JP2012000072A - Novel antifibrin antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel antibody binding with fibrin and not binding with fibrinogen, and a use thereof.SOLUTION: There is disclosed an antibody or an antigen-binding fragment thereof, comprising H strand CDR1, H strand CDR2, H strand CDR3, L strand CDR1, L strand CDR2, L strand CDR3, wherein these CDRs include an amino acid sequence composed of a specific sequence or an amino acid sequence containing one or several conservative amino acid substitutions in the amino acid sequence. The antibody characteristically binds with human fibrin and murine fibrin.

Description

  The present invention relates to a novel anti-fibrin antibody, and a reagent and method for detecting fibrin. The present invention also relates to a reagent and method for determining a thrombosis-related disease using an anti-fibrin antibody. The present invention further relates to a complex of an anti-fibrin antibody and an antitumor moiety, and a preventive or therapeutic agent for a tumor containing the complex.

  The relationship between cancer and blood clotting is described by French surgeon Torso in the 1800s, “Edema due to thrombosis of the extremities in patients with gastric cancer”. Recent clinical epidemiological data also reveal that the frequency of thrombosis due to hypercoagulation is significantly higher than that of healthy individuals in most carcinomas including pancreatic cancer, gastric cancer, and brain tumor (Non-patent Document 1). It is considered that fibrin accumulation, coagulative necrosis, and angiogenesis accompanying the abnormal coagulation occur repeatedly with the progress of the tumor in the cancer tissue.

  Fibrin is not present in tissues under normal physiological conditions, unlike the precursor fibrinogen that is widely found in the body. When activated thrombin leaks out of the blood vessel and cuts out the C-terminal peptide of fibrinogen, a fibrin monomer is formed, and the fibrin monomer is polymerized to form fibrin fibers. It is specific to tissues with pathological conditions such as inflammation, and is formed when pathological conditions accompanied by coagulation such as cancer, myocardial infarction, and cerebral infarction occur. Therefore, fibrin present in cancer tissues in the absence of cerebral circulatory diseases such as myocardial infarction and cerebral infarction is just a cancer-specific molecule.

  On the other hand, antibodies have been developed as means for detecting fibrin. For example, Patent Documents 1 to 6 describe anti-fibrin antibodies that recognize human fibrin but not fibrinogen.

  In addition, since the late 1970s, when monoclonal antibody production methods were established, research on “missile therapy”, in which toxins and anticancer agents are added to monoclonal antibodies to selectively attack cancerous parts, has been active. However, with the exception of approval for some lymphomas and leukemias, the clinical usefulness of missile therapy in normal solid cancers such as lung cancer, colorectal cancer, breast cancer, and gastric cancer has not been proven and has been achieved to date. Problems with so-called missile therapy using monoclonal antibodies against cancer-specific antigens include: (1) The application of monoclonal antibodies is limited to cancers in which specific antigens are expressed on the membrane surface of cancer cells; (2) Monoclonal antibodies May be neutralized with the antigen released in the blood and not delivered to the local cancer site; (3) Even if the monoclonal antibody is delivered to the local cancer site, It is possible that the stroma becomes a barrier before reaching the cancer cell, and the monoclonal antibody may not reach the site of the important cancer cell, especially pancreatic cancer, Skills gastric cancer, colon cancer, lung cancer and other intractable cancers Is known to be rich in interstitium.

  On the other hand, the research group of the present inventor has increased tumor blood vessel permeability as a vascular characteristic of cancer, and high molecular substances that are difficult to leak from normal blood vessels can easily leak from cancer blood vessels. Also, because of the poor lymphangiogenesis compared to angiogenesis, the EPR effect (enhanced permeation and retention effect) was found that the macromolecular substance once leaked in the cancer tissue could not drain into the lymph vessel and stayed in the cancer tissue for a long time Yes.

  As described above, since fibrin has also been shown to be associated with thrombus formation and important diseases, there is a development of antibodies that specifically react with fibrin, and means and methods that can specifically detect fibrin. Still desired.

JP 2001-354700 A JP 2009-149686 JP 2008-29353 A Japanese Unexamined Patent Publication No. 9-127108 JP-A-9-104700 JP-A-8-301900

PD Stein et al Incidence of venous thromboembolism in patients hospitalized with cancer.American J Med 116, 60-68, 2006

  Therefore, an object of the present invention is to provide a novel antibody that binds to fibrin and does not bind to fibrinogen, and uses thereof.

  As a result of intensive studies to solve the above problems, the present inventor has produced mouse antibodies using the pulverized product of fibrin clots as an immunogen, and binds to human and mouse fibrin. It was found that an antibody that does not bind was obtained, and that this anti-fibrin antibody can be used to detect fibrin and to determine fibrin-related diseases. The present inventor also prepared a human chimeric antibody based on the mouse anti-fibrin antibody, and confirmed that this chimeric antibody retains the same reactivity as the mouse anti-fibrin antibody. Furthermore, it was confirmed that fibrin present in a tumor can be visualized using an anti-fibrin antibody and that an antitumor compound can be delivered to the tumor. The present invention has been made based on the above findings.

That is, the present invention includes the following [1] to [29].
[1] Below:
(A) an H chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence comprising one or several conservative amino acid substitutions in the amino acid sequence,
(B) the H chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence containing one or several conservative amino acid substitutions in the amino acid sequence,
(C) the H chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence containing one or several conservative amino acid substitutions in the amino acid sequence,
(D) an L chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence containing one or several conservative amino acid substitutions in the amino acid sequence,
(E) the L chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence containing one or several conservative amino acid substitutions in the amino acid sequence, and (f) 1 or 2 in the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence L chain CDR3 consisting of amino acid sequence containing several conservative amino acid substitutions
An antibody or an antigen-binding fragment thereof, which comprises binding to human fibrin.

[2] The antibody or antigen-binding fragment according to [1], which binds to human fibrin and mouse fibrin.
[3] The antibody or antigen-binding fragment according to [1] or [2], which does not bind to human fibrinogen and mouse fibrinogen.
[4] The antibody or antigen-binding fragment according to any one of [1] to [3], which is a monoclonal antibody.

[5] The antibody or antigen-binding fragment according to any one of [1] to [4], which is an antibody produced by a hybridoma cell having the accession number NITE P-923.
[6] The antibody or antigen-binding fragment according to any one of [1] to [4], which is an antibody produced by a hybridoma cell having the accession number NITE P-923.
[7] The antibody or antigen-binding fragment according to any one of [1] to [4], which is a chimeric antibody or a humanized antibody.

[8] The antibody or antigen-binding fragment thereof according to [7], which is a chimeric antibody comprising an H chain variable region consisting of the amino acid sequence of SEQ ID NO: 8 and an L chain variable region consisting of the amino acid sequence of SEQ ID NO: 10.
[9] The antibody or antigen-binding fragment according to any one of [1] to [8], which is labeled.
[10] A nucleic acid comprising a base sequence encoding the antibody or antigen-binding fragment according to any one of [1] to [8].
[11] An expression vector comprising the nucleic acid according to [10].
[12] A transformant comprising the nucleic acid according to [10] or the expression vector according to [11], and producing the antibody or antigen-binding fragment according to any one of [1] to [8].

[13] The antibody or antigen according to any one of [1] to [8], comprising the step of culturing the transformant according to [12] in a medium and collecting the antibody or antigen-binding fragment from the culture. A method for producing a binding fragment.
[14] A cell that produces the antibody or antigen-binding fragment according to any one of [1] to [8].
[15] The cell according to [14], which is a hybridoma cell having a deposit number of NITE P-923.
[16] A reagent for immunoassay of fibrin, comprising the antibody or antigen-binding fragment according to any one of [1] to [9].

[17] A reagent for determining a thrombus-related disease, comprising the antibody or antigen-binding fragment according to any one of [1] to [9].
[18] The reagent according to [17], wherein the thrombosis-related disease is infarction or cancer.
[19] A thrombus visualization agent comprising the labeled antibody or antigen-binding fragment according to [9].
[20] The thrombus visualization agent according to [19] for visualizing infarction or cancer.

[21] A method for detecting fibrin in a sample,
(A) contacting the sample with the antibody or antigen-binding fragment according to any one of [1] to [9];
(B) a method comprising detecting whether the antibody or antigen-binding fragment has bound to fibrin in a sample.

[22] A method for determining a thrombus-related disease in a subject comprising:
(A) contacting the antibody or antigen-binding fragment according to any one of [1] to [9] with a sample derived from a subject;
(B) a method comprising detecting whether the antibody or antigen-binding fragment has bound to fibrin in a sample.
[23] The method according to [22], wherein the thrombus-related disease is infarction or cancer.
[24] The method according to any one of [21] to [23], wherein the sample is selected from the group consisting of a cell and tissue sample, and a biological fluid sample.

[25] A method for producing a modified anti-fibrin antibody or antigen-binding fragment,
(A) preparing an antibody or antigen-binding fragment having an amino acid sequence modified from the amino acid sequence of the antibody or antigen-binding fragment according to any one of [1] to [8];
(B) A method comprising the step of determining whether the obtained antibody or antigen-binding fragment binds to fibrin.
[26] A complex of the antibody or antigen-binding fragment according to any one of [1] to [9] and an antitumor moiety.
[27] The complex according to [26], wherein the antibody or antigen-binding fragment and the antitumor moiety are bound via a linker.
[28] The complex according to [26] or [27], wherein the antitumor moiety is an anticancer agent.
[29] A preventive or therapeutic agent for tumors comprising the complex according to any one of [26] to [28].

  The present invention provides antibodies against fibrin and antigen-binding fragments thereof. By using the anti-fibrin antibody of the present invention, it is possible to detect the presence of fibrin and a thrombus with high sensitivity, reliability, and simpleness, and as a result, a thrombus-related disease can be determined. In addition, by using the anti-fibrin antibody of the present invention, it becomes possible to deliver an appropriate compound or molecule to a site where a thrombus exists, for example, a tumor. In particular, since the anti-fibrin antibody of the present invention binds to human and mouse fibrin and does not bind to human and mouse fibrinogen, it is considered useful in the medical diagnostic field and the pharmaceutical field.

Figure 3 shows the results of an ELISA that tested for reactivity of mouse anti-fibrin IgM antibody with mouse and human fibrin, and mouse and human fibrinogen. A and B are results of using a commercially available anti-fibrin antibody as a control, and C is a result of using the mouse anti-fibrin IgM antibody of the present invention. The amino acid sequence and base sequence of the H chain (A) and L chain (B) of mouse anti-fibrin IgM antibody are shown. Results of ELISA tested for reactivity of chimeric antibodies with mouse and human fibrin, and mouse and human fibrinogen are shown. It is a photograph which shows the immuno-staining result of the human pancreatic cancer tissue using the antibody of this invention. It is a photograph which shows the immuno-staining result of the human brain tumor tissue using the antibody of this invention. A shows a human brain tumor (glioma) surgical tissue section, and B shows the result of immunostaining the section. It is a photograph which shows the immuno-staining result of the cancer tissue using the antibody of this invention. A shows the tissue stained with hematoxylin and eosin, B shows the immunostained tissue with anti-fibrin antibody IgM, and C shows the immunostained tissue of fluorescently labeled human chimeric anti-fibrin IgG. 2 is a photograph showing in vivo imaging of cancer tissue using the antibody of the present invention. A shows tumor formation in mice bearing human breast cancer, and B and C show accumulation of labeled human chimeric antibody at the site of tumor formation. Shows the synthesis scheme of branched linkers (A to H), the coupling scheme of anti-tumor compound SN-38 and PEG (I to K), and the binding of branched linkers to PEG-SN-38 complex . The binding method of the anti-fibrin antibody of the present invention and the antitumor compound SN-38 is shown. Cell growth of SUIT2 cells in the presence of SN-38-antibody complex via linear linker (A) or SN-38-antibody complex via branched linker (B) It is a graph which shows a rate (%). It is a graph which shows the anti-tumor effect of the anti- fibrin antibody-SN-38 complex with respect to the tumor in a carcinogenesis model mouse. It is a photograph which shows the antitumor effect of the anti- fibrin antibody-SN-38 complex with respect to the tumor in a carcinogenesis model mouse.

Hereinafter, the present invention will be described in detail.
The present invention provides novel antibodies against fibrin. Fibrin is a protein involved in blood coagulation, and fibrin monomer is produced by the action of thrombin from fibrinogen present in plasma. This fibrin monomer forms a polymer and gels, thereby forming an insoluble fibrin clot, that is, a thrombus. There are numerous reports on the structure and function of fibrin, which are known in the art. The present invention relates to antibodies and antigen-binding fragments that specifically bind to fibrin and their uses.

1. Antibody to fibrin and antigen-binding fragment The antibody according to the present invention is characterized by binding to fibrin and not binding to fibrinogen. The antibodies and antigen-binding fragments according to the present invention comprise the following H chain (heavy chain) complementarity determining region (CDR) and L chain (light chain) CDR:
(A) H chain CDR1 consisting of the amino acid sequence of FTNYGMN (SEQ ID NO: 1) or an amino acid sequence containing one or several conservative amino acid substitutions in the amino acid sequence,
(B) H chain CDR2 consisting of the amino acid sequence of WINTYTGEATYA (SEQ ID NO: 2) or an amino acid sequence containing one or several conservative amino acid substitutions in the amino acid sequence,
(C) H chain CDR3 consisting of the amino acid sequence of LMDY (SEQ ID NO: 3) or an amino acid sequence containing one or several conservative amino acid substitutions in the amino acid sequence,
(D) an L chain CDR1 consisting of the amino acid sequence of KASQDINKYIA (SEQ ID NO: 4) or an amino acid sequence containing one or several conservative amino acid substitutions in the amino acid sequence,
(E) an amino acid sequence of YTSTLQP (SEQ ID NO: 5) or an L chain CDR2 comprising an amino acid sequence containing one or several conservative amino acid substitutions in the amino acid sequence, and (f) an amino acid sequence of LQYDNLTW (SEQ ID NO: 6) or L chain CDR3 consisting of an amino acid sequence comprising one or several conservative amino acid substitutions in the amino acid sequence.

In the present invention, “antibody” and “antigen-binding fragment” refer to an entire antibody molecule that specifically binds to fibrin or a fragment thereof (eg, a fragment such as Fab, Fab ′, F (ab ′) ₂ , Fv). Meaning, it may be a polyclonal antibody or a monoclonal antibody. In the present invention, “antibody” and “antigen-binding fragment” also include chimeric antibodies, humanized antibodies and human antibodies, and fragments thereof.

In the present invention, an antibody “specifically binds” to fibrin means that it binds to fibrin with a higher affinity than its affinity for other peptides or proteins. As used herein, “high affinity” means an affinity that is high enough to allow fibrin to be detected separately from other peptides or proteins by methods known in the art. It means a binding affinity such that the binding constant (K _a ) is at least 10 ⁷ M ⁻¹ , preferably at least 10 ⁸ M ⁻¹ , more preferably 10 ⁹ M ⁻¹ or higher.

  In the present invention, the binding (reactivity) to fibrin and the binding (reactivity) to fibrinogen can be determined by a method known in the art, for example, by using a known ELISA method. (Wu, Sau-Ching et al. Applied and Environmental Microbiology 68: 3261-3269, 2002).

  In addition, the antibody and antigen-binding fragment according to the present invention can bind to mouse and human fibrin but not to mouse and human fibrinogen. Therefore, test data obtained using this antibody in mice can be extrapolated to humans.

  In the present invention, “conservative amino acid substitution” is known in the art and refers to substitution of an amino acid with an amino acid having similar properties to that amino acid. It is known in the art that a protein containing an amino acid sequence containing one or several conservative amino acid substitutions in a specific amino acid sequence retains the same activity as a protein containing that specific amino acid sequence. Therefore, in the present invention, an antibody and an antigen-binding fragment containing an amino acid sequence having such a conservative amino acid substitution can be used in the antibody of the present invention as long as the desired activity, that is, binding to fibrin is retained. included. For example, neutral (polar) amino acids (Asn, Ser, Gln, Thr, Tyr, Cys), neutral (nonpolar, ie hydrophobic) amino acids (Gly, Trp, Met, Pro, Phe, Ala, Val, Leu, Ile), acidic (polar) amino acids (Asp, Glu), basic (polar) amino acids (Arg, His, Lys) are replaced with amino acids having the same properties.

  Hereinafter, a method for producing the antibody or antigen-binding fragment thereof according to the present invention will be described in detail.

  The antibody of the present invention can be prepared by using an immunogen obtained by dissolving a pulverized fibrin clot in a buffer and, if necessary, adding an adjuvant for effective immunization. Examples of the adjuvant include commercially available Freund's complete adjuvant (FCA) and Freund's incomplete adjuvant (FIA). These adjuvants can be used alone or in combination.

  When producing monoclonal antibodies, the immunogen is administered to mammals such as mice, rabbits, rats and the like. Immunization is performed mainly by injecting intravenously, subcutaneously, intraperitoneally, or into the footpad. The interval between immunizations is not particularly limited, and immunization is performed 1 to 5 times at intervals of several days to several weeks. Then, antibody-producing cells are collected 3 to 20 days after the final immunization day. Examples of antibody-producing cells include lymph node cells, spleen cells, peripheral blood cells and the like.

  In order to obtain a hybridoma, cell fusion between antibody-producing cells and myeloma cells is performed. Generally available cell lines can be used as myeloma cells to be fused with antibody-producing cells. The cell line used has drug selectivity and cannot survive in a HAT selection medium (including hypoxanthine, aminopterin, and thymidine) in an unfused state, but can survive only in a state fused with antibody-producing cells. Those having the following are preferred. Examples of myeloma cells include mouse myeloma cell lines such as P3X63-Ag.8.U1 (P3U1) and NS-I.

  Next, the myeloma cell and the antibody-producing cell are fused. Cell fusion is performed by mixing antibody-producing cells and myeloma cells in animal cell culture media such as serum-free DMEM and RPMI-1640 media, and in the presence of a cell fusion promoter (eg, polyethylene glycol). Perform the reaction. Alternatively, cell fusion can be performed using a commercially available cell fusion device utilizing electroporation.

  The target hybridoma is selected from the cells after cell fusion treatment. For example, the cell suspension is appropriately diluted with a fetal bovine serum-containing RPMI-1640 medium or the like and then spread on a microtiter plate. Selective medium (for example, HAT medium) is added to each well, and thereafter cell culture is performed by appropriately replacing the selective medium. As a result, cells that grow in about 10 to 30 days after the start of culture in a selective medium can be obtained as hybridomas.

  Next, the culture supernatant of the hybridoma that has grown is screened for the presence of antibodies that react with fibrin. The screening of hybridomas may be carried out in accordance with a usual method, and for example, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), or radioimmunoassay (RIA) can be employed. Cloning of the fused cells is performed by a limiting dilution method or the like to establish a hybridoma that produces the target monoclonal antibody.

  As a method for collecting the monoclonal antibody from the established hybridoma, a normal cell culture method or ascites formation method can be employed. When antibody purification is required in the antibody collection method, known methods such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, affinity chromatography are appropriately selected, or a combination thereof is used. Can be purified.

  The globulin type of the monoclonal antibody that can be used in the present invention is not particularly limited as long as it has a specific binding activity to fibrin, and may be any of IgG, IgM, IgA, IgE, and IgD. And IgM are preferred.

  According to the above-described method for producing a monoclonal antibody, the present inventors established a hybridoma cell producing a mouse anti-fibrin IgM antibody. In the present invention, it is preferable to use an antibody produced from hybridoma cell 102-10 having the accession number NITE P-923. The antibody produced from this hybridoma cell 102-10 includes an H chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8 and an L chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 10. In addition, the H chain CDR1 to 3 of this antibody have the amino acid sequences shown in SEQ ID NOs: 1 to 3, respectively, and the L chain CDR1 to 3 have the amino acid sequences shown in SEQ ID NOs: 4 to 6, respectively.

  The antibody of the present invention may be an antibody that binds to an epitope to which the antibody produced by the hybridoma cell 102-10 binds.

The antibody of the present invention can also be produced by splicing a gene from an antibody molecule having antigen specificity to fibrin prepared as described above together with a gene from a human antibody molecule having an appropriate biological activity. Chimeric antibodies (Morrison et al., 1984, Proc. Natl. Acad. Sci., 81: 6851-6855; Neuberger et al., 1984, Nature, 312: 604-608; Takeda et al., 1985, Nature, 314 : 452-454). Also, single chain antibodies (US Pat. No. 4,946,778; Bird, 1988, Science 242: 423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; Ward et al. , 1989, Nature 334: 544-546), F (ab ′) ₂ fragments, Fab fragments, single chain antibodies and the like are also produced using techniques known in the art (eg, digestion with papain or trypsin). be able to.

  The above-described antibodies and antigen-binding fragments of the present invention can also be prepared using genetic engineering techniques.

  For example, the monoclonal antibody is prepared from the nucleic acid of the hybridoma cell line prepared as described above, the nucleic acid containing the base sequence encoding the antibody containing the H chain or L chain of the monoclonal antibody produced by the hybridoma. can do. These nucleic acids can be obtained from hybridomas by ordinary genetic engineering techniques, and their base sequences can also be determined by known base sequencing methods. For example, mRNA is extracted from a hybridoma cell line to synthesize cDNA. The synthesized cDNA is inserted into a vector such as a phage or a plasmid to prepare a cDNA library. From the library, a recombinant phage or recombinant plasmid having cDNA encoding the heavy chain variable region (VH) using a non-human animal antibody such as a mouse antibody constant region or variable region as a probe, and Recombinant phage or recombinant plasmid having cDNA encoding VL is isolated, respectively. Determine the entire nucleotide sequence of the H chain variable region (VH) and L chain variable region (VL) of the target antibody on the recombinant phage or recombinant plasmid, and estimate the entire amino acid sequence of VH and VL from the nucleotide sequence. . As an example, the nucleotide sequences of nucleic acids encoding the H chain variable region and L chain variable region of the monoclonal antibody produced by the hybridoma cell line 102-10 are shown in SEQ ID NO: 7 and SEQ ID NO: 9, respectively.

  The nucleic acid encoding the H chain variable region and the L chain variable region may be a mutant of the above base sequence (natural mutant or artificial mutant). For example, in the nucleotide sequence of the nucleic acid encoding the H chain or L chain variable region, a mutant having a deletion, substitution, addition or insertion of one or several bases and encoding a protein that binds to fibrin. Can be used. Here, “one or several” refers to 1 to 20, preferably 1 to 15, more preferably 1 to 10. Further, for example, it can be used as a variant that encodes a protein that hybridizes under stringent conditions to a complementary sequence of a base sequence of a nucleic acid encoding an H chain or L chain variable region and binds to fibrin. Here, the “stringent conditions” are not limited thereto, but for example, at 30 ° C. to 50 ° C., 3-4 × SSC (150 mM sodium chloride, 15 mM sodium citrate, pH 7.2), Hybridization in 0.1-0.5% SDS for 1-24 hours, more preferably at 40 ° C.-45 ° C., 3.4 × SSC, hybridization in 0.3% SDS for 1-24 hours, and subsequent washing. Examples of washing conditions include conditions such as continuous washing at room temperature with a solution containing 2 × SSC and 0.1% SDS, and a 1 × SSC solution and a 0.2 × SSC solution. However, the combinations of the above conditions are exemplary, and those skilled in the art will understand the above or other factors that determine the stringency of hybridization (eg, the concentration, length and GC content of the hybridization probe, the hybridization reaction). It is possible to achieve the same stringency as above by appropriately combining the time and the like.

  Chimeric antibodies are described, for example, by Morrison et al., 1984, Proc. Natl. Acad. Sci., 81: 6851-6855; Neuberger et al., 1984, Nature, 312: 604-608; Takeda et al., 1985, Nature 314: 452-454. For example, a chimeric antibody can be prepared by splicing the gene of a mouse monoclonal antibody of the above hybridoma cell line together with the gene of an antibody molecule derived from another mammal. Examples of the chimeric antibody include a human chimeric antibody having the H chain and / or L chain variable region of the mouse monoclonal antibody and a human immunoglobulin constant region. In the present invention, for the production of a chimeric antibody, nucleic acid base sequences (SEQ ID NO: 7 and SEQ ID NO: 9 respectively) encoding an H chain variable region and / or an L chain variable region, and variants of the base sequences are used. Can be used.

The humanized antibody has, for example, a part of a variable region including a variable region or a hypervariable region derived from a mouse monoclonal antibody, and a constant region of a human immunoglobulin, or a part of a variable region of a human immunoglobulin and a constant region. In the case of a humanized antibody, the antibody-derived antibody region is preferably less than about 10%. In the present invention, the humanized antibody has, for example, an amino acid sequence comprising at least one complementarity determining region (CDR1, 2 and 3) in the amino acid sequence of the H chain variable region and / or the L chain variable region of the mouse monoclonal antibody. Can be included. Specifically, the variable region derived from a mouse monoclonal antibody has the following CDR:
(A) H chain CDR1 consisting of the amino acid sequence of FTNYGMN (SEQ ID NO: 1) or an amino acid sequence containing one or several conservative amino acid substitutions in the amino acid sequence,
(B) H chain CDR2 consisting of the amino acid sequence of WINTYTGEATYA (SEQ ID NO: 2) or an amino acid sequence containing one or several conservative amino acid substitutions in the amino acid sequence,
(C) H chain CDR3 consisting of the amino acid sequence of LMDY (SEQ ID NO: 3) or an amino acid sequence containing one or several conservative amino acid substitutions in the amino acid sequence,
(D) an L chain CDR1 consisting of the amino acid sequence of KASQDINKYIA (SEQ ID NO: 4) or an amino acid sequence containing one or several conservative amino acid substitutions in the amino acid sequence,
(E) an amino acid sequence of YTSTLQP (SEQ ID NO: 5) or an L chain CDR2 comprising an amino acid sequence containing one or several conservative amino acid substitutions in the amino acid sequence, or (f) an amino acid sequence of LQYDNLTW (SEQ ID NO: 6) or L chain CDR3 consisting of an amino acid sequence containing one or several conservative amino acid substitutions in the amino acid sequence
At least one of them.

  SEQ ID NOs: 1 to 3 correspond to amino acids 48 to 54, 69 to 80, and 118 to 121 of the heavy chain variable region amino acid sequence shown in SEQ ID NO: 8, respectively. These correspond to amino acids 44 to 54, 70 to 76 and 109 to 116 in the L chain variable region amino acid sequence shown in SEQ ID NO: 10, respectively.

  A human acceptor antibody sequence suitable for the mouse donor sequence (CDR sequence) can be identified by computer comparison between the amino acid sequence of the mouse variable region and the H chain or L chain sequence of a known human antibody. A variable domain from a human antibody whose framework sequence exhibits high sequence identity with the framework regions of the mouse L chain variable region and the H chain variable region is a Kabat that utilizes NCBI BLAST (USA) using the mouse framework sequence. Can be identified by querying the database. At this time, an acceptor sequence that shares 80% or more, preferably 90% or more of the sequence identity with the mouse donor sequence can be selected. Based on the nucleotide sequences encoding the human acceptor antibody H chain and L chain sequences thus identified, recombination is performed so that a part of the variable region is replaced with that of the mouse antibody.

In addition, expression vectors for antigen-binding fragments such as Fab, F (ab ′) ₂ , Fv fragments, single chain antibodies and the like can be appropriately designed by those skilled in the art.

  Subsequently, the nucleic acid encoding the H chain and / or L chain of the antibody or antigen-binding fragment or a variant thereof is cloned and incorporated into an appropriate expression vector. Examples of expression vectors include pAGE107 (Cytotechnology, 3, 133 (1990)), pAGE103 (J. Biochem., 101, 1307 (1987)), pQCxID (Clontech) and pQCxIH (Clontech). In addition to the nucleic acid encoding the antibody or antigen-binding fragment, a promoter and enhancer, a selectable marker gene, and the like may be inserted into the expression vector. Examples of such promoters and enhancers include SV40 early promoter and enhancer, Moloney murine leukemia virus LTR promoter and enhancer, and immunoglobulin H chain promoter and enhancer. Examples of the selection marker gene include a neomycin resistance gene, an ampicillin resistance gene, and a chloramphenicol resistance gene.

  The expression vector may be one in which both nucleic acids encoding the H chain and L chain are incorporated into a single expression vector, or an expression vector and / or L chain in which a nucleic acid encoding the H chain is incorporated. It may be an expression vector incorporating a nucleic acid to be encoded.

  When constructing an expression vector for a human chimeric antibody, for example, a restriction enzyme is previously added upstream of the gene encoding the H chain constant region (CH) and L chain constant region (CL) of the human antibody of the chimeric antibody expression vector. A recognition sequence is provided, and a cDNA encoding a variable region of a mouse antibody is inserted into a cloning site comprising this recognition sequence.

  When constructing an expression vector for a humanized antibody, the DNA sequence encoding the amino acid sequence of the framework region (FR) of the variable region of the human antibody and the DNA sequence encoding the amino acid sequence of the CDR of the variable region of the mouse antibody By ligating, a DNA sequence encoding the amino acid sequence of VH and the amino acid sequence of VL is designed. Subsequently, cDNAs encoding the humanized antibody VH and VL can be inserted upstream of the gene encoding the human antibody CH and CL to construct an expression vector for a humanized antibody.

  The expression vector constructed as described above is introduced into an appropriate host cell to obtain a transformant. The host to be used is not particularly limited as long as it can express a nucleic acid on an introduced expression vector and produce an antibody or an antigen-binding fragment. Examples include bacteria (such as E. coli), yeast (such as Saccharomyces cerevisiae), animal cells (such as COS cells and CHO cells), and insects (such as silkworms, Sf9 cells, and Sf21 cells).

  The method for introducing a nucleic acid or expression vector into bacteria or yeast is not particularly limited as long as it is a method for introducing DNA into bacteria or yeast, and examples thereof include an electroporation method, a spheroplast method, and a lithium acetate method. Examples of the method for introducing a nucleic acid or expression vector into animal cells or insect cells include an electroporation method, a calcium phosphate method, and a lipofection method.

  A transformant is selected by utilizing the property of a marker gene constituted in the nucleic acid to be introduced. For example, when a neomycin resistance gene is used, cells showing resistance to the G418 drug are selected.

  The antibody or antigen-binding fragment of the present invention can be obtained by culturing the transformant introduced with the nucleic acid encoding it in a medium and collecting it from the culture. “Culture” means any of culture supernatant, cultured cells, or cell lysate. The method of culturing the transformant in a medium is performed according to a usual method used for culturing a host.

As a medium for cultivating a transformant obtained using bacteria or yeast as a host, as long as it contains a carbon source, a nitrogen source, inorganic salts, and the like and can culture the transformant efficiently, Either a natural medium or a synthetic medium may be used. The culture is usually performed at about 20 to 40 ° C. for about 1 to 24 hours under aerobic conditions such as shaking culture or aeration and stirring culture. During the culture period, the pH is kept near neutral. During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary. As a medium for culturing a transformant obtained using an animal cell as a host, a generally used RPMI1640 medium, DMEM medium, a medium obtained by adding fetal bovine serum or the like to these mediums, or the like is used. The culture is usually performed at about 37 ° C. for about 1 to 7 days in the presence of 5% CO ₂ . During culture, antibiotics such as kanamycin and penicillin may be added to the medium as necessary.

  After the culture, when the antibody or antigen-binding fragment is produced intracellularly or in cells, the protein is extracted by disrupting the cells or cells. When the antibody or antigen-binding fragment is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like.

  The collected antibody or antigen-binding fragment is obtained by methods well known in the art, such as chromatography using a protein A or protein G column, ion exchange chromatography, hydrophobic chromatography, ammonium sulfate salting out method, gel filtration, affinity chromatography. It can refine | purify by combining etc. suitably.

  The molecular weight of the purified antibody or antigen-binding fragment can be measured by polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting or the like. Further, the reactivity of the obtained antibody or antigen-binding fragment, that is, the binding activity to fibrin and optionally the binding activity to fibrinogen can be measured by the above-described method or the like.

2. Reagent for immunological measurement of fibrin It is possible to detect fibrin in a sample using the antibody prepared as described above. This detection can be performed based on any method as long as it is a measurement method using an antibody, that is, an immunological measurement method. For example, fibrin detection can be performed by immunohistochemical staining and immunoelectron microscopy, and immunoassay (enzyme immunoassay (ELISA, EIA), fluorescent immunoassay, radioimmunoassay (RIA), immunochromatography, Western blotting, etc.) Etc. can be implemented.

  The target sample is not particularly limited. For example, a tissue or cell sample (cancer tissue or cell such as stomach, duodenum, large intestine, pancreas, gallbladder, bile duct, bronchi, lung, etc.), biological fluid sample ( Gastric mucus, duodenal juice, pancreatic juice, bile, ascites, sputum, bronchoalveolar lavage fluid, blood, serum, plasma, etc.). For example, in the case of immunostaining, it is preferable to use a tissue sample (biopsy specimen, excised specimen) or cytodiagnosis sample as a sample.

  In the immunological measurement method of the present invention, fibrin is detected by binding fibrin in a sample to the antibody or antigen-binding fragment of the present invention and detecting the binding. In the present invention, “detection” includes not only detecting the presence or absence of fibrin but also quantitatively detecting fibrin and immunostaining fibrin.

  An immunoassay for fibrin typically involves contacting a sample to be tested with an antibody or antigen-binding fragment according to the present invention and binding the bound antibody or antigen-binding fragment using techniques known in the art. Including detecting. “Contact” means that the fibrin present in the sample and the antibody or antigen-binding fragment according to the present invention can be brought into close proximity so as to be able to bind. Operations such as applying a solution, immersing a solid sample in an antibody-containing solution, and mixing a liquid sample and an antibody-containing solution are included.

  The immunoassay may be performed in either a liquid phase system or a solid phase system. Further, the format of the immunoassay is not limited, and a direct solid phase method, a sandwich method, a competitive method, or the like may be used.

  The antibody or antigen-binding fragment according to the present invention can also be used histologically for in situ detection of fibrin, such as immunohistochemical staining (eg immunostaining) or immunoelectron microscopy. is there. In situ detection can be performed by excising a histological sample from a subject (such as a biopsy tissue sample, a paraffin-embedded section of tissue) and contacting it with a labeled antibody or antigen-binding fragment.

  The immunoassay can be manipulated by a known method (Ausubel, F.M. et al., Short Protocols in Molecular Biology, Chapter 11 “immunology” John Wiley & Sons, Inc. 1995). Alternatively, the complex of fibrin and antibody may be separated by a known separation means (chromatography, salting-out method, alcohol precipitation method, enzyme method, solid phase method, etc.) and the label signal may be detected. .

  As an example of an immunoassay, for example, when a solid phase system is used, an antibody or an antigen-binding fragment may be immobilized on a solid support or carrier (resin plate, membrane, bead, etc.), or a sample is immobilized. May be. For example, an antibody or antigen-binding fragment is immobilized on a solid support, and the support is washed with an appropriate buffer, and then treated with a sample. Next, the solid support is washed a second time with a buffer to remove unbound antibody or antigen-binding fragment. The binding of the fibrin in the sample to the antibody or antigen-binding fragment can be detected by detecting the bound antibody or antigen-binding fragment on the solid support by a conventional means. Alternatively, the solid sample is treated with a solution containing the antibody or antigen-binding fragment, followed by washing with a buffer to remove unbound antibody or antigen-binding fragment, and then binding on the solid sample. Antibodies or antigen-binding fragments can be detected by conventional means.

  The binding activity of the antibody can be measured according to a well-known method. A person skilled in the art can determine an effective and optimal measurement method for each assay according to the type and format of the immunoassay employed, the type of label used, the target of the label, and the like.

  In the present invention, in order to easily detect the reaction between the antibody or antigen-binding fragment of the present invention and fibrin present in a sample, the reaction is carried out by labeling the antibody or antigen-binding fragment of the present invention. Detection is performed directly or indirectly by using a labeled secondary antibody or a biotin-avidin complex. Examples of labels that can be used in the present invention and detection methods thereof are described below.

  In the case of enzyme immunoassay, for example, peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase, lactate dehydrogenase, amylase and the like can be used. Moreover, an enzyme inhibitor, a coenzyme, etc. can also be used. These enzymes and antibodies can be bound by a known method using a cross-linking agent such as glutaraldehyde or maleimide compound.

  In the case of a fluorescence immunoassay, for example, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC) or the like can be used. These fluorescent labels can be bound to antibodies by conventional techniques.

In the case of a radioimmunoassay, for example, tritium, iodine ¹²⁵ , iodine ^{131 and the} like can be used. The radioactive label can be bound to the antibody by a known method such as the chloramine T method or the Bolton Hunter method.

  For example, when the antibody or antigen-binding fragment of the present invention is directly labeled with a label as described above, the sample is contacted with the labeled antibody or antigen-binding fragment of the present invention, and the fibrin-antibody complex is prepared. Let it form. In the case of quantification, the amount of fibrin in the sample can be measured from the amount of bound labeled antibody or the amount of unbound labeled antibody after separating unbound labeled antibody.

  Further, for example, when a labeled secondary antibody is used, the antibody or antigen-binding fragment of the present invention is reacted with a sample (primary reaction), and the resulting complex is further reacted with a labeled secondary antibody (2 Next reaction). The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at different times. By the primary reaction and the secondary reaction, a fibrin-antibody-labeled secondary antibody complex of the present invention or an antibody-fibrin-labeled secondary antibody complex of the present invention is formed. When quantification is performed, unbound labeled secondary antibody is separated, and the amount of fibrin in the sample can be measured from the amount of bound labeled secondary antibody or the amount of unbound labeled secondary antibody.

  When the biotin-avidin complex system is used, a biotinylated antibody or antigen-binding fragment is reacted with the sample, and the resulting complex is reacted with avidin. Since avidin can specifically bind to biotin, the binding between the antibody and fibrin can be measured by detecting the signal of the label added to avidin. Although the label added to avidin is not particularly limited, for example, an enzyme label (peroxidase, alkaline phosphatase, etc.) is preferable.

  Detection of the labeled signal can also be performed according to methods known in the art. For example, in the case of using an enzyme label, a substrate that develops color by degradation by enzymatic action is added, and the enzyme activity is obtained by optically measuring the amount of degradation of the substrate. The amount of antibody is calculated from the comparison. The substrate varies depending on the type of enzyme used. For example, when peroxidase is used as the enzyme, 3,3 ′, 5,5′-tetramethylbenzidine (TMB), diaminobenzidine (DAB), etc. When alkaline phosphatase is used as the enzyme, paranitrophenol or the like can be used. The fluorescent label can be detected and quantified using, for example, a fluorescence microscope or a plate reader. When a radioactive label is used, the radiation dose emitted by the radioactive label is measured with a scintillation counter or the like.

  The present invention also relates to a reagent for immunoassay of fibrin comprising the antibody or antigen-binding fragment of the present invention. In the immunoassay reagent of the present invention, the antibody or antigen-binding fragment may be labeled. The antibody or antigen-binding fragment may be in a free form, or may be immobilized on a solid support (for example, a membrane, a bead, etc.).

In addition to the antibody or antigen-binding fragment of the present invention, the immunoassay reagent may contain components useful for carrying out the immunoassay method. Such components include, for example, buffers for use in immunoassays, sample processing reagents, labels, competitors, secondary antibodies, and the like.
By using the immunological measurement reagent of the present invention, fibrin in a sample can be detected easily and simply.

3. Determination of thrombosis-related diseases Since the antibody or antigen-binding fragment of the present invention specifically reacts with human fibrin as described above, it is used in a reagent for determining a fibrin-related disease or disorder, such as a thrombosis-related disease. Can do. In the present invention, “thrombosis-related disease” means a disease or disorder in which there is a correlation between the state of the disease or disorder and the presence of a thrombus. Such thrombosis-related diseases include, but are not limited to, infarcts such as myocardial infarction, cerebral infarction, cerebral hemorrhage, cerebral embolism, cerebral thrombosis, subarachnoid hemorrhage, pulmonary infarction and the like, and cancers such as pancreatic cancer, gastric cancer, esophagus Cancer, colorectal cancer, colon cancer, ovarian cancer, breast cancer and lung cancer are included. By detecting the presence of fibrin, it is possible to determine the presence or absence of a thrombus-related disease and specify the position of the thrombus-related disease.

  The antibody or antigen-binding fragment of the present invention is particularly useful for detecting cancer cells. For example, cancer cells can be stained by using for immunostaining, and cancer diagnosis can be performed reliably and reliably.

  The determination reagent of the present invention contains the above-described antibody or antigen-binding fragment of the present invention. Therefore, by using the determination reagent of the present invention, the subject is detected by detecting fibrin contained in a sample collected from a subject suspected of suffering from or suspected of having a thrombus-related disease (for example, infarction or cancer). The presence of a thrombus-related disease and the position of the thrombus-related disease in the subject can be determined quickly and easily. Reagents for determining diseases or disorders using such immunological measurement methods are well known, and those skilled in the art can easily select appropriate components other than antibodies. Further, the determination reagent of the present invention can be used in any technique as long as it is a technique for performing an immunological measurement method.

4). Thrombus Visualization / In Vivo Imaging, Delivery to Thrombus Site The antibodies and antigen-binding fragments of the present invention bind to fibrin in a subject when administered to the subject. Therefore, it is possible to visualize fibrin, ie, a thrombus in a subject, using the antibody and antigenic fragment of the present invention. It is also possible to deliver a compound or molecule to a fibrin in a subject, ie, a thrombus site, by binding the compound or molecule to an antibody or antigenic fragment of the invention.

The thrombus visualization agent of the present invention contains a labeled antibody or antigen-binding fragment of the present invention. As the label, any label known in the field of in vivo imaging can be used. Such labels include fluorescent materials such as IRDye800 series, fluorescein, FITC, fluorescent emitting metals ( ¹⁵² Eu, lanthanum series, etc.); chemical or bioluminescent materials such as luminol, imidazole, luciferin, luciferase, green fluorescent protein ( GFP); radioisotopes such as ^99m Tc, ¹²³ I, ¹³¹ I, ⁹⁷ Ru, ⁶⁷ Cu, ¹¹ C, ¹³ N; paramagnetic isotopes such as ¹⁵³ Gd, ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵² Cr, ⁵⁶ Fe, etc .; contrast agents such as gadolinium, gadolinium complexes, iodine contrast agents, etc. The antibody or the antigen-binding fragment can be bound to the label by a method known in the art, for example, it can be directly chemically bound or indirectly bound through an appropriate linker. May be.

  In the present invention, a compound or molecule such as a drug or prodrug is bound to the antibody or antigen-binding fragment of the present invention in place of the label, so that the compound or molecule is present in the fibrin-existing site of the subject, that is, the thrombus site. Can be delivered to. Such drugs or prodrugs include known thrombolytic agents (for example, urokinase, streptokinase, tissue type plasminogen activator) and the like. Such drug or prodrug targeting agents to the thrombus are also encompassed by the present invention.

The present invention further provides a complex of the antibody or antigen-binding fragment of the present invention and an antitumor moiety. Since the antibody or antigen-binding fragment of the present invention binds to a thrombus site (fibrin) in a tumor as described above, the anti-tumor moiety can be delivered to the tumor by binding to the anti-tumor moiety. The antitumor moiety that can be bound to the antibody or antigen-binding fragment of the present invention is not particularly limited as long as it is an antitumor moiety known in the art. Antitumor moieties include anticancer drugs such as irinotecan (CPT-11), irinotecan metabolite SN-38 (10-hydroxy-7-ethylcamptothecin), adriamycin, taxol, 5-fluorouracil, nimustine, laministin, etc. Drugs, antimetabolites such as gemcitabine and hydroxycarbamide, plant alkaloids such as etoposide and vincristine, anticancer antibiotics such as mitomycin and bleomycin, platinum preparations such as cisplatin, molecular targeting agents such as sorafenib and erlotinib, methotrexate, cytosine arabi Noside, 6-thioguanine, 6-mercaptopurine, cyclophosphamide, ifosfamide, busulfan, etc .; radioisotopes such as boron 10 ( ¹⁰ B), indium 111 ( ¹¹¹ In) and yttrium 90 ( ⁹⁰ Y) It is done. The antitumor moiety is preferably of a molecular weight such that after the complex of the present invention is delivered to the thrombus site in the tumor tissue, it is released from the complex at that site and can reach the entire tumor tissue.

  Further, the antibody or antigen-binding fragment can be bound to the anti-tumor moiety by a method known in the art, and either direct binding or indirect binding may be used. For example, a covalent bond can be used as a direct bond. As an indirect bond, a bond via a linker can be used.

  In the present invention, the antibody or antigen-binding fragment and the antitumor moiety are preferably bound via a linker. The binding of two molecules via a linker can reduce the antigenicity of the anti-tumor moiety and is preferred for administration to a subject. General techniques for linkers are described, for example, in Hermanson, GT Bioconjugate Techniques, Academic Press, 1996; Harris, JM and Zalipsky, S., Poly (ethylene glycol), Chemistry and Biological Applications, ACS Symposium Series, 1997; Veronese, F. and Harris, edited by JM, Peptide and protein PEGylation. Advanced Drug Delivery Review 54 (4), 2002.

  The linker means a divalent or higher valent group that connects two compounds. The linker that can be used in the present invention is not particularly limited, and examples thereof include polyalkylene glycol linkers, alkylene groups, peptides, sugar chains, and other polymer carriers. The alkylene part of the alkylene glycol which is a constituent unit of the polyalkylene glycol linker has 1 to 3000 carbon atoms, preferably 2 to 1000 carbon atoms, and more preferably 2 to 100 carbon atoms. The molecular weight of the polyalkylene glycol linker is usually 30 to 50000 Da, preferably 500 to 30000 Da. The polyalkylene glycol linker is preferably a polyethylene glycol (PEG) linker. The alkylene group may be linear or branched.

  The linker includes a linear linker (bivalent linker) and a branched linker (trivalent or higher linker). The linear linker has an anti-tumor moiety at one end and an anti-fibrin antibody or antigen-binding fragment at the other end. Branched linkers usually have an anti-tumor moiety in each branch (each chain) and an anti-fibrin antibody or antigen-binding fragment at the other end.

〔式中、PEGはポリエチレングリコール鎖であり、n及びmはエチレングリコール単位の数であり、独立して5〜100の整数を表す。〕
が挙げられる。式Iのリンカーは、通常、スクシンイミジル基を有する末端において抗体又は抗原結合性フラグメントと連結し、他の末端において抗腫瘍性部分と連結する。 Specific examples of linear linkers include those of formula I:

[Wherein, PEG is a polyethylene glycol chain, n and m are the number of ethylene glycol units, and independently represent an integer of 5 to 100. ]
Is mentioned. The linker of formula I is usually linked to the antibody or antigen-binding fragment at the end having a succinimidyl group and to the antitumor moiety at the other end.

〔式中、PEGはポリエチレングリコール鎖であり、xはエチレングリコール単位の数であり、5〜100の整数を表す。〕
が挙げられる。式IIのリンカーは、通常、スクシンイミジル基を有する末端において抗体又は抗原結合性フラグメントと連結し、他の末端において抗腫瘍性部分と連結する。 Further specific examples of linear linkers include linkers of formula II:

[Wherein, PEG is a polyethylene glycol chain, x is the number of ethylene glycol units, and represents an integer of 5 to 100. ]
Is mentioned. The linker of formula II is usually linked to the antibody or antigen-binding fragment at the end having a succinimidyl group and to the antitumor moiety at the other end.

〔式中、PEGはポリエチレングリコール鎖であり、n、m及びqはエチレングリコール単位の数であり、独立して5〜100の整数を表す。〕
が挙げられる。式IIIのリンカーは、通常、スクシンイミジル基を有する末端において抗体又は抗原結合性フラグメントと連結し、他の複数の末端において抗腫瘍性部分と連結する。この分枝状のリンカーは、例えば参考例１に記載のように調製することができる。 Specific examples of branched linkers include linkers of formula III:

[Wherein, PEG is a polyethylene glycol chain, n, m, and q are the number of ethylene glycol units, and independently represent an integer of 5 to 100. ]
Is mentioned. The linker of formula III is usually linked to the antibody or antigen-binding fragment at the end having a succinimidyl group and to the antitumor moiety at the other multiple ends. This branched linker can be prepared, for example, as described in Reference Example 1.

  Techniques for linking an antibody or antigen-binding fragment to an anti-tumor moiety via a linker are known in the art. For example, the bond between the antibody or antigen-binding fragment and the linker is a covalent bond or a non-covalent bond (ionic bond, hydrophobic bond, etc.), preferably a covalent bond. This bond is preferably a bond that hardly releases an antitumor moiety in blood when the complex of the present invention is administered to a subject. Examples of such bonds include bonds between maleimide groups and thiol groups, bonds obtained by reacting haloesters with thiols, amide bonds between carboxyl groups and amino groups, disulfide bonds between thiol groups and thiol groups, amino acids. Schiff base by aldehyde group and thioester bond between thiol group and carboxylic acid, ester bond between hydroxyl group and carboxyl group, bond by amino group and squaric acid derivative (for example, dimethyl squaric acid), dienylaldehyde group and amino group And the like. Specific examples of such a bond include a bond between a maleimide group present at one end of the linker and a thiol group contained in a cysteine residue on the antibody or antigen-binding fragment, a succinimide group present at one end of the linker, Dehydration substitution bond with amino group contained in lysine residue on antibody or antigen-binding fragment (for example, WO2008 / 096760), amino group present at one end of linker and aspartic acid on antibody or antigen-binding fragment or Examples include dehydration condensation bonds with carboxylic acids contained in glutamic acid (for example, using WSCDI). See, for example, WO / 2010/055950 for a specific method for binding an antibody or antigen-binding fragment to a linker.

  On the other hand, the bond between the antibody or antigen-binding fragment or linker and the anti-tumor moiety is a covalent bond or a non-covalent bond (ionic bond, hydrophobic bond, etc.), preferably a covalent bond. In particular, when an antitumor compound is used as the antitumor moiety, the binding should be such that the antitumor moiety is not easily released in blood when the complex of the present invention is administered to a subject. Is preferred. From this point of view, the bond between the antibody or antigen-binding fragment or linker and the antitumor moiety is not limited, but is preferably an ester bond, a carbamate bond, a carbonate bond, a thiocarbamate bond, An ester bond is preferable. In the case of an ester bond, it is expected that the antitumor moiety is released from the complex of the present invention in a sustained release by the hydrolysis of the bond by carboxylesterase in the tumor tissue or non-enzymatically. In the case of carbamate binding, it is expected that the antibody complex in the cell is endocytosed, then cleaved by intracellular carboxylesterase, and the antitumor moiety is released from the complex of the present invention in a sustained release manner. Is done. In the case of a carbonate bond, it is expected that the bond is hydrolyzed non-enzymatically and the antitumor moiety is released from the complex of the present invention in a sustained manner. In the case of a thiocarbamate bond, the bond is hydrolyzed non-enzymatically, and the antitumor moiety is expected to be released from the complex of the present invention in a sustained manner.

  When an antitumor compound is used as the antitumor moiety in the complex of the present invention, the number of antitumor compounds that bind to one molecule of antibody or antigen-binding fragment is not particularly limited in theory. From the viewpoints of stability and ease of production, the number is usually 1 to 10, preferably 1 to 8.

  Illustratively, the case where SN-38 (10-hydroxy-7-ethylcamptothecin) is used as the antitumor moiety and the polyethylene glycol linker is used as the linker will be described specifically. Those skilled in the art can produce the desired complex of the present invention by appropriately changing the reaction conditions.

  (I) First, a polyethylene glycol having a carboxyl group at one end and an amino group protected by Boc, Fmoc, etc. at the other end and SN-38 are subjected to dehydration condensation, and the hydroxyl group of SN-38 Introducing a polyethylene glycol linker.

  (II) A polyethylene glycol having a succinimide group at one end and a maleimide group at the other end is mixed with the product of (I), and the succinimide group and the amino group of the product of (I) To introduce a maleimide group into the polyethylene glycol linker.

  (III) The product of (II) and an antibody or antigen-binding fragment are mixed, the maleimide group in the product of (II) is reacted with the thiol group in the antibody or fragment, and the product of (II) The conjugate of the present invention is obtained by binding an antibody or fragment.

  The conjugates of the present invention can bind to fibrin in tumor tissue and deliver an anti-tumor moiety to the tumor and remain in the tumor tissue for a long period of time and continue to exert an anti-tumor effect for a long period of time. It has the effect. Therefore, the complex of the present invention can be used as a preventive or therapeutic agent for tumors. That is, a tumor in the mammal can be prevented or treated by administering an effective amount of the complex of the present invention to the subject. Furthermore, the complex of the present invention can also exert an anti-tumor effect over a long period of time by inhibiting the formation of blood vessels that remain in the tumor tissue for a long period of time and nourish the tumor in the border region of the tumor.

  The tumor to be treated or prevented in the present invention is not limited and includes solid cancers such as pancreatic cancer, gastric cancer, esophageal cancer, colorectal cancer, colon cancer, ovarian cancer, breast cancer and lung cancer.

  The complex of the present invention is not targeted to the cancer cell itself, but to fibrin present where it leaks from the tumor blood vessels. As described above, the antibody of the present invention does not react with fibrinogen and has an extremely high affinity for fibrin. Therefore, antibody-anti-tumor partial complex with high biocompatibility selectively leaks from tumor blood vessels due to EPR effect (enhanced permeation and retention effect) (after leakage from normal blood vessels because it is a polymer) It binds to fibrin present in the tumor stroma and forms a scaffold there. That is, for a long period of time, the complex of the present invention continues to exist in the interstitial fibrin. As described in Example 6 below, when an anti-tumor moiety SN-38 is conjugated with an antibody of the present invention via an ester bond, SN-38 is released from the complex by carboxylesterase in the tumor or naturally. Releases slowly. Since SN-38 is a small molecule, it can move relatively freely in cancer tissue and can spread throughout the cancer or attack cancer cells efficiently. Because SN-38 is time-dependent in its anti-tumor effect, such prolonged exposure of cancer cells to SN-38 can effectively kill cancer cells.

  The agent of the present invention containing the above-mentioned thrombus visualization agent and tumor preventive or therapeutic agent may contain both a pharmaceutically acceptable carrier or additive in addition to the antibody or the antigen-binding fragment. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, Examples include gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, mannitol, sorbitol, and lactose. The additive to be used is appropriately or in combination selected from the above depending on the dosage form.

  The administration method of the drug of the present invention is not particularly limited, and oral administration or parenteral administration, for example, subcutaneous administration, intradermal administration, intramuscular administration, intravenous administration, transdermal administration, rectal administration, nasal administration Etc.

  When orally administering the drug of the present invention, tablets, capsules (hard capsules, soft capsules, microcapsules, etc.), granules, powders, pills, troches, liquids for internal use, solutions, suspensions, It may be any emulsion, syrup, etc., and may be a dried product that is redissolved when used. In addition, when the drug of the present invention is administered parenterally, for example, intravenous injection (including infusion), intramuscular injection, intraperitoneal injection and injection for subcutaneous injection (eg, solution, emulsion, suspension), ointment Preparations such as topical agents (especially eye ointments), creams, suppositories, cataplasms, eye drops, nasal drops, inhalants, liniments, aerosols, etc. In some cases, they are provided in unit dose ampoules or in multi-dose containers.

  These various preparations are excipients, extenders, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, pH adjusters, preservatives, solubilizers commonly used in medicine. Agents, preservatives, flavoring agents, absorption promoters, soothing agents, stabilizers, tonicity agents and the like can be appropriately selected and produced by conventional methods.

  The antibody or antigen-binding fragment or complex to be blended with the drug of the present invention varies depending on the type of antibody, the type of complex and the antitumor part contained in the complex, its use, dosage form, administration route, etc. For example, it may be 1 to 99% by weight, preferably 5 to 90% based on the total weight.

  In addition, the dosage and administration interval of the drug of the present invention include the type of antibody or antigen-binding fragment contained in the drug, the type of anti-tumor part contained in the complex, the subject to be administered, the age and weight of the subject, administration It depends on the route and the number of administrations, and can be changed over a wide range.

  The subject to which the agent of the present invention is administered is not particularly limited, and mammals such as humans, domestic animals (cattle, pigs, etc.), pets (dogs, cats, etc.), laboratory animals (mouse, rats, monkeys). Etc.). In particular, it is preferably used for a subject suspected of having a thrombus-related disease and a subject having a thrombus-related disease. The drug containing the complex of the present invention is particularly preferably used for a subject suspected of having a tumor and a subject having a tumor.

  In the case of a thrombus visualization agent, after administration of a drug, the presence or position of an antibody or antigen-binding fragment in a subject is visualized using a label as an indicator. Preferably, the presence or location of the antibody or antigen binding fragment is visualized by known imaging techniques. Imaging methods vary depending on the label used, the type of subject, the site to be imaged, etc., but computed tomography (CT), positron tomography (PET), nuclear magnetic resonance imaging (MRI), and other in An in vivo imaging system can be used. This makes it possible to visualize the presence or location of a thrombus in a subject based on the label of the antibody or antigen-binding fragment.

5). Production of modified anti-fibrin antibody or antigen-binding fragment Based on the anti-fibrin antibody or antigen-binding fragment of the present invention, a modified antibody or antigen-binding fragment exhibiting a desired activity can be produced. Specifically, the amino acid sequence of the anti-fibrin antibody or antigen-binding fragment of the present invention is modified to prepare a modified antibody or antigen-binding fragment, and the activity of the obtained modified antibody or antigen-binding fragment is measured. judge. The modification of the amino acid sequence is not particularly limited, and includes deletion, substitution, addition or insertion of 1 to 100, for example, 1 to 50 amino acids in the amino acid sequence, fusion of another peptide, and the like. Such modification of the amino acid sequence and preparation of an antibody or antigen-binding fragment having the modified amino acid sequence can be performed by methods known in the art.

  Whether or not the obtained modified antibody or antigen-binding fragment binds to fibrin can be determined by a known method as described above. In some cases, the fibrin neutralizing activity of the modified antibody or the platen-binding fragment and the binding property to fibrinogen may be examined. The modified antibody or antigen-binding fragment thus obtained also has a binding activity to fibrin, and thus is useful for the above-described uses.

Hereinafter, the present embodiment will be described in more detail with reference to examples. The present invention is not limited to these.
[Example 1]
Preparation of mouse anti-fibrin antibody As an immunogen, a fibrin clot was prepared by allowing 200 IU thrombin to act on a 20 mg / ml fibrinogen (Sigma) solution in the presence of 0.1 M calcium chloride. The prepared fibrin clot was transferred to a mortar, added with liquid nitrogen, frozen and crushed with a pestle, and finally made into a 1 mg / ml fibrin suspension in a phosphate buffer and used for mouse sensitization.

  The Balb / c mouse strain was injected 3 times in total with 25 μg of fibrin crushed solution on the sole of the foot 2 days later. At that time, TiterMAX emulsion (TiterMAX USA Inc.) was used as an adjuvant. Three days after the final immunization, lymph node cells were collected from the mice.

The resulting lymph node cells and myeloma cells (P3U1) were fused, and further selected by ELISA using fibrin protein. Specifically, fibrinogen (Sigma) plates and fibrin plates were prepared, and hybridomas showing ELISA positive on the fibrin plate and negative on the fibrinogen plate were selected. For fibrinogen plates, add 50 μg / ml fibrinogen-dissolved TBS (pH 8.5) solution to a 96-well plate (Nunc468667) at 100 μl / well, and leave at 4 ° C. for 18 hours. Washed 3 times with 0.01% Tween 80, dried overnight at 37 ° C, and stored at 4 ° C until use. The fibrin plate was dried until the same as the fibrinogen plate, and then a TBS solution containing 10 NIH U / ml human thrombin (containing 10 mM CaCl ₂ and 7 mM L-cysteine) was added to the 96-well plate at 100 μl / well. After 1 hour at 37 ° C, the supernatant was removed, washed 3 times with PBS (containing 0.01% Tween80), dried overnight at 37 ° C, and stored at 4 ° C until use.

  ELISA was performed by adding the sampled hybridoma culture supernatant (stock solution) to a fibrinogen plate or fibrin plate at 50 μL / well and reacting at room temperature for 60 minutes. After washing 3 times with PBS, goat anti-mouse IgG-POD label (MBL product, Code.330) diluted 10,000-fold with dilution buffer (MBL) was added at 50 μL / well and allowed to react at room temperature for 60 minutes. After washing three times, a color developing solution (manufactured by MBL) was added at 50 μL / well for color development for 15 minutes, and 1.5 mol / L phosphoric acid was added at 50 μL / well to stop the reaction. After stopping the reaction, absorbance was measured at a measurement wavelength of 450 nm and a reference wavelength of 620 nm.

  A hybridoma cell that produces a monoclonal antibody that binds to human fibrin and does not bind to human fibrinogen was selected, and a cell line (clone 102-10) was established. The antibody produced by this cell line was mouse IgM. This hybridoma cell line is “102-10”, the National Institute for Product Evaluation and Technology (NITE) Patent Microorganism Depositary Center (NPMD) (Nise Biotechnology Headquarters, 2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture) Was deposited on April 1, 2010 and assigned the deposit number NITE P-923.

  In addition, the culture supernatant of hybridoma cell line 102-10, commercially available anti-fibrin antibody NYB-T2G1 (anti-fibrin II antibody, ACCURATE CHEMICAL & SCIENTIFIC CORPORATION) and MH-1 (American Biogenetic Sciences, Inc., Japanese Patent Laid-Open No. 11-80200) The reactivity with mouse and human fibrin, and mouse and human fibrinogen was examined by ELISA using the fibrin plate and fibrinogen plate described above.

  The result is shown in FIG. As shown in FIG. 1, it was confirmed that the mouse IgM antibody derived from the hybridoma cell line 102-10 specifically reacts with mouse and human fibrin but does not react with mouse and human fibrinogen.

[Example 2]
Preparation of Chimeric Antibody Total RNA was extracted from the hybridoma cell line 102-10 prepared in Example 1, and cDNAs of the antibody H chain variable region and L chain variable region were amplified using the adapter ligation RT-PCR method. . The primer sequences used are as follows:

For H chain Forward primer: 5'-CGACTGGAGCACGAGGACACTGA-3 '(SEQ ID NO: 11)
Reverse primer: 5'-GTTGTTCTGGTAGTTCCAGGTG-3 '(SEQ ID NO: 12)
For L chain Forward primer: 5'-CGACTGGAGCACGAGGACACTGA-3 '(SEQ ID NO: 13)
Reverse primer: 5′-CCGCTTAATTAACTAACACTCATTCCTGTTGAAGCTCT-3 ′ (SEQ ID NO: 14).

  Each amplified cDNA was cloned into pT7Blue (Promega). The H chain variable region (396 bp) and L chain variable region (381 bp) cDNAs are shown in FIGS. 2A shows the base sequence and amino acid sequence of the antibody H chain, and corresponds to the variable region (1 to 396 bp) and a part of the constant region. The base sequence and amino acid sequence of the variable region of the H chain are shown in SEQ ID NOs: 7 and 8, respectively. B of FIG. 2 shows the base sequence and amino acid sequence of the antibody L chain, and corresponds to the entire length of the protein coding region (variable region is 1 to 381 bp). The base sequence and amino acid sequence of the variable region of the L chain are shown in SEQ ID NOs: 9 and 10, respectively. In FIG. 2, complementarity determining regions (CDR) 1 to 3 of the H chain and the L chain are indicated by squares.

  After amplification of the H and L chain variable regions by PCR, the H chain variable region is inserted into pQCxIP (Clontech) incorporating the constant region, and the L chain variable region is inserted into pQCxIH (Clontech) incorporating the constant region for expression. Completed the vector. The expression vector was transfected into CHO cells (RIKEN BioResource Center) using Lipofectamine 2000 (Invitrogen). Human anti-fibrin chimeric antibody constant expression cell line (human IgG clone 102-10Hu) is selected with puromycin (Sigma) 10μg / mL and hygromycin B (Invitrogen) 500μg / mL to obtain both resistant strains It was established by that. The established cell lines were maintenance cultured with F12 (Sigma) 10% FBS, 1 mM HEPES (Sigma), 1% penicillin streptomycin (Invitrogen), puromycin 10 μg / mL, and hygromycin B 500 μg / mL.

Established cell lines using 10% FBS medium for master cells (F12 [Sigma: N6658], 10% FBS [Hyclone: lot.APM22733] (FBS is Bovine IgG) using 175 cm ² flasks (for adherent cells) 1% Penicillin-Streptomycin [Invitrogen], 1 mM HEPES [Sigma: H4034-500G], 50 mg / mL Hygromycin B 5.7 mL [WAKO: 085-06153], and 2 mg / The cells were cultured to confluence in 2.9 mL of puromycin hydrochloride (WAKO: 533-71593)). The culture supernatant was removed by aspiration, and the flask was washed with 10 mL of PBS. This washing was performed twice. After removing PBS, 5 mL of TRYPSIN / 0.5% EDTA [Invitrogen: 25300054 CAMP] was added, and the mixture was allowed to stand in a CO ₂ incubator for 5 minutes. After confirming that the cells were detached from the bottom surface, the enzyme reaction was stopped by adding 10 mL of 10% FBS medium for master cells. Thereafter, the cells were removed by pipetting, and all the liquid in the flask was collected and centrifuged at 1000 rpm for 5 minutes. The supernatant after centrifugation was removed, and the pellet was suspended in 30 mL of 10% FBS medium for master cells. Next, 29 mL of 10% FBS medium for master cells was added to the flask, and 1 mL of the cell suspension after centrifugation was added.

Subsequently, the following operation was performed for mass culture. 29 mL of 5% FBS medium F12 (Sigma) for mass culture was added to 30 175 cm ² flasks (for floating cells). 1 mL of each of the cell suspension obtained above was added to a flask for mass culture, and statically cultured in a CO ₂ incubator. About 1 week after the passage, 5 mL of 5% FBS medium for mass culture was added. Under the condition that cells floating under a microscope were confluent, 10 mL of 5% FBS medium for mass culture was added every 3 to 4 days thereafter, and the volume was increased to 50 mL. Microscope at the time of medium addition. If cell aggregates are almost gone and the ratio of dead cells is over 80%, the culture is stopped at that point and 3-4 days after the last addition of the medium. The supernatant was collected and centrifuged at 3000 rpm for 5 minutes. 0.05% amount of sodium azide was added to the culture supernatant and stored refrigerated until purification.

  Antibody purification was performed as follows. That is, the culture supernatant obtained above was filtered through a 0.22 um filter to remove insolubles. The culture supernatant was applied to a protein G Sepharose (registered trademark) 4B packed column equilibrated with a binding buffer (phosphate buffer pH 7). The column was washed 4 times with a rinse buffer (phosphate buffer pH 7) 5 times the column volume, and an elution buffer (glycine buffer) pH 4 was passed 1.8 times the column volume. Next, elution buffer (glycine buffer) pH 3 was flowed 6 times the column volume and collected for each fraction, and the eluate was quickly neutralized with 1M Tris HCl buffer (pH 7). The absorbance of the fraction was measured, and the fraction containing the antibody was collected and proceeded to the next purification of hydroxyapatite.

  In the purification of hydroxyapatite, the sample was first dialyzed against 10 mM Na-PB (pH 6.5) and 300 mM NaCl. After equilibrating a hydroxyapatite type II packed column with 10 mM Na-PB (pH 6.5), 300 mM NaCl, add all samples, wash with 10 mM Na-PB (pH 6.5), 300 mM NaCl, Elution was performed with 10 mM Na-PB (pH 6.5) and 2 M NaCl. Fractions were collected, each fraction was confirmed by SDS-PAGE, and a sample was collected. After dialysis against PBS, it was concentrated by ultrafiltration to obtain a purified human anti-fibrin chimeric antibody. The reactivity of the obtained chimeric antibody was confirmed using ELISA as in Example 1. The result is shown in FIG.

  As shown in FIG. 3, it was shown that the human chimeric antibody specifically reacts with mouse and human fibrin but does not react with mouse and human fibrinogen. Therefore, human antibodies derived from mouse IgM retain the reactivity of mouse IgM and can be more suitably applied to humans. In addition, since it has binding properties with mouse and human fibrin, it is possible to extrapolate experimental results in mice to humans.

[Example 3]
In this example, human pancreatic cancer surgical specimens and human brain tumors (gliomas) were prepared using the mouse IgM antibody and human chimeric antibody prepared in Examples 1 and 2. ) Immunostaining of surgical specimens was performed.

Human pancreatic cancer surgical specimens were obtained from Fukushima Medical University Pathology, and paraffin sections were prepared. In addition, human brain tumor (glioma) surgery specimens were donated by Kumamoto University Hospital Neurosurgery, and paraffin sections were prepared. Paraffin sections were treated with xylene and ethanol to deparaffinize and immersed in demineralized water. Sections were blocked in 0.3% H ₂ O ₂ / MeOH for 20 minutes and washed with TBST (Tris buffer, Tween 20) for 5 minutes. Then, the section was immersed in an antigen activation solution (10 mM citrate buffer, pH 6.0), treated with microwave (MW) at 93 ° C. for 20 minutes, allowed to stand for 30 minutes, and then 5 times with TBST for 5 minutes. Washed.

  Sections were processed at 4 ° C. using 10 μg / ml mouse IgG antibody (Example 1) or 2 μg / ml human chimeric antibody (Example 2) as the primary antibody. Next, after washing 3 times with TBST for 5 minutes, several drops of anti-mouse secondary antibody (Code No. K4001, DAKO) or 200-fold diluted anti-human secondary antibody (Code No. 206, MBL) The reaction was allowed to proceed for 1 hour at room temperature. The sections were washed 3 times with TSBT for 5 minutes, and DAB (Code No. K4007, DAKO) was added dropwise as a chromogenic substrate to react for 5 minutes. The sections were washed with demineralized water, immersed in hematoxylin for 20 seconds, washed with water for 10 minutes, and then treated with ethanol and xylene.

  FIG. 4 shows the result of immunostaining of a human pancreatic cancer surgical specimen section using the chimeric antibody. Further, FIG. 5 shows the result of immunostaining of a human brain tumor surgical specimen section using mouse IgM antibody. As shown in FIGS. 4 and 5, it was found that human pancreatic cancer tissue can be stained using this antibody.

[Example 4]
Immunostaining of human tissue using mouse IgM antibody and human chimeric antibody Immunostaining of invasive squamous cell carcinoma was performed in the same manner as in Example 3. The result is shown in FIG. In FIG. 6, A shows a tissue subjected to hematoxylin and eosin staining, and invasive squamous cell carcinoma surrounded by cancer stroma is observed. B shows an immunostained tissue with anti-fibrin antibody IgM, and it is confirmed that fibrin is significantly present in the cancer stroma. C shows an intratumoral accumulation image of fluorescently labeled human chimeric anti-fibrin IgG, and it can be confirmed that the antibody is specifically accumulated in the stroma in the tumor and coincides with the fibrin existing site of B .

[Example 5]
In vivo imaging using antibodies In this example, the tumor accumulation of anti-fibrin antibodies was examined.

  The human breast cancer cell line MCF7 was transplanted to the back of BALB / c nude mice (female, 6 weeks old). As shown in FIG. 7A, cancer formation was observed in mice. Ten days after transplantation, a human chimeric antibody labeled with IRDye800 (Li-Cor) was administered via the mouse tail vein. The distribution of antibodies 1 day, 3 days and 7 days after the administration was analyzed using a biological imaging apparatus OV110 (Olympus) and NightOWL II LB 983 (Berthold).

  The results on day 7 after administration are shown in FIGS. From these results, it was shown that the anti-fibrin antibody selectively accumulates in the tumor tissue and accumulates in the tumor tissue for a long time.

[Reference Example 1]
Binding of branched linker and antitumor compound SN-38 According to the scheme shown in FIG. 8, the branched linker and antitumor compound SN-38 (10-hydroxy-7-ethylcamptothecin) were bound. In the following reaction, “DMAP” represents N, N-dimethyl-4-aminopyridine, “DMF” represents N, N-dimethylformamide, and “THF” represents tetrahydrofuran.

(1) Combination of SN-38 and PEG
In a DMF solution (1 mL) of 10-hydroxy-7-ethylcamptothecin (102.1 mg, 0.260 mmol), Boc-PEG ₂₇ -COOH (407.1 mg, 0.286 mmol), DMAP (15.9 mg, 0.130 mmol), WSCDI (water soluble carbodiimide: 54.8 mg, 0.286 mmol) was added at 0 ° C. The mixture was stirred at room temperature for 19 hours, and the reaction mixture was purified by gel filtration column chromatography (LH20 CHCl ₃ : MeOH = 1: 1), silica gel column chromatography (CHCl ₃ : MeOH = 15: 1 to 9: 1). Ester I was obtained as a colorless oil (420.1 mg, 90%).
¹ H-NMR δ (CD ₃ OD) 8.20 (d, J = 9.2 Hz, 1H), 7.99 (s, 1H), 7.66 (m, 2H), 5.60 (d, J = 16.5 Hz, 1H), 5.40 ( d, J = 16.5 Hz, 1H), 5.32 (M, 2H), 3.93 (t, J = 6.4 Hz, 2H), 2.96 (t, J = 6.4 Hz, 2H), 1.97 (m, 2H), 1.43 ( s, 9H), 1.40 (t, J = 7.6 Hz, 3H), 1,02 (t, J = 7.8 Hz, 3H); ¹³ C-NMR (DMSO-d ₆ ) δ164.8, 161.9, 149.2, 148.5 , 143.1, 142.7, 141.3, 138.2, 137.7, 137.5, 122.4, 119.5, 118.9, 117.2, 110.7, 106.5, 89.7, 70.4, 64.6, 62.3, 62.0, 62.0, 62.0, 61.9, 61.8, 61.8, 61.7, 61.5, 58.1 , 58.0, 57.1, 41.2, 40.1, 31.8, 28.1, 26.3, 19.4, 14.4, 4.9, -1.1.

(2) Branched linker synthesis
To a solution of 3- (allyloxy) -2- (allyloxymethyl) -2-((but-3-enyloxy) methyl) propan-1-ol (14.85 g, 31.17 mmol) in DMF (30 mL) was added NaH (21.87 g, 46.78 mmol) was added at room temperature. After 1 hour, bromide 1-((5-bromopentyloxy) methyl) -4-methoxybenzene (16.7 g, 58.04 mmol) was added. The flask was washed twice with DMF (3 mL). The mixture was stirred at 55 ° C. for 1 day. After cooling to room temperature, N, N-dimethyl 1,3-propanediamine (10 mL) was added. After 1 hour, the mixture was diluted with saturated NH ₄ Cl and EtOAc. The aqueous layer was extracted with EtOAc. The mixed layer was washed with saturated brine. The organic layer was dried over Na ₂ SO ₄ and filtered. After evaporation, the residue was purified on a silica gel column (hexane: EtOAc 9: 1 to 4: 1) to give compound A [1-((5- (3- (allyloxy) -2- (allyloxymethyl)] as a colorless oil. The ether compound (8.60 g, 58%) of -2-((but-3-enyloxy) methyl) propyl) pentyloxy) methyl) -4-methoxybenzene] was obtained.
¹ H-NMR δ 7.23 (dd, J = 6.4 Hz, 2.4 Hz, 2H), 6.5 (dd, J = 6.4 Hz, 2.4 Hz, 2H), 5.85 (m, 3H), 5.23 (d, J = 17.2 Hz , 3H), 5.11 (d, J = 10.4 Hz, 3H), 4.41 (s, 2H), 3.93 (m, 6H), 3.78 (s, 3H), 3.42 (s, 8H) 3.39-3.36 (m, 4H ), 1.60-1.51 (m, 4H), 1.38 (m, 2H), ¹³ C-NMR δ 135.21, 129.11, 115.97, 113.69, 72.53, 72.26, 71.33, 7.13, 69.60, 69.42, 55.30, 45.44, 29.65, 29.50 , 22.94.

Triaryl compound 1-((5- (3- (allyloxy) -2- (allyloxymethyl) -2-((but-3-enyloxy) methyl) propoxy) pentyloxy) methyl) -4-methoxybenzene ( 8.60 g, 18.06 mmol) in CH ₂ Cl ₂ (200 mL) and MeOH (200 mL) was bubbled with ozone at −78 ° C. until the reaction mixture turned light blue. After replacing ozone with oxygen gas, NaBH ₄ (8.60 g, 210 mmol) was added in several portions. The reaction mixture was gradually warmed and then stirred overnight at room temperature. After concentrating the reaction, the mixture was partitioned between CHCl ₃ and saturated NH ₄ Cl. The aqueous layer was extracted with CHCl ₃ . The combined layers were washed with saturated brine, dried over Na ₂ SO ₄ and filtered. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (CHCl ₃ : MeOH 9: 1) to obtain compound B [2,2 ′-(2-((2-hydroxyethoxy) methyl) -2-(( 5- (4-Methoxybenzyloxy) pentyloxy) methyl) propane-1,3-diyl) bis (oxy) diethanol] triol compound (8.56 g quant.) Was obtained.
¹ H-NMR δ 7.23 (d, J = 8.8 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H), 4.41 (s, 2H), 3.84 (s, 3H), 3.79 (s like, 6H) , 3.53 (m, 6H), 3.48 (s like, 8H), 3.43-3.37 (m, 4H), 2.86 (s, 3H), 1.58-1.53 (m, 4H), 1.39 (m, 2H); ¹³ C -NMR δ 129.16, 113.69, 72.56, 72.50, 71.67, 70.46, 70.05, 70.00, 61.4, 55.30, 45.40, 29.56, 29.32, 22.92.

Triol compound B (2,2 ′-(2-((2-hydroxyethoxy) methyl) -2-((5- (4-methoxybenzyloxy) pentyloxy) methyl) propane-1,3-diyl) CBr ₄ (8.14 g, 24.51 mmol) was added in several portions at 0 ° C. to a solution of bis (oxy) diethanol (2.91 g, 6.14 mmol) and PPh ₃ (6.43 g, 24.51 mmol) in THF (50 mL). The mixture was stirred overnight at room temperature, diethyl ether was added to the mixture, the precipitate was filtered, the filtrate was concentrated and purified by silica gel column chromatography (hexane: EtOAc 9: 1 to 4: 1) to give the compound C [1-((5- (3- (2-Bromoethoxy) -2,2-bis ((2-bromoethoxy) methyl) propoxy) pentyloxy) methyl) -4-methoxybenzene] tribromide (3.48 g, 86%).
¹ H-NMR d 7.25 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.4 Hz, 2H), 4.42 (s, 2H), 3.80 (s, 3H), 3.74-3.71 (m, 6H ), 3.47 (s, 8H), 3.47-3.38 (m, 6H), 1.7-1.50 (m, 4H), 1.40 (m, 2H); ¹³ C-NMR δ 129.12, 113.66, 72.53, 71.33, 71.14, 70.08 , 69.38, 68.88, 55.29, 45.72, 30.82, 29.63, 22.97.

Tribromide C (1-((5- (3- (2-bromoethoxy) -2,2-bis ((2-bromoethoxy) methyl) propoxy) pentyloxy) methyl) -4-methoxybenzene (3.48 g, 5.27 mmol) in DMF (10 mL) was added NaN ₃ (5.27 g, 81.08 mmol) and the mixture was stirred for 4 h at 50 ° C. The mixture was diluted with EtOAc and saturated NaHCO ₃ . The mixture was washed with saturated brine, dried over Na ₂ SO ₄ , and the solvent was concentrated under reduced pressure.The residue was purified by silica gel column chromatography (hexane: EtOAc 7: 3), Compound D [1-((5- (3- (2-azidoethoxy) -2,2-bis ((2-azidoethoxy) methyl) propoxy) pentyloxy) methyl) -4-methoxybenzene] triazide compound ( 2.84 g, 98%).
¹ H-NMR δ 7.23 (d, J = 8.8 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H), 4.41 (s, 2H), 3.78 (s, 3H), 3.59 (t, J = 4.8 Hz, 6H), 3.47 (s, 8H), 3.44-3.28 (m, 10H), 1.61-1.55 (m, 4H), 1.39 (m, 2H); ¹³ C-NMR δ 129.04, 113.63, 72.53, 71.32, 70.46, 70.13, 69.71, 68.85, 55.32, 50.86, 45.12, 29.70, 23.04.

To a solution of the above PMB ether (2.63 g, 4.7 mmol) in CH ₂ Cl ₂ (30 mL) and H ₂ O (20 mL) was added DDQ (1.30 g, 5.75 mmol) at 0 ° C. After the addition, the ice bath was removed and stirred at room temperature. After 5 hours, the reaction was quenched with citrate buffer and the aqueous layer was extracted with EtOAc. The combined layers were washed with saturated NaHCO ₃ and saturated brine. The organic layer was dried over Na ₂ SO ₄ . After filtration, the solvent was removed. The residue was purified by silica gel column chromatography (hexane: EtOAc 1: 1) and compound E [5- (3- (2-azidoethoxy) -2,2-bis ((2-azidoethoxy) methyl) propoxy) pentane 1-ol] alcohol compound (1.55 g, 75%) was obtained.
¹ H-NMR δ 3.64-3.58 (m, 8H), 3.46 (s, 6H), 3.41-3.38 (m, 4H), 3.31-3.28 (m, 6H), 1.57-1.55 (m, 4H), 1.41 ( m, 2H); ¹³ C-NMR δ 71.20, 70.46, 69.67, 68.84, 62.92, 50.86, 45.08, 32.57, 29.36, 22.56.

Jones reagent was added at 0 ° C. to a solution of the alcohol compound (1.55 g, 3.61 mmol) obtained above in acetone (20 mL). Excess Jones reagent was destroyed with isopropyl alcohol iPrOH and the precipitate was filtered. After concentration, the residue was purified by silica gel column chromatography (CHCl ₃ : EtOAc 7: _{3 to} 1: 1), and compound F [5- (3- (2-azidoethoxy) -2,2-bis ((2- Azidoethoxy) methyl) propoxy) pentanoic acid] acid (1.37 g, 86%) was obtained.
¹ H-NMR δ 3.72 (t, J = 6.0 Hz, 3H), 3.60 (t, J = 4.4 Hz, 4H), 3.47 (s, 8H), 3.44-3.40 (m, 4H), 3.32 (t, J = 4.8 Hz, 4H), 2.83 (t, J = 7.6 Hz, 2H), 1,70 (m, 2H), 1.60 (m, 2H); ¹³ C-NMR δ 177.69, 77.21, 70.77, 70.48, 69.68, 68.96, 50.87, 45.11, 33.55, 28.93, 21.81.

The above-obtained acid (1.65 g, 3.85 mmol), N- (tert-butoxycarbonyl) -1,5-diaminopentane (1.56 g, 7.69 mmol) and HOBt (1.03 g, 7.60 mmol) in CH ₂ Cl ₂ ( 30 mL) To the solution was added water soluble carbodiimide WSCDI (1.47 g, 7.69 mmol) at 0 ° C. The mixture was stirred overnight at room temperature and diluted with CHCl ₃ and saturated NH ₄ Cl. The aqueous layer was extracted with CHCl ₃ . The combined layers were washed with saturated NaHCO ₃ and saturated brine, dried over Na ₂ SO ₄ and the solvent was concentrated. The residue was purified by silica gel column chromatography (CHCl ₃ : EtOAc 1: 1 to EtOAc only) and compound G [tert-butyl 5- (5- (3- (2-azidoethoxy) -2,2-bis (( 2-Azidoethoxy) methyl) propoxy) pentanamide) pentylcarbamate] amide compound (2.18 g, 90%) was obtained.
¹ H-NMR δ 5.60 (bs, 1H), 4.55 (bs, 1H), 3.72 (t, J = 6.0 Hz, 2H), 3.62 (t, J = 4.8 Hz, 4H), 3.47-3.41 (m, 12H ), 3.30 (t, J = 4.8 Hz, 4H), 3.22 (q, J = 6.4 H, 2H), 3.10 (m, 2H), 2.17 (t, J = 7.2 Hz, 2H), 1.70-1.46 (m , 7H), 1.43 (s, 9H), 1.34 (m, 3H), ¹³ C-NMR δ 172.64, 155.87, 77.21, 71.15, 71.13, 70.99, 70.94, 70.50, 69.73, 69.69, 69.36, 68.97, 50.91, 45.35 , 45.11, 40.35, 39.36, 36.56, 31.00, 29.88, 20.40, 29.23, 28.55, 24.09, 22.77.

(3) Linkage between branched linker and SN-38 Dissolve SN38-PEG Boc compound (0.71 g, 0.396 mmol) of Compound J in CH ₂ Cl ₂ (20 mL) and add 4.0 M dioxane (20 mL). Stir at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, toluene was added, and the mixture was further concentrated and dried under vacuum. This residue was dissolved in CH ₂ Cl ₂ (20 mL), iPr ₂ NEt (0.57 mmol, 3.24 mmol) was added at 0 ° C., anhydrous succinimide (60 mg, 0.594 mmol) was added, and the mixture was stirred at room temperature overnight. The reaction solution was purified by LH20 (CHCl ₃ : MeOH 1: 1) and silica gel column chromatography (CHCl ₃ : MeOH 9: 1 to 4: 1) to obtain K.

  On the other hand, trimethylphosphine (0.5 mL, 1M solution) was added to a solution of triazide H (63 mg, 0.10 mmol) in dioxane (1 mL) and water (0.5 mL), and the mixture was stirred overnight at room temperature under a nitrogen atmosphere.

Concentrate the reaction mixture, add the above carboxylic acid K in CH ₂ Cl ₂ (10 mL), add HOBt (213 mg, 1.58 mmol), WSCDI (308 mg, 1.58 mmol), iPr ₂ NEt (0.5 mL). It was. The reaction solution was stirred overnight at room temperature, and the reaction solution was purified with LH20 (CHCl ₃ : MeOH 1: 1) to obtain 0.71 g. This was dissolved in dioxane (20 mL) and 4.0 M hydrochloric acid-dioxane solution (20 mL), stirred for 3 hours at room temperature, and then concentrated under reduced pressure. This was dissolved in CH ₂ Cl ₂ (1 mL), iPr ₂ NEt (0.1 mL) was added, and N-succimidyl PEG ₁₂ -Mal (100 mg) and iPr ₂ NEt (0.2 mL) were added. The mixture was stirred overnight at room temperature, and purified with LH20 (CHCl ₃ : MeOH 1: 1) to obtain the target product L.

[Reference Example 2]
Bonding of linear linker and antitumor compound SN-38 The linear linker and antitumor compound SN-38 (10-hydroxy-7-ethylcamptothecin) were bound as described below. In the following reaction, “DMAP” represents N, N-dimethyl-4-aminopyridine, “DMF” represents N, N-dimethylformamide, and “TFA” represents trifluoroacetic acid.

(1) Combination of SN-38 and PEG

In a DMF solution (1 mL) of 10-hydroxy-7-ethylcamptothecin (102.1 mg, 0.260 mmol), Boc-PEG ₂₇ -COOH (407.1 mg, 0.286 mmol), DMAP (15.9 mg, 0.130 mmol), WSCDI (water soluble carbodiimide: 54.8 mg, 0.286 mmol) was added at 0 ° C. The mixture was stirred at room temperature for 19 hours, and the reaction mixture was purified by gel filtration column chromatography (LH20 CHCl ₃ : MeOH = 1: 1), silica gel column chromatography (CHCl ₃ : MeOH = 15: 1 to 9: 1). The ester was obtained as a colorless oil (420.1 mg, 90%).
¹ H-NMR δ (CD ₃ OD) 8.20 (d, J = 9.2 Hz, 1H), 7.99 (s, 1H), 7.66 (m, 2H), 5.60 (d, J = 16.5 Hz, 1H), 5.40 ( d, J = 16.5 Hz, 1H), 5.32 (M, 2H), 3.93 (t, J = 6.4 Hz, 2H), 2.96 (t, J = 6.4 Hz, 2H), 1.97 (m, 2H), 1.43 ( s, 9H), 1.40 (t, J = 7.6 Hz, 3H), 1,02 (t, J = 7.8 Hz, 3H); ¹³ C-NMR (DMSO-d ₆ ) δ164.8, 161.9, 149.2, 148.5 , 143.1, 142.7, 141.3, 138.2, 137.7, 137.5, 122.4, 119.5, 118.9, 117.2, 110.7, 106.5, 89.7, 70.4, 64.6, 62.3, 62.0, 62.0, 62.0, 61.9, 61.8, 61.8, 61.7, 61.5, 58.1 , 58.0, 57.1, 41.2, 40.1, 31.8, 28.1, 26.3, 19.4, 14.4, 4.9, -1.1.

(2) Introduction of maleimide group into linear linker

To a solution of Boc-PEG ₂₇ -camptothecin (221.5 mg, 0.123 mmol) in CH ₂ Cl ₂ (10 mL) was added TFA (1 mL) at room temperature. The mixture was stirred for 1.5 hours, then removed in vacuo and toluene was added. After evaporation, the residue was dried under high vacuum. The residue was dissolved in CH ₂ Cl ₂ (5 mL) and MAL-PEG ₁₂ -NHS (128.2 mg, 0.148 mmol) and iPr ₂ NEt (48 μL, 0.246 mmol) was added at 0 ° C. After 30 minutes, the mixture was purified by gel filtration column chromatography (LH-20, CHCl ₃ : CH ₃ OH = 1: 1) and silica gel column chromatography to obtain the desired product as a colorless oil (276.0 mg, 91 %).
¹ H-NMR (CD ₃ OD) δ 8.20 (d, J = 9.2 Hz, 1H), 8.00 (s, 1H), 7.66-7.64 (m, 2H), 6.83 (s, 2H), 5.60 (d, J = 16.0 Hz, 1H), 5.41 (d, J = 16.0 Hz, 1H), 5.34 (s, 1H), 3.93 (t, J = 6.0 Hz, 2H), 2.97 (t, J = 6.0 Hz, 2H), 2.49-2.45 (m, 4H), 2.00-1.98 (m, 2H), 1.41 (t, J = 7.6 Hz, 3H), 1.02 (t, J = 7.6 Hz, 3H); ¹³ C-NMR (DMSO-d ₆ ) δ 172.1, 170.4, 169.8, 169.7, 169.1, 156.5, 151.7, 149.7, 148.9, 146.2, 145.6, 145.0, 134.3, 131.1, 128.4, 126.8, 125.3, 118.8, 115.0, 96.5, 72.3, 69.9, 69.6, 69.5 69.4, 69.0, 68.9, 66.7, 65.8, 65.1, 49.5, 36.0, 34.8, 34.1, 33.9, 31.6, 30.3, 25.4, 22.3, 13.9, 7.8.

[Example 6]
Binding of SN-38-Linker Conjugate and Antibody As shown in FIG. 9, PEG-SN-38 conjugate bound to a branched linker (Reference Example 1), or PEG bound to a linear linker -SN-38 conjugate (Reference Example 2) was conjugated with an anti-fibrin antibody.

  Specifically, the antibody was prepared at a concentration of 1.0 mg / ml in PBS. Dithiothreitol (DTT) was added to the antibody to a final concentration of 10 mM and reacted at 37 ° C. for 30 minutes. The reaction reagent was removed with Amicon Ultra. Absorption measurement showed that the antibody recovery was about 80%. Moreover, 7 SH groups were obtained per antibody from the quantification results of SH groups by (5,5′-dithiobis (2-nitrobenzoic acid)) (DNTB).

  Next, it is dissolved in 100 mM phosphate buffer + 150 mM NaCl + 5 mM EDTA (pH 6.0) so that the protein concentration becomes 0.5 mg / ml, and L (maleimide compound) 4 of FIG. After mixing at a ratio, the mixture was reacted at room temperature for 1 hour and then at 4 ° C. overnight.

  After removing the reaction reagent with Amicon Ultra, it was replaced with PBS. When the protein was recovered with Amicon Ultra, the recovery rate was 90%, and 21 SN-38s were added per antibody in the antibody to which the branched linker was bound.

  In addition, the amount of SN-38 bound to the antibody when using a linear linker and a branched linker was examined using HPLC. The results are shown in Table 1. In the table, the “AVERAGE” column shows the average amount of SN-38 binding per antibody molecule.

  As shown in Table 1, the SN-38-antibody complex via a branched linker has a SN-38 binding amount per antibody as compared with the case where a conventional linear linker is used. It was found that the average was about 3 times more.

[Example 7]
In vitro cell killing effect In this example, the in vitro cell killing effect of the antibody-SN-38 complex prepared in Example 6 on pancreatic cancer cell line SUIT2 was confirmed. Specifically, 3000 SUIT2 cancer cells were seeded in a 96-well plate, and 24 hours later, a complex of SN-38 and antibody via a linear linker or SN-38 and antibody via a branched linker And the number of cells 48 hours later was measured by the WST-8 method using Cell Counting Kit-8 (Dojindo). As controls, free SN-38 and the known anticancer drug irinotecan (CPT11) were used.

  The results are shown in FIGS. 10A and 10B. In FIG. 10, SUIT2 in the presence of a complex of SN-38 and an antibody via a linear linker (A) or a complex of SN-38 and an antibody via a branched linker (B). The cell growth rate (%) of the cells is shown. As shown in FIG. 10, the IC50 in terms of SN-38 concentration is 0.028 μM when a branched linker is used (B in FIG. 10), and 0.034 μM when a linear linker is used (FIG. 10). A). Therefore, the antitumor effect was confirmed even when any complex was used. In addition, when a branched linker was used, the dose of the antibody conjugate could be reduced to about 1/3.

  SN-38 has already been clinically applied as a prodrug called irinotecan CPT-11, but because it is a small molecule, its distribution is not distinguished between normal tissue and cancer tissue, and it does not stay long in cancer. Therefore, it has been pointed out clinically that the characteristic of the time-dependent antitumor effect of SN-38, which is the active substance, is not utilized and the side effects are strong. As shown in this Example, the selective cancer toxicity was enhanced by increasing the amount of SN-38 added to the antibody by about 3 times by using a branched linker.

[Example 8]
In Vivo Antitumor Effect In this example, the influence of the antibody-SN-38 complex prepared in Example 6 on a mouse chemical carcinogenesis model was examined.

  In the mouse chemical carcinogenesis model, approximately 200 ml of 1 mg / ml DMBA (7,12-dimethylbenzanthracene) is applied to the back of the mouse as an initiator and then PMA (phorbol myristic acid acetate) is applied once a week. About half a year later, we established a natural chemical carcinogenesis model mouse that exhibits a pathological condition close to that of humans. When the tumor diameter of a mouse chemical carcinogenesis model reaches 4 mm, CPT-11 is intravenously converted to SN-38 at 23.2 mg / kg, or anti-fibrin antibody-SN-38 complex is converted to SN-38 at 13.5 mg / kg. Administered.

  The results are shown in FIGS. As shown in FIG. 11, anti-fibrin antibody-SN-38 showed a stronger antitumor effect than CPT-11. Further, as shown in FIG. 12, in case 1, the tumor is remarkably reduced. In Case 2, the color of the tumor has changed from red (positive blood flow) to white (negative blood flow) due to the effect of the drug, indicating the strength of the anti-tumor effect.

  Therefore, it was confirmed that the anti-fibrin antibody of the present invention can deliver an antitumor compound to a tumor site and exert an antitumor effect.

  The present invention provides antibodies against fibrin and antigen-binding fragments thereof. By using the anti-fibrin antibody of the present invention, it is possible to detect the presence of fibrin and a thrombus with high sensitivity, reliability, and simpleness, and as a result, a thrombus-related disease can be determined. In addition, by using the anti-fibrin antibody of the present invention, it becomes possible to deliver an appropriate compound or molecule to a site where a thrombus exists, for example, a tumor. In particular, since the anti-fibrin antibody of the present invention binds to human and mouse fibrin and does not bind to human and mouse fibrinogen, it is considered useful in the medical diagnostic field and the pharmaceutical field.

  Accession number NITE P-923 (Hybridoma 102-10, deposited on April 1, 2010)

SEQ ID NOs: 1 to 6: Artificial sequence (antibody CDR sequence)
SEQ ID NOs: 7 to 10: Artificial sequences (antibody variable region sequences)
Sequence numbers 11-14: Artificial sequence (primer)

Claims (29)

(A) an H chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence comprising one or several conservative amino acid substitutions in the amino acid sequence,
(B) the H chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence containing one or several conservative amino acid substitutions in the amino acid sequence,
(C) the H chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence containing one or several conservative amino acid substitutions in the amino acid sequence,
(D) an L chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence containing one or several conservative amino acid substitutions in the amino acid sequence,
(E) the L chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence containing one or several conservative amino acid substitutions in the amino acid sequence, and (f) 1 or 2 in the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence L chain CDR3 consisting of amino acid sequence containing several conservative amino acid substitutions
An antibody or an antigen-binding fragment thereof, which comprises binding to human fibrin.   The antibody or antigen-binding fragment according to claim 1, which binds to human fibrin and mouse fibrin.   The antibody or antigen-binding fragment according to claim 1 or 2, which does not bind to human fibrinogen and mouse fibrinogen.   The antibody or antigen-binding fragment according to any one of claims 1 to 3, which is a monoclonal antibody.   The antibody or antigen-binding fragment according to any one of claims 1 to 4, which is an antibody produced by a hybridoma cell having the accession number NITE P-923.   The antibody or antigen-binding fragment according to any one of claims 1 to 4, which is an antibody that binds to an epitope to which an antibody produced by a hybridoma cell having the deposit number NITE P-923 binds.   The antibody or antigen-binding fragment according to any one of claims 1 to 4, which is a chimeric antibody or a humanized antibody.   The antibody or antigen-binding fragment thereof according to claim 7, which is a chimeric antibody comprising an H chain variable region comprising the amino acid sequence of SEQ ID NO: 8 and an L chain variable region comprising the amino acid sequence of SEQ ID NO: 10.   The antibody or antigen-binding fragment according to any one of claims 1 to 8, which is labeled.   A nucleic acid comprising a base sequence encoding the antibody or antigen-binding fragment according to any one of claims 1 to 8.   An expression vector comprising the nucleic acid according to claim 10.   A transformant comprising the nucleic acid according to claim 10 or the expression vector according to claim 11, and producing the antibody or antigen-binding fragment according to any one of claims 1 to 8.   The antibody or antigen-binding fragment according to any one of claims 1 to 8, comprising a step of culturing the transformant according to claim 12 in a medium and collecting the antibody or antigen-binding fragment from the culture. Production method.   A cell that produces the antibody or antigen-binding fragment according to claim 1.   The cell according to claim 14, which is a hybridoma cell having a deposit number of NITE P-923.   A reagent for immunological measurement of fibrin, comprising the antibody or antigen-binding fragment according to any one of claims 1 to 9.   A reagent for determining a thrombus-related disease, comprising the antibody or antigen-binding fragment according to any one of claims 1 to 9.   The reagent according to claim 17, wherein the thrombosis-related disease is infarction or cancer.   A thrombus visualization agent comprising the labeled antibody or antigen-binding fragment according to claim 9.   The thrombus visualization agent according to claim 19 for visualizing infarction or cancer. (A) contacting the antibody or antigen-binding fragment according to any one of claims 1 to 9 with a sample;
(B) A method for detecting fibrin in a sample, comprising detecting whether the antibody or antigen-binding fragment has bound to fibrin in the sample. (A) contacting the antibody or antigen-binding fragment according to any one of claims 1 to 9 with a sample derived from a subject;
(B) A method for determining a thrombosis-related disease in a subject comprising detecting whether the antibody or antigen-binding fragment has bound to fibrin in a sample.   The method according to claim 22, wherein the thrombosis-related disease is infarction or cancer.   24. The method of any one of claims 21 to 23, wherein the sample is selected from the group consisting of a cell and tissue sample, and a biological fluid sample. (A) preparing an antibody or antigen-binding fragment having an amino acid sequence modified from the amino acid sequence of the antibody or antigen-binding fragment according to any one of claims 1 to 8,
(B) A method for producing a modified anti-fibrin antibody or antigen-binding fragment, comprising the step of determining whether or not the obtained antibody or antigen-binding fragment binds to fibrin.   A complex of the antibody or antigen-binding fragment according to any one of claims 1 to 9 and an antitumor moiety.   27. The complex according to claim 26, wherein the antibody or antigen-binding fragment and the antitumor moiety are linked via a linker.   28. The complex according to claim 26 or 27, wherein the antitumor moiety is an anticancer agent.   A preventive or therapeutic agent for tumors comprising the complex according to any one of claims 26 to 28.

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