactured by ASONE Co., Ltd.) to screen lymphocytes that produce antibodies that bind to extracellular vesicles. The results are as follows: Figure 1 As shown, a clone PEMb14 secreted by pancreatic cancer cells from a patient with early-stage pancreatic cancer and producing antibodies reactive with extracellular vesicles was obtained.

[0129] (d) Determination of PEMb14 antibody gene After the clone PEMb14 was isolated using the AS ONE single cell selection analysis system, the antibody gene of the lymphocyte was amplified using the single cell PCR method (Invitrogen, catalog number: 12574-035). The amplified heavy chain and light chain gene fragments were inserted into the vector pCEC3.2 rabbit (rabbit) (Invitrogen, catalog number: 12574-035) for cloning.

[0130] The CDR base sequences of the heavy and light chains of the PEMb14 antibody were determined by a DNA sequencer, and the amino acid sequences of CDR1 to 3 of the heavy and light chains shown in Table 1 and the base sequences of CDR1 to 3 of the heavy and light chains shown in Table 2 were identified. In addition, the amino acid sequence and base sequence of the entire heavy chain variable region and light chain variable region are shown below.

[0131] [Table 1] SEQ ID NO. sequence name Amino acid sequence 1 Heavy chain CDR1 EFSFSSSYY 2 Heavy chain CDR2 LYAGSGGVT 3 Heavy chain CDR3 AREVPADAAYGYFNL 4 Light chain CDR1 QSVYNNNN Light chain CDR2 SAS 6 Light chain CDR3 LGDFGGGIRA

[0132] [Table 2]

[0133] Heavy chain variable region (amino acid sequence) METGLRWLLLVAVLKGVQCEQLEESGGDLVKPGASLTLTCTASEFSFSSSYYMCW VRQAPGKGLEWIACLYAGSGGVTYYASWAKGRFTISKTSSTTVTLQMTSLTAADTATY FCAREVPADAAYGYFNL (SEQ ID NO: 13).

[0134] Light chain variable region (amino acid sequence) MDTRAPTQLLGLLLLWLPGATIAQVLTQTPSPVSAAVGGTVTINCQASQSVYNNNN LAWFQQKPGQPPKQLIYSASTLASGVSSRFKGSGSGTQFTLTISGVQCDDAATYYCLGD FGGGIRA(SEQ ID NO:14).

[0135] Heavy chain variable region (base sequence) ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTGAGCAGCTGGAGGAGTCCGGGGGAGACCTGGTCAAGCCTGGGGCATCCCTGACACTCACCTGCACAGCCTCTGAATTCTCCTTCAGTAGCAGCTACTACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCTTGCCTTTATGCTGGTAGTGGTGGTGTCACTTACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGCGAGAGAGGTCCCTGCTGATGCTGCTTATGGATACTTTAACTTA(SEQ ID NO:15).

[0136] Light chain variable region (base sequence) ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACAATTGCTCAAGTGCTGACCCAGACTCCATCCCCTGTGTCTGCCGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATAATAACAACAACTTAGCCTGGTTTCAGCA GAAACCAGGGCAGCCTCCCAAGCAACTGATCTATTCTGCATCCACTCTGGCATCTGGGGTGTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTATTACTGTCTAGGCGATTTTGGTGGTGGTATCCGGGCT(SEQ IDNO:16).

[0137] (e) Preparation of PEMb14 Antibodies (Rabbit IgG, κ) Each 100 mm culture plate contains 1.2 × 10 7 30 HEK293 cells were prepared, and the two rabbit antibody (IgG) expression vectors (heavy chain and light chain) obtained in Example 1 (d) were introduced into the gene using PEI (Merck, catalog number: 408727-100ML) at 8.50 μg per culture plate and cultured for 7 days, and the culture supernatant containing the PEMb14 antibody was collected. The obtained culture supernatant was provided to Ab-Capcher (ProteNova, catalog number: P-002-200), washed with TBS buffer, and eluted with Gentle Ag / Ab Elution buffer (pH 6.6) (ThermoFisher, catalog number: #21027). Then, it was dialyzed in TBS buffer containing 0.2 M arginine, PBS buffer containing 0.2 M arginine, and PBS buffer in sequence. The antibody concentration was measured using a BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific, catalog number: 23225).

[0138] Example 2. Blood test of early pancreatic cancer patients using PEMb14 antibody (a) Peroxidase labeling of PEMb14 antibody 10 μg of rat anti-human CD63 monoclonal antibody (manufactured by the Institute of Cell Engineering, clone name: 1C8-2B11) was peroxidase-labeled using Ab-10 Rapid Peroxidase Labeling Kit (manufactured by Dojindo Chemical Research Institute, catalog number: LK33) according to the protocol.

[0139] (b) Preparation of serum samples Serum was collected as a biological sample at the Osaka International Cancer Center, and the CA19-9 value was measured. The staging of pancreatic cancer is based on the UICC TNM classification (8th edition, 2017). Each serum was diluted 10 times with PBS as a serum sample.

[0140] (c) Determination of Antigenic Values ​​of PEMb14 Antibodies in Serum of Healthy People and Pancreatic Cancer Patients Add 100 μl of 10 μg / ml PEMb14 antibody (rabbit IgG) to each well of the MaxiSorp 96-well plate and let stand at room temperature for 1 hour. Next, wash each well three times with 200 μl of washing solution (50 mM Tris-HCl (pH 8.0), 140 mM NaCl containing 0.05% Triton-X 100), add 200 μl of blocking solution (washing solution containing 5% BSA) and let stand at room temperature for 1 hour. Remove the blocking solution, add 100 μl of serum sample and shake at room temperature for 2 hours to react, then wash each well 5 times. Next, add 100 μl of peroxidase-labeled rat anti-human CD63 monoclonal antibody diluted 1,000 times with blocking solution and let stand at room temperature for 1 hour. After washing each well five times, 100 μl of peroxidase substrate solution (SeraCare LifeSciences, code number: 5120-0053) was added to each well and reacted at room temperature for 20 minutes with shaking. After the reaction, 50 μl of 2% sulfuric acid was added to terminate the reaction, and the absorbance at 450 nm of each well was measured using a microplate reader.

[0141] The results are shown in Table 3. In patients with pancreatic cancer, especially early pancreatic cancer (stage 0 and stage 1), the positive rate of the antigen value based on the PEMb14 antibody in the serum was higher than the positive rate of CA19-9. This shows that the possibility of having pancreatic cancer, especially early pancreatic cancer, can be determined based on the antigen value of the PEMb14 antibody.

[0142] [Table 3] Staging Number of serum samples PEMb14 / positive number (positive rate) CA19-9 / positive number (positive rate) 0 5 3(60％) 0(0％) 1 8 5(63％) 5(63％) 0+1 13 8(62％) 5(38％)

[0143] Comparative Example 1. Rabbit monoclonal antibody binding to extracellular vesicles derived from the culture supernatant of pancreatic cancer cell lines Manufacturing (a) Preparation of extracellular vesicles from culture supernatant of pancreatic cancer cell lines Commercially available human pancreatic cancer cell lines (KP-3, KP-3L, BxPC3, Mia-PaCa2, and SUIT-2) were cultured in RPMI 1640 medium containing 10% FBS for 3 days and the culture supernatants were collected. Extracellular vesicles were isolated from the collected supernatants using the MagCapture Exosome Extraction Kit PS according to the operating procedures, and the resulting five types of extracellular vesicles were mixed. The protein concentration of extracellular vesicles was quantified using the BCA protein assay kit with BSA solution as the standard.

[0144] (b) Screening of lymphocytes producing antibodies that bind to extracellular vesicles In the same manner as in Example 1(c), the extracellular vesicles of (a) were immobilized in a microchamber and the lymphocytes (2×10 5 Cells) were screened for lymphocytes that produced antibodies that bind to extracellular vesicles. As a result, six clones of antibodies secreted by pancreatic cancer cell lines that reacted with extracellular vesicles were obtained. Then, antibodies (D5, D13, D15, D17, D19, D29) were prepared from each clone in the same manner as in Example 1(e).

[0145] (c) Determination of Antigen Values ​​in Serum of Healthy People and Pancreatic Cancer Patients As biological samples, serum (manufactured by Proteogenex, 13 healthy subjects / 15 pancreatic cancer (stage 1) patients) was diluted 10-fold with PBS to prepare serum samples. Each antigen value was measured in the same manner as in Example 2(c).

[0146] The results are shown in Table 4. In patients with early pancreatic cancer (stage 1), the positive rate based on each antigen value in the serum was lower than that of PEMb14. This indicates that antibodies that bind to extracellular vesicles derived from a medium obtained by culturing cells of artificially formed cell lines are more practical than antibodies that bind to extracellular vesicles derived from a medium obtained by culturing cells of artificially formed cell lines.

[0147] [Table 4] Antibody Name Pancreatic Cancer Staging Number of serum samples Positive number (positive rate) d5 1 15 1(7％) d13 1 15 1(7％) d15 1 15 0(0％) d17 1 15 2(13％) d19 1 15 3(20％) d29 1 15 0(0％)

[0148] Comparative Example 2. Preparation of rabbit monoclonal antibodies that bind to extracellular vesicles derived from serum of pancreatic cancer patients (a) Preparation of extracellular vesicles from serum of pancreatic cancer patients Using MagCapture Exosome Extraction Kit PS, extracellular vesicles were isolated from 1 mL of serum from 10 pancreatic cancer patients (stage 1) (produced by Proteogenex, 3 males / 7 females, aged 40-65 years) according to the operating procedures, and the 10 types of extracellular vesicles obtained were mixed. The protein concentration of extracellular vesicles was quantified using the BCA protein detection kit with BSA solution as the standard.

[0149] (b) Screening of lymphocytes producing antibodies that bind to extracellular vesicles In the same manner as in Example 1(c), the extracellular vesicles of (a) were immobilized in a microchamber and the lymphocytes (2×10 5 Cells) were screened for lymphocytes that produced antibodies that bind to extracellular vesicles. As a result, five clones that produced antibodies that reacted with extracellular vesicles and were contained in the serum of patients with early pancreatic cancer (stage 1) were obtained. Then, antibodies (hs8, hs15, hs22, hs28, and hs39) were prepared from each clone in the same manner as in Example 1(e).

[0150] (c) Determination of Antigen Values ​​in Serum of Healthy People and Pancreatic Cancer Patients As biological samples, serum (manufactured by Proteogenex, 13 healthy subjects / 15 pancreatic cancer (stage 1) patients) was diluted 10-fold with PBS to prepare serum samples. Each antigen value was measured in the same manner as in Example 2(c).

[0151] The results are shown in Table 5. In patients with early pancreatic cancer (stage 1), the positive rate based on each antigen value in the serum was lower than that of PEMb14. This indicates that antibodies that bind to extracellular vesicles derived from the culture medium obtained by culturing a biological sample (at least one selected from the group consisting of biological tissues and cells isolated from the biological tissues) are more practical than antibodies that bind to extracellular vesicles derived from the serum of pancreatic cancer patients.

[0152] [Table 5] Antibody Name Pancreatic Cancer Staging Number of serum samples Positive number (positive rate) hs8 1 15 2(13％) hs15 1 15 0(0％) hs22 1 15 0(0％) hs28 1 15 1(7％) hs39 1 15 0(0％)

Claims

1. An antibody or an antigen-binding fragment thereof, comprising: A heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, A heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, A heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, A light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence represented by SAS, and A light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody is a monoclonal antibody.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody is IgG.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antigen binding fragment is Fab, F(ab')2, miniantibody, scFv-Fc, Fv, scFv, diabody, triabody or tetrabody. 5 . A polynucleotide comprising a coding sequence for the antibody or antigen-binding fragment thereof according to claim 1 . A cell comprising the polynucleotide according to claim 5.

7. A cancer testing agent comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

8. The inspection agent according to claim 7, wherein: The cancer is metastatic cancer.

9. The inspection agent according to claim 7, wherein: The cancer is pancreatic cancer.

10. The inspection agent according to claim 7, wherein: The cancer is early pancreatic cancer.

11. A pharmaceutical composition for diagnosing cancer, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 labeled with a diagnostic agent.

12. The pharmaceutical composition according to claim 11, wherein The diagnostic reagent is a radioactive substance.

13. The pharmaceutical composition according to claim 11, wherein The cancer is pancreatic cancer.

14. A pharmaceutical composition for treating cancer, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

15. The pharmaceutical composition for treating cancer according to claim 14, wherein: The cancer is pancreatic cancer.

16. A pharmaceutical composition for inhibiting cancer metastasis, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

17. The pharmaceutical composition for inhibiting cancer metastasis according to claim 16, wherein: The cancer is pancreatic cancer.

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