WO2014133093A1 - 不溶性フィブリンに対する抗体 - Google Patents
不溶性フィブリンに対する抗体 Download PDFInfo
- Publication number
- WO2014133093A1 WO2014133093A1 PCT/JP2014/054925 JP2014054925W WO2014133093A1 WO 2014133093 A1 WO2014133093 A1 WO 2014133093A1 JP 2014054925 W JP2014054925 W JP 2014054925W WO 2014133093 A1 WO2014133093 A1 WO 2014133093A1
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- WO
- WIPO (PCT)
- Prior art keywords
- antibody
- amino acid
- fibrinogen
- acid sequence
- fibrin
- Prior art date
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/22—Haematology
- G01N2800/226—Thrombotic disorders, i.e. thrombo-embolism irrespective of location/organ involved, e.g. renal vein thrombosis, venous thrombosis
Definitions
- the present invention relates to an antibody that binds to insoluble fibrin and does not bind to fibrinogen, and a reagent and method for detecting insoluble fibrin containing the antibody.
- the present invention also relates to a reagent and method for determining a thrombosis-related disease using the antibody.
- the present invention relates to a complex of the antibody and an antitumor moiety.
- Insoluble fibrin does not exist in tissues under normal physiological conditions, unlike the precursor fibrinogen widely recognized in the living body.
- the thrombin that has leaked out of the blood vessel and activated cleaves fibrinogen to form a fibrin monomer, and the fibrin monomer is polymerized and crosslinked to form fibrin fibers. Therefore, insoluble fibrin is specific to tissues in a pathological state such as bleeding and inflammation, and is formed when a pathological condition accompanied by coagulation such as cancer, myocardial infarction, and cerebral infarction occurs.
- insoluble fibrin is a marker molecule for such thrombosis-related diseases
- insoluble fibrin present in cancer tissues in the absence of cerebral circulatory diseases such as myocardial infarction and cerebral infarction can be said to be a cancer-specific molecule.
- cerebral circulatory diseases such as myocardial infarction and cerebral infarction
- Patent Documents 1 to 6 antibodies have been developed as means for detecting fibrin.
- the present inventors have also developed an antibody that binds to fibrin and does not bind to fibrinogen, and clarifies its usefulness (Patent Document 7).
- insoluble fibrin is formed by cleaving the end of fibrinogen, which is a precursor, the amino acid sequences of insoluble fibrin and fibrinogen are completely identical except for the presence or absence of a portion that is cleaved away. Yes. Furthermore, there may be soluble fibrin (FDP, fibrin degradation product) generated by the degradation of insoluble fibrin by plasmin or the like in the living body. Therefore, in the presence of molecules having very high homology in amino acid sequence and structure (fibrinogen, FDP, etc.), in order to detect insoluble fibrin, an antibody having higher affinity and specificity for insoluble fibrin is required. ing.
- FDP fibrin degradation product
- the present invention has been made in view of the above-described problems of the prior art, and an object thereof is to provide an antibody that does not bind to fibrinogen and has high affinity and specificity for insoluble fibrin.
- the present inventor has intensively studied the properties and functions of an antibody (102-10 antibody) that binds to fibrin and does not bind to fibrinogen described in Patent Document 7.
- the 102-10 antibody can detect thrombus generated in cancer, cerebral infarction, myocardial infarction, inflammatory disease and the like in vitro and in vivo.
- the 102-10 antibody does not bind to fibrinogen, but can bind to the B ⁇ chain among the three types of polypeptide chains (A ⁇ chain, B ⁇ chain, and ⁇ chain) constituting fibrinogen.
- the epitope of the 102-10 antibody is a site consisting of amino acids at positions 231 to 246 of the fibrinogen B ⁇ chain.
- Fibrinogen becomes a fibrin monomer when the amino terminus of the fibrinogen B ⁇ chain constituting the fibrinogen is cleaved to form a fibrinogen ⁇ chain, and further, the monomer is polymerized and crosslinked to form insoluble fibrin.
- the epitope is a site that is bound to a fibrinogen ⁇ chain in the fibrinogen B ⁇ chain.
- the present inventors thought that the 102-10 antibody could not bind to fibrinogen because the epitope of the antibody was hidden in the molecule.
- the 102-10 antibody can bind to insoluble fibrin when the fibrin monomer is polymerized and cross-linked to expose the epitope in the fibrinogen ⁇ chain or fibrinogen B ⁇ chain in the fibrin monomer.
- the amino acid sequences of the variable region and complementarity determining region were determined.
- the present invention has been made based on the above findings. That is, the present invention provides the following ⁇ 1> to ⁇ 11>.
- a light chain variable region comprising the amino acid sequence of SEQ ID NOs: 4 to 6 or an amino acid sequence in which at least one amino acid is substituted, deleted, added and / or inserted in at least one of the amino acid sequences
- the amino acid sequence shown in SEQ ID NOs: 8 to 10 or a heavy chain variable region containing an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequences The antibody according to ⁇ 1>.
- the light chain variable region comprising the amino acid sequence of SEQ ID NOs: 12 to 14 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequences
- the amino acid sequence shown in SEQ ID NOs: 16 to 18 or a heavy chain variable region containing an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequences The antibody according to ⁇ 1>.
- ⁇ 4> a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence;
- a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence, ⁇ 1> The antibody described.
- ⁇ 5> a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence;
- a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence, ⁇ 1>
- An immunological measurement reagent comprising the antibody according to any one of ⁇ 1> to ⁇ 5>.
- a reagent for determining a thrombus-related disease comprising the antibody according to any one of ⁇ 1> to ⁇ 5>.
- a thrombus visualization agent comprising the antibody according to any one of ⁇ 1> to ⁇ 5>.
- ⁇ 9> (a) a step of bringing the antibody according to any one of ⁇ 1> to ⁇ 5> into contact with a sample, and (b) a step of detecting whether or not the antibody has bound to insoluble fibrin in the sample.
- a method for detecting insoluble fibrin in a sample comprising: ⁇ 10> (a) a step of bringing the antibody according to any one of ⁇ 1> to ⁇ 5> into contact with a sample derived from a subject, (b) whether the antibody has bound to insoluble fibrin in the sample
- a method for determining a thrombus-related disease in a subject comprising the step of: ⁇ 11> A complex of the antibody according to any one of ⁇ 1> to ⁇ 5> and an antitumor moiety.
- an antibody that does not bind to fibrinogen and has high affinity and specificity for insoluble fibrin.
- the presence of insoluble fibrin and thrombus can be detected with high sensitivity, reliability, and ease, and as a result, a thrombus-related disease can be determined.
- FIG. 6 is a photomicrograph showing the result of immunostaining a tissue of colorectal cancer, colorectal adenoma or reactive lymphadenitis with the 102-10 antibody.
- FIG. 5 is a photomicrograph showing the result of immunostaining cerebral infarction, arthritis or damaged skin tissue with 102-10 antibody.
- the number of days in the figure indicates the number of days that have elapsed since onset or wounding.
- An arrow indicates an insoluble fibrin (thrombus) formation site.
- 1 shows a schematic diagram of a PET probe consisting of a 102-10 antibody, p-isothiocyanate benzyl-desferrioxamine B, and 89 Zr nuclides.
- FIG. 9 is a photograph showing a result of observing a cross section of a tumor isolated from a chemical carcinogenic mouse administered with the PET probe shown in FIG. 8.
- HE shows the result of observing the tumor cross section by HE staining
- Fibrin shows the result of observing the tumor cross section by immunostaining with the 102-10 antibody
- Autoradiogram The result of having detected the radiation emitted from a PET probe is shown. It is a microscope picture which shows the result of having immunostained the fibrin clot with the 102-10 antibody.
- the amino acid sequence marked in the figure is a region consisting of histidine at position 179 to lysine at position 264 of the fibrinogen B ⁇ chain (86 amino acids), which was suggested by amino acid sequence analysis that the epitope of antibody 102-10 exists. ).
- 10 is a graph showing the results of a competitive inhibition experiment in the binding to the 102-10 antibody using a synthetic peptide consisting of a partial sequence of the 86 amino acid region. It is a figure which shows the result of having analyzed the binding state of the fibrinogen B ⁇ chain and the fibrinogen ⁇ chain in the fibrinogen molecule by computer simulation.
- the arrow indicates the epitope of the 102-10 antibody (site consisting of amino acids at positions 231 to 246 of the fibrinogen B ⁇ chain).
- the affinity of 102-10 antibody, anti- ⁇ chain antibody (Fib-0355 antibody, Fib-3435 antibody) or anti- ⁇ chain antibody (13-30 antibody, 34-105 antibody) to fibrin or fibrinogen was analyzed by ELISA. It is a graph which shows the result.
- Anti- ⁇ chain antibody indicates the result of analyzing a monoclonal antibody obtained by immunizing a mouse with a polypeptide consisting of amino acids 231 to 246 of fibrinogen B ⁇ chain.
- FIG. 3 is a view showing a light chain (L chain) variable region and a heavy chain (H chain) variable region of an anti- ⁇ chain antibody (Fib-0355 antibody) and amino acid sequences of CDR1 to CDR3 thereof.
- FIG. 3 is a view showing a light chain (L chain) variable region and a heavy chain (H chain) variable region of an anti- ⁇ chain antibody (34-105 antibody), and amino acid sequences of these CDR1 to CDR3.
- ⁇ Antibodies to insoluble fibrin> As shown in the Examples below, the present inventors have made a site consisting of amino acids 231 to 246 of the fibrinogen B ⁇ chain or a site consisting of amino acids 187 to 202 of the fibrinogen ⁇ chain (both in SEQ ID NO: 1). Antibody that binds to insoluble fibrin but cannot bind to fibrinogen, although the epitope is the site shown in the figure) or the site consisting of amino acids 232 to 246 of the fibrinogen ⁇ chain (site shown in SEQ ID NO: 2). was revealed.
- the present invention provides an antibody that binds to insoluble fibrin and does not bind to fibrinogen, and binds to the site shown in SEQ ID NO: 1 or the site shown in SEQ ID NO: 2.
- insoluble fibrin means that fibrin monomer is produced from fibrinogen by the action of thrombin, and the fibrin monomer is polymerized to form a hardly soluble fibrin polymer, and the fibrin polymer is cross-linked by factor XIII. It means what has been converted.
- the “insoluble fibrin” in the present invention includes the fibrin monomer and the fibrin polymer constituting the insoluble fibrin, but includes insoluble fibrin degraded by plasmin and solubilized (FDP, fibrin degradation product). Absent.
- “fibrinogen” means a complex formed by crosslinking two fibrinogen A ⁇ chains, two fibrinogen B ⁇ chains, and two fibrinogen ⁇ chains.
- the “fibrinogen B ⁇ chain” is typically a protein having the amino acid sequence set forth in SEQ ID NO: 19 if derived from human, and the “fibrinogen ⁇ chain” is typically derived from human. Is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 20.
- “fibrinogen ⁇ chain” means a protein from which fibrinogen B ⁇ chain is cleaved by the action of thrombin and fibrinopeptide B, which is the N-terminal portion thereof, is removed. Typically, it is a protein consisting of the amino acid sequence at positions 45 to 491 set forth in SEQ ID NO: 19.
- the amino acid sequence described in SEQ ID NO: 1 is the amino acid sequence of positions 231 to 246 of the human fibrinogen B ⁇ chain or the amino acid sequence of positions 187 to 202 of the human fibrinogen ⁇ chain, and the amino acid sequence described in SEQ ID NO: 2 Is the amino acid sequence of positions 232 to 246 of the human fibrinogen ⁇ chain.
- Antibody in the present invention includes all classes and subclasses of immunoglobulins. “Antibody” includes polyclonal antibodies and monoclonal antibodies, and also includes forms of functional fragments of antibodies. “Polyclonal antibodies” are antibody preparations comprising different antibodies directed against different epitopes. The “monoclonal antibody” means an antibody (including an antibody fragment) obtained from a substantially homogeneous antibody population. In contrast to polyclonal antibodies, monoclonal antibodies are those that recognize a single determinant on an antigen. The antibody of the present invention is preferably a monoclonal antibody. The antibodies of the present invention are antibodies that have been separated and / or recovered (ie, isolated) from components of the natural environment.
- whether an antibody binds to insoluble fibrin or does not bind to fibrinogen can be determined by a method known in the art. Examples of such a known method are shown in Examples described later. An ELISA method using a plate on which insoluble fibrin or fibrinogen is immobilized is mentioned.
- an antibody having high specificity for insoluble fibrin means an antibody that binds to fibrin with higher affinity than its affinity for other peptides or proteins.
- the high affinity means that the affinity is high enough that insoluble fibrin can be detected separately from other peptides or proteins by a method known in the art.
- the antibody of the present invention can bind to insoluble fibrin derived from humans and mice and does not bind to fibrinogen derived from humans and mice, as shown in Examples described later. Therefore, test data obtained using this antibody in mice can be extrapolated to humans.
- preferred embodiments of the monoclonal antibody of the present invention include the monoclonal antibody (Fib-0355 antibody) produced by the hybridoma Fib-0355 and the monoclonal antibody (Fib-3435 antibody produced by the hybridoma Fib-3435, as described in Examples below. ), A monoclonal antibody (13-30 antibody) produced by hybridoma 13-30, and a monoclonal antibody (34-105 antibody) produced by hybridoma 34-105.
- the monoclonal antibody produced from the hybridoma has a light chain variable region comprising light chains CDR1 to CDR3 and a heavy chain variable region comprising heavy chains CDR1 to CDR3. Or an amino acid sequence variant thereof.
- a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence;
- a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence;
- an antibody having the heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15 or the amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence.
- another preferred embodiment of the antibody of the present invention is an ELISA described in Example 2 described later, wherein the affinity for insoluble fibrin (absorbance at a wavelength of 492 nm) is 0.5 or more (more preferably 1.0 or more, The antibody is preferably 1.5 or more, particularly preferably 2.0 or more.
- another preferred embodiment of the antibody of the present invention is such that the affinity for insoluble fibrin (absorbance at a wavelength of 492 nm) in the ELISA described in Example 2 described later is higher than that of the 102-10 antibody described in Patent Document 7.
- the antibody is 5 times or more (more preferably 10 times or more).
- Another preferable embodiment of the antibody of the present invention is that the ratio of insoluble fibrin to affinity for fibrinogen (absorbance at 492 nm) in the ELISA described in Example 2 described later is 10 times or more (more preferably 20). Antibody).
- the antibodies of the present invention include mouse antibodies, chimeric antibodies, humanized antibodies, human antibodies, and functional fragments of these antibodies.
- a human as a pharmaceutical
- a chimeric antibody, a humanized antibody, or a human antibody is desirable from the viewpoint of reducing side effects.
- a “chimeric antibody” is an antibody in which a variable region of a certain antibody is linked to a constant region of a heterogeneous antibody.
- a chimeric antibody for example, immunizes a mouse with an antigen, cuts out an antibody variable region (variable region) that binds to the antigen from the mouse monoclonal antibody gene, and binds to a human bone marrow-derived antibody constant region (constant region) gene. This can be obtained by incorporating it into an expression vector and introducing it into a host for production (for example, JP-A-8-280387, US Pat. No. 4,816,397, US Pat. No. 4,816,567, US Pat. 5807715).
- the “humanized antibody” is an antibody obtained by grafting (CDR grafting) the gene sequence of the antigen binding site (CDR) of a non-human-derived antibody to a human antibody gene, and its production method is publicly known. (See, for example, EP239400, EP125503, WO90 / 07861, WO96 / 02576).
- a “human antibody” is an antibody derived from all regions. In the production of human antibodies, a method for screening production of active antibodies from human B cells, a phage display method, and a transgenic animal (for example, a mouse) capable of producing a repertoire of human antibodies by immunization. It can be used.
- the “functional fragment” of an antibody means a part (partial fragment) of an antibody that binds to the site shown in SEQ ID NO: 1 or the site shown in SEQ ID NO: 2.
- Fab, Fab ′, F (ab ′) 2 variable region fragment (Fv), disulfide bond Fv, single chain Fv (scFv), sc (Fv) 2, diabody, multispecific antibody, And polymers thereof.
- Fab means a monovalent antigen-binding fragment of an immunoglobulin composed of one light chain and part of a heavy chain. It can be obtained by papain digestion of antibodies and by recombinant methods. “Fab ′” differs from Fab by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines in the hinge region of the antibody. “F (ab ') 2” means a divalent antigen-binding fragment of an immunoglobulin that consists of both light chains and parts of both heavy chains.
- “Variable region fragment (Fv)” is the smallest antibody fragment with complete antigen recognition and binding sites. Fv is a dimer in which a heavy chain variable region and a light chain variable region are strongly linked by a noncovalent bond. “Single-chain Fv (scFv)” comprises the heavy and light chain variable regions of an antibody, and these regions are present in a single polypeptide chain. “Sc (Fv) 2” is a chain formed by joining two heavy chain variable regions and two light chain variable regions with a linker or the like.
- a “diabody” is a small antibody fragment having two antigen-binding sites, the fragment comprising a heavy chain variable region bound to a light chain variable region in the same polypeptide chain, each region comprising a separate It forms a pair with the complementary region of the strand.
- a “multispecific antibody” is a monoclonal antibody that has binding specificities for at least two different antigens. For example, it can be prepared by co-expression of two immunoglobulin heavy / light chain pairs where the two heavy chains have different specificities.
- the antibodies of the present invention include antibodies whose amino acid sequence has been modified without reducing the desired activity (affinity for antigen, specificity for antigen, and / or other biological properties).
- Amino acid sequence variants of the antibodies of the invention can be made by introducing mutations into the DNA encoding the antibody chains of the invention or by peptide synthesis. Such modifications include, for example, residue substitutions, deletions, additions and / or insertions within the amino acid sequences of the antibodies of the invention.
- the site where the amino acid sequence of the antibody is altered may be the constant region of the heavy or light chain of the antibody, as long as it has an activity equivalent to that of the antibody before the alteration, and the variable region (framework region and CDR).
- Modification of amino acids other than CDR is considered to have a relatively small effect on the binding affinity with the antigen, but at present, the amino acid of the CDR is modified to screen for an antibody having an increased affinity for the antigen.
- Methods are known (PNAS, 102: 8466-8471 (2005), Protein Engineering, Design & Selection, 21: 485-493 (2008), International Publication No. 2002/051870, J. Biol. Chem., 280: 24880- 24887 (2005), Protein Engineering, Design & Selection, 21: 345-351 (2008)).
- the number of amino acids to be modified is preferably within 10 amino acids, more preferably within 5 amino acids, and most preferably within 3 amino acids (for example, within 2 amino acids, 1 amino acid).
- the amino acid modification is preferably a conservative substitution.
- conservative substitution means substitution with another amino acid residue having a chemically similar side chain. Groups of amino acid residues having chemically similar amino acid side chains are well known in the technical field to which the present invention belongs.
- acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine, arginine, histidine), neutral amino acids, amino acids having a hydrocarbon chain (glycine, alanine, valine, leucine, isoleucine, proline), hydroxy group Amino acids with amino acids (serine / threonine), amino acids with sulfur (cysteine / methionine), amino acids with amide groups (asparagine / glutamine), amino acids with imino groups (proline), amino acids with aromatic groups (phenylalanine / tyrosine / (Tryptophan).
- “having equivalent activity” means that the affinity for the antigen is the target antibody (typically, the Fib-0355 antibody, the Fib-3435 antibody, the 13-30 antibody, the 34-105 shown in the Examples described later).
- Antibody for example, 70% or more, preferably 80% or more, more preferably 90% or more.
- the affinity for an antigen can be evaluated by the ELISA method.
- deamidation is suppressed by substituting the amino acid adjacent to the amino acid deamidated or deamidated with another amino acid for the purpose of increasing the stability of the antibody. May be.
- glutamic acid can be substituted with other amino acids to increase antibody stability.
- the present invention also provides the antibody thus stabilized.
- the antibody of the present invention is a polyclonal antibody
- an immunized animal is immunized with an antigen (polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, its partial peptide, or a cell expressing these).
- the antiserum can be purified and obtained by conventional means (eg, salting out, centrifugation, dialysis, column chromatography, etc.).
- Monoclonal antibodies can be prepared by a hybridoma method or a recombinant DNA method.
- a typical example of the hybridoma method is the method of Kohler and Milstein (Kohler & Milstein, Nature, 256: 495 (1975)).
- the antibody-producing cells used in the cell fusion step in this method are spleen cells, lymph node cells, peripheral blood leukocytes, etc. of animals immunized with the antigen (eg, mouse, rat, hamster, rabbit, monkey, goat). is there. It is also possible to use antibody-producing cells obtained by allowing an antigen to act in the medium on the above-mentioned cells or lymphocytes previously isolated from non-immunized animals.
- the myeloma cells various known cell lines can be used.
- the antibody-producing cells and myeloma cells may be of different animal species as long as they can be fused, but are preferably of the same animal species.
- the hybridoma is produced, for example, by cell fusion between a spleen cell obtained from a mouse immunized with an antigen and a mouse myeloma cell, and is then specific to the site shown in SEQ ID NO: 1 or 2 by subsequent screening.
- Hybridomas that produce monoclonal antibodies can be obtained.
- a monoclonal antibody specific for the site shown in SEQ ID NO: 1 or 2 can be obtained by culturing the hybridoma or from the ascites of the mammal to which the hybridoma has been administered.
- the DNA encoding the antibody of the present invention is cloned from a hybridoma, a B cell or the like and incorporated into an appropriate vector, which is then introduced into a host cell (for example, a mammalian cell line, E. coli, yeast cell, insect cell, It is a technique for introducing an antibody of the present invention into a recombinant antibody by introducing it into a plant cell or the like (for example, PJ Delves, Antibody Production: Essential Technologies, 1997 WILEY, P. Shepherd and C. Dean Monoclonal. 2000 OXFORD UNIVERSITY PRESS, Vandame AM et al., Eur. J. Biochem.
- the DNA encoding the heavy chain or the light chain may be separately incorporated into an expression vector to transform the host cell.
- a host cell may be transformed by incorporating it into a single expression vector (see International Publication No. 94/11523).
- the antibody of the present invention can be obtained in a substantially pure and uniform form by culturing the above host cell, separating and purifying it from the host cell or culture medium. For the separation and purification of the antibody, the methods used in the usual purification of polypeptides can be used.
- transgenic animal bovine, goat, sheep, pig, etc.
- a transgenic animal production technology a large amount of monoclonal antibody derived from the antibody gene is produced from the milk of the transgenic animal. It is also possible to obtain.
- the present invention comprises a DNA encoding the antibody of the present invention, an expression vector comprising the DNA, a transformant comprising the DNA or the expression vector and producing the antibody of the present invention, culturing the transformant, Providing a method for producing the antibody of the present invention, comprising a step of separating and purifying the antibody from the transformant or culture medium, and a hybridoma that produces the antibody of the present invention or contains DNA encoding the antibody of the present invention. it can.
- the present invention also relates to a method for producing an antibody that binds to immunity, insoluble fibrin and does not bind to fibrinogen with a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2. Can also be provided. Furthermore, an antigen having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 for producing an antibody that binds to insoluble fibrin and does not bind to fibrinogen can be provided.
- insoluble fibrin can be detected by immunohistochemical staining and immunoelectron microscopy, as well as immunoassay (enzyme immunoassay (ELISA, EIA), fluorescent immunoassay, radioimmunoassay (RIA), immunochromatography, Western blotting, etc. ) Etc. can be implemented.
- the target sample is not particularly limited.
- 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.
- tissue sample biopsy specimen, excised specimen
- cytodiagnosis sample as a sample.
- insoluble fibrin is detected by binding insoluble fibrin in a sample to the antibody of the present invention and detecting the binding.
- detection includes not only detecting the presence or absence of insoluble fibrin but also quantitatively detecting insoluble fibrin and immunostaining insoluble fibrin.
- Immunoassays for insoluble fibrin typically involve contacting a sample to be tested with an antibody of the present invention and detecting the bound antibody using techniques known in the art.
- Contact means that the insoluble fibrin present in the sample and the antibody of the present invention can be brought into close proximity so that the antibody can be bound.
- the antibody-containing solution is applied to the solid sample. Such operations as immersing the solid sample in the antibody-containing solution and mixing the liquid sample and the 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 of the present invention can also be used histologically for in situ detection of insoluble fibrin, such as immunohistochemical staining (for example, immunostaining) or immunoelectron microscopy.
- 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.
- the immunoassay can be operated by a known method (Ausubel, FM et al., Short Protocols in Molecular Biology, Chapter 11 “immunology” John Wiley & Sons, Inc. 1995).
- the complex of the insoluble fibrin and the 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.
- chromatography, salting-out method, alcohol precipitation method, enzyme method, solid phase method, etc. chromatography, salting-out method, alcohol precipitation method, enzyme method, solid phase method, etc.
- an antibody may be immobilized on a solid support or carrier (resin plate, membrane, beads, etc.), or a sample may be immobilized.
- a solid support or carrier for example, the antibody is immobilized on a solid support, 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. Then, by detecting the bound antibody on the solid support by a conventional means, the binding between the insoluble fibrin in the sample and the antibody can be detected.
- the solid sample is treated with a solution containing the antibody followed by a buffer wash to remove unbound antibody, and then the bound antibody on the solid sample is detected by conventional means. Can do.
- Antibody binding activity can be measured according to a 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.
- the reaction in order to easily detect the reaction between the antibody of the present invention and insoluble fibrin present in a sample, the reaction is directly detected by labeling the antibody of the present invention, or a labeled secondary Indirect detection is performed by using an antibody or a biotin-avidin complex. Examples of labels that can be used in the present invention and detection methods thereof are described below.
- enzyme immunoassay for example, peroxidase, ⁇ -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.
- a cross-linking agent such as glutaraldehyde or maleimide compound.
- fluorescent 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.
- FITC fluorescein isothiocyanate
- TRITC tetramethylrhodamine isothiocyanate
- 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.
- the sample is brought into contact with the labeled antibody of the present invention to form an insoluble fibrin-antibody complex.
- the amount of insoluble 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.
- the antibody of the present invention and the sample are reacted (primary reaction), and the labeled complex is further reacted with the obtained complex (secondary 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.
- 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.
- quantification is performed, unbound labeled secondary antibody is separated, and the amount of insoluble fibrin in the sample can be measured from the amount of bound labeled secondary antibody or the amount of unbound labeled secondary antibody.
- the biotinylated antibody and the sample are reacted, and the resulting complex is reacted with avidin. Since avidin can specifically bind to biotin, the binding between the antibody and insoluble fibrin can be measured by detecting the signal of the label added to avidin.
- the label added to avidin is not particularly limited, for example, an enzyme label (peroxidase, alkaline phosphatase, etc.) is preferable.
- the detection of the label signal can also be performed according to a method known in the art.
- 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.
- TMB 5,5′-tetramethylbenzidine
- DAB diaminobenzidine
- alkaline phosphatase 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 immunological measurement of insoluble fibrin comprising the antibody of the present invention.
- the antibody may be labeled.
- the antibody may be in a free form, or may be immobilized on a solid support (for example, a membrane, a bead, etc.).
- the reagent for immunological measurement may contain components useful for carrying out the immunological measurement method in addition to the antibody of the present invention.
- Such components include, for example, buffers for use in immunoassays, sample processing reagents, labels, competitors, secondary antibodies, and the like.
- 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.
- insoluble fibrin By detecting the presence of insoluble fibrin, it is possible to determine the presence or absence of a thrombus-related disease, to determine the progress (deterioration or improvement) of the thrombus-related disease, and to specify the position of the thrombus-related disease.
- the determination reagent of the present invention contains the above-described antibody of the present invention. Therefore, the test reagent of the present invention is used to detect insoluble fibrin contained in a sample collected from a subject who is suspected of having or suspected to have a thrombosis-related disease (for example, infarction or cancer). The presence of a thrombus-related disease in the body, the progress of the thrombus-related disease in the subject, 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.
- the antibody of the present invention binds to insoluble fibrin in a subject when administered to the subject. Therefore, it is possible to visualize insoluble fibrin, ie, a thrombus in a subject, using the antibody of the present invention. In addition, by binding a compound or molecule to the antibody of the present invention, it is possible to deliver the compound or molecule to insoluble fibrin in a subject, ie, a thrombus site.
- the thrombus visualization agent of the present invention preferably contains a labeled antibody of the present invention.
- any label known in the field of in vivo imaging can be used.
- 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), etc .; radioisotopes, such as 89 Zr, 99 mTc, 123 I, 131 I, 97 Ru, 67 Cu, 11 C, 13 N, etc .; 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 and the label can be bound by a method known in the art.
- the antibody and the label can be directly bonded chemically or indirectly via an appropriate linker.
- linker include p-isothiocyanate benzyl desferrioxamine B, thiocarbamate, amide, carbamate, and maleimide.
- a compound or molecule such as a drug or prodrug is bound to the antibody of the present invention in place of the label, thereby delivering the compound or molecule to the site of insoluble fibrin present in the subject, that is, the thrombus site.
- drugs or prodrugs include known thrombolytic agents (for example, urokinase, streptokinase, tissue type plasminogen activator) and the like.
- thrombolytic agents for example, urokinase, streptokinase, tissue type plasminogen activator
- the present invention further provides a complex of the antibody of the present invention and an antitumor moiety. Since the antibody of the present invention binds to a thrombus site (fibrin) in a tumor as described above, the antitumor moiety can be delivered to the tumor by binding to the antitumor moiety.
- the antitumor moiety that can be bound to the antibody of the present invention is not particularly limited as long as it is an antitumor moiety known in the art.
- Antitumor moieties include alkylation of anticancer agents such as irinotecan (CPT-11), irinotecan metabolite SN-38 (10-hydroxy-7-ethylcamptothecin), adriamycin, taxol, 5-fluorouracil, nimustine, laministin and the like.
- anticancer agents such as irinotecan (CPT-11), irinotecan metabolite SN-38 (10-hydroxy-7-ethylcamptothecin), adriamycin, taxol, 5-fluorouracil, nimustine, laministin and the like.
- 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, MMAE (monomethyl auristatin E), DM-1 (mertansine), calikiama Thin like; radioisotopes, such as boron 10 (10 B), indium 111 (111 In) and yttrium 90 (90 Y), and the like.
- 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
- the binding between the antibody and the antitumor moiety can be performed by a method known in the art, and may be either direct binding or indirect binding.
- a covalent bond can be used as a direct bond.
- a bond via a linker can be used as an indirect bond.
- the antibody and the antitumor moiety are 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, G .; T.A. Bioconjugate Technologies, Academic Press, 1996; Harris, J. et al. M.M. And Zalipsky, S. Ed., Poly (ethylene glycol), Chemistry and Biological Applications, ACS Symposium Series, 1997; Veronese, F .; And Harris, J.M. M.M. Hen, Peptide and protein PEGylation. It is described in Advanced Drug Delivery Review 54 (4), 2002.
- the linker means a divalent or higher group connecting 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, 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 the antibody of the present invention at another end.
- Branched linkers usually have an antitumor moiety in each branch (each chain) and an antibody at another end.
- linear linkers include linkers of the following formula I:
- 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.
- the linker of formula I is usually linked to the antibody at the end with a succinimidyl group and to the antitumor moiety at the other end.
- linear linkers include linkers of formula II:
- PEG is a polyethylene glycol chain
- x is the number of ethylene glycol units, and represents an integer of 5 to 100.
- the linker of formula II is usually linked to the antibody at the end with the succinimidyl group and to the antitumor moiety at the other end.
- branched linkers include linkers of formula III:
- 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.
- the linker of formula III is usually linked to the antibody at the end with the succinimidyl group and to the antitumor moiety at the other ends. This branched linker can be prepared as described in Reference Example 1 of Patent Document 7, for example.
- the bond between the antibody 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.
- 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.
- 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, a succinimide group present at one end of the linker, and a lysine residue on the antibody.
- Dehydration substitution bond with an amino group contained in the group for example, WO 2008/096760
- dehydration condensation bond between an amino group present at one end of the linker and a carboxylic acid contained in aspartic acid or glutamic acid on the antibody for example, WSCDI Used. See, for example, WO / 2010/055950 for a specific method for binding an antibody to a linker.
- the bond between the antibody or the linker and the antitumor moiety is a covalent bond or a non-covalent bond (ionic bond, hydrophobic bond, etc.), preferably a covalent bond.
- 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.
- the bond between the antibody or linker and the antitumor moiety is not limited, but is preferably an ester bond, a carbamate bond, a carbonate bond, or a thiocarbamate bond, and more preferably an ester bond. is there.
- 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.
- 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.
- 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.
- 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.
- the number of antitumor compounds bound to one antibody molecule is not particularly limited in theory, but the stability of the complex and the ease of production From the viewpoint of properties and the like, the number is usually 1 to 10, preferably 1 to 8.
- SN-38 (10-hydroxy-7-ethylcamptothecin) is used as an antitumor moiety and a polyethylene glycol linker is used as a linker
- SN-38 is dehydrated and condensed with a polyethylene glycol having a carboxyl group at one end and an amino group protected by Boc, Fmoc or the like at the other end, and the hydroxyl group of SN-38 Introducing a polyethylene glycol linker.
- the conjugates of the present invention can bind to insoluble fibrin in tumor tissue and deliver the anti-tumor moiety to the tumor and remain in the tumor tissue for a long period of time and exert an anti-tumor effect for a long period of time. Has the effect of continuing. 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 is a solid cancer 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 is targeted to insoluble fibrin existing at the leaked place from the tumor blood vessel.
- the antibody of the present invention does not react with fibrinogen and has an extremely high affinity for insoluble fibrin. Therefore, highly biocompatible antibody-anti-tumor partial complexes are selectively leaked from tumor blood vessels by the EPR effect (enhanced permeation and retention effect) (they do not leak from normal blood vessels because they are macromolecules). It binds to insoluble 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.
- Example 6 described in Patent Document 7 when the antitumor moiety SN-38 is bound to the antibody of the present invention by an ester bond, the complex is produced by carboxylesterase in the tumor or naturally. SN-38 is released in a sustained manner. Since SN-38 is a small molecule, it has a very high possibility of moving around in cancer tissue relatively freely, going to the whole cancer, and attacking cancer cells efficiently. In addition, since SN-38 has a time-dependent antitumor effect, such long-term 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.
- a pharmaceutically acceptable carrier or additive 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 Can be performed.
- 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.
- intravenous injection including infusion
- intramuscular injection intraperitoneal injection and injection for subcutaneous injection
- ointment Preparations such as topical agents (especially eye ointments), creams, suppositories, eye drops, eye drops, nasal drops, inhalants, liniments, aerosols, etc.
- topical agents especially eye ointments
- creams especially eye ointments
- suppositories especially eye ointments
- eye drops especially eye drops
- eye drops nasal drops
- inhalants liniments
- aerosols etc.
- they are provided in unit dose ampoules or in multi-dose containers.
- compositions are excipients, extenders, binders, wetting agents, disintegrating agents, lubricants, surfactants, dispersants, buffering agents, pH adjusting agents, 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 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. 1 to 99% by weight, preferably 5 to 90%.
- the dose and administration interval of the drug of the present invention depend on the type of antibody contained in the drug, the type of anti-tumor part contained in the complex, the subject of administration, the age and weight of the subject, the administration route, and the number of administrations Unlike, it can vary widely.
- 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.
- a thrombus visualization agent after administration of a drug, the presence or position of an antibody in a subject is visualized using a label as an index.
- the presence or position of the antibody is visualized by a known imaging technique.
- the imaging method varies 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 A vivo imaging system can be used.
- CT computed tomography
- PET positron tomography
- MRI nuclear magnetic resonance imaging
- ⁇ ELISA> A plate in which fibrinogen is immobilized on a well (fibrinogen plate) is a Maxi soap plate (manufactured by nunc) and human or mouse-derived fibrinogen (manufactured by Sigma, diluted with PBS) at 1 ⁇ g / 100 ⁇ L / well. And the plate was sealed and allowed to stand at 4 ° C. overnight.
- the fibrin plate was prepared by adding 100 ⁇ L of TBS containing 0.05 NIHU / mL thrombin (Sigma), 1 mM CaCl 2 and 7 mM L-cysteine (Merck) to the wells of the fibrinogen plate and washing with TBS-T. This was prepared by blocking with N102 (manufactured by NOF Corporation).
- peroxidase was labeled on the 102-10 antibody using a peroxidase labeling kit NH 2 (manufactured by DOJINDO). Further, this labeled 102-10 antibody was prepared to 1 ⁇ g / mL with Block Ace (manufactured by DS PharmaBio Medical).
- a fibrin clot was created by incubating TBS containing 10 mg fibrinogen, 0.02 M CaCl 2 , 2.5 NIHU / mL thrombin and 7 mM L-cysteine in a 1.5 mL tube at 37 ° C. for 1 hour.
- the 102-10 antibody showed high affinity for insoluble fibrin, but no affinity for fibrinogen was observed in both human and mouse.
- the 102-10 antibody binds to insoluble fibrin, but does not bind to soluble fibrin (FDP: fibrin degradation product) obtained by treating insoluble fibrin with various enzymes. It became clear.
- NYB-T2G1 had low affinity for insoluble fibrin and D-dimer because its epitope was covered by fibrin polymerization.
- MH-1 showed high affinity for soluble fibrin (D-dimer), but had low affinity for deposited fibrin (insoluble fibrin) like NYB-T2G1.
- the antibody 102-10 showed no affinity for fibrinogen and D-dimer, and showed high affinity only for deposited insoluble fibrin.
- fibrin formation was not observed in the normal tissue by the 102-10 antibody.
- fibrin was detected by the 102-10 antibody in colon cancer and colon adenocarcinoma as well as the brain tumor, lung cancer and pancreatic cancer.
- no fibrin formation was observed in reactive lymphadenitis (lymphoma).
- the 102-10 antibody can detect cancer-specific fibrin formation (fibrin formed continuously under no symptoms).
- fibrin was detected by the antibody 102-10 at the onset of cerebral infarction and myocardial infarction.
- fibrin formation was not observed 3 weeks after the onset of both cerebral infarction and myocardial infarction, and the disappearance of fibrin was confirmed.
- fibrin was detected by antibody 102-10 in acute pancreatitis, but such fibrin formation was not observed in chronic pancreatitis.
- the wound healing model was prepared by anesthetizing female FVB / N mice with isoflurane and attaching a 1 cm wound to the back of the mice. And the state of healing was observed every day without treating the wound.
- the model animals (cerebral infarction model, inflammation model and wound healing model) in which the above three types of thrombus formation occurred were also analyzed by tissue immunostaining using the 102-10 antibody. The obtained results are shown in FIG.
- PET probe 102-10 antibody and p-isothiocyanate benzyl-desferrioxamine B (p-isothiocyanotobenzyl-desferrioxamine B, DF, manufactured by Macrocyclics) at a ratio of DF to antibody (1: 1 to 1: 3) And purified with Sephadex G-50 (GE). Also, 89 Zr-oxalate was prepared with a cyclotron.
- DF-conjugated 102-10 antibody 100 ⁇ g / 20 ⁇ L PBS
- 5.0-5.6 M Bq 89 Zr-oxalate (3.7-5.6 GBq / mL, pH 7-9) for 1 hour
- the PET probe thus obtained (see FIG. 8) achieved a radiochemical yield of 73-96%, a purity of 96-98%, and specific activity by thin layer chromatography using 50 mM DTPA was not observed. 37-44 kBq / ⁇ g.
- the tumor was taken out and embedded at ⁇ 20 ° C. using a frozen tissue embedding agent (optimal-cutting-temperature (OCT) compound, manufactured by Sakura Finetech).
- OCT optical-cutting-temperature
- a 20- ⁇ m-thick dry section was exposed with an imaging plate (Fuji Film) and stained with hematoxylin and eosin (Muto Chemical) (H & E staining).
- the removed tumor was also analyzed by immunostaining with 102-10 antibody and autoradiography using the antibody as a probe. The obtained result is shown in FIG.
- fibrin was detected by the 102-10 antibody at the time of onset or wounding in cerebral infarction, arthritis and skin injury. In the disease model and the wound model, it was confirmed that fibrin disappeared after 2 to 3 weeks from the onset.
- the 102-10 antibody can specifically detect fibrin not only in vitro but also in vivo.
- fibrin through the detection of fibrin, it became clear that the antibody 102-10 can also be used to detect cancer in vivo.
- benign diseases such as cerebral infarction, myocardial infarction and inflammatory disease, it has also been clarified that the state of the disease (course information) can be obtained through detection of such fibrin.
- a fibrin clot prepared by the same method as described in Reference Example 1 was frozen using OCT compound to prepare a 6 ⁇ m-thick frozen section. These frozen sections were air-dried, washed with PBS, and blocked with 5% skim milk / PBS. Subsequently, the antibody 102-10 labeled with Alexa Follow 647 protein labeling kit (manufactured by Invitrogen) was added, incubated for 1 hour, and then encapsulated with Fluoromount G (manufactured by Southern Biotec). And the sample prepared in this way was observed with the fluorescence microscope. The obtained results are shown in FIG.
- the molecular weight of the three chains constituting fibrinogen is about 67 KDa for A ⁇ chain, about 56 KDa for B ⁇ chain, and about 48 KDa for ⁇ chain.
- the fibrin clot was stained in a mesh form with the 102-10 antibody, reflecting the state in which the fibrin molecules were cross-linked in the fibrin clot.
- fibrinogen in a non-reduced state could not be detected by the 102-10 antibody.
- the B ⁇ chain (molecular weight: about 56 kDa) of fibrinogen in which the complex formation was eliminated in the reduced state by the antibody 102-10 can be detected. Therefore, it was revealed that the epitope of the 102-10 antibody is located at a site in the fibrinogen B ⁇ chain that is exposed by being denatured (reduced).
- ⁇ Amino acid sequence> The fibrinogen reduced and heat-treated by the method described in Reference Example 4 was developed on SDS-PAGE. Subsequently, fibrinogen B ⁇ chain was extracted from the gel using Easy Stain Reverse and Atprep MF (both manufactured by Atto). The extracted fibrinogen B ⁇ chain was cleaved with lysyl endopeptidase. The peptide fragment having the minimum molecular weight that binds to the 102-10 antibody was extracted from the cleaved fibrinogen B ⁇ chain peptide by Western blotting. Subsequently, the peptide fragment was subjected to amino acid sequencing.
- the epitope of the 102-10 antibody exists in the region (86 amino acids) consisting of histidine at position 179 to lysine at position 264 of the fibrinogen B ⁇ chain (SEQ ID NO: 19) (Fig. 14).
- an experiment was conducted by the following method.
- ⁇ Competition inhibition experiment> The 86 amino acid region was further divided into 5 to produce a synthetic peptide. Competition inhibition experiments were performed using these peptides. That is, the synthetic peptide serially diluted at 0.01 to 100 ⁇ M was added to 1 ⁇ g / mL 102-10 antibody and incubated at room temperature for 30 minutes. 100 ⁇ L of the mixed solution of antibody and peptide was added to the fibrin plate and incubated at room temperature for 30 minutes. Thereafter, the plate was washed with TBS-T, 100 ⁇ L of 1 step slow TMB (Thermo) was added, and colorimetry was performed for 5 minutes. The stop reaction was performed by adding 100 ⁇ L of 2N H 2 SO 4 .
- the arrangement of 5 is as follows. Fib-1: NIPVVSGKEECEEIIRKGETS (positions 179 to 252 of the fibrinogen B ⁇ chain, SEQ ID NO: 21) Fib-3: CNIPVVSGKE (positions 231 to 240 of the fibrinogen B ⁇ chain, SEQ ID NO: 22) No.
- HQLYIDETVNSNIPTNLRVLRSILENLRSK positions 179-208 of the fibrinogen B ⁇ chain, SEQ ID NO: 23
- No. 5 CNIPVVSGCKEEEIIR (positions 231 to 246 of the fibrinogen B ⁇ chain, SEQ ID NO: 1)
- KLH carrier protein conjugates (Fib-1 KLH and Fib-3 KLH) were also prepared and used for competition inhibition experiments.
- the antibody 102-10 is a site consisting of amino acids 231 to 246 (No. 5) only, and in addition, No. 5 5 did not bind to the site (Fib-3 KLH) consisting of amino acids at positions 231 to 240 included in 5. Therefore, the epitope of the 102-10 antibody was revealed to be a site consisting of amino acids at positions 231 to 246 of the fibrinogen B ⁇ chain (amino acid described in SEQ ID NO: 1).
- the fibrinogen B ⁇ chain is cleaved by thrombin between the 44th arginine and the 45th glycine to remove fibrinopeptide B (polypeptide consisting of amino acids 1 to 44 of the fibrinogen B ⁇ chain), etc.
- fibrinogen becomes a fibrin monomer, and the monomer is polymerized and crosslinked to form insoluble fibrin. Therefore, the epitope of the antibody 102-10 is a site consisting of amino acids at positions 187 to 202 of the fibrinogen ⁇ chain (protein obtained by removing fibrinopeptide B from the fibrinogen B ⁇ chain) in insoluble fibrin.
- fibrinogen A ⁇ chain is cleaved while fibrinogen B ⁇ chain is retained and fibrinopeptide A is removed, a fibrin monomer that can be polymerized from fibrinogen is produced. Therefore, even in insoluble fibrin, the site consisting of amino acids at positions 231 to 246 of the fibrinogen B ⁇ chain can be an epitope of the 102-10 antibody.
- the epitope of the identified 102-10 antibody was analyzed by computer simulation. As a result, it was revealed that the epitope is a region bound to the ⁇ chain in the fibrinogen molecule (see FIG. 16, the arrow in the figure indicates the epitope of the 102-10 antibody).
- the site of the ⁇ chain bound to the B ⁇ chain is a region (KNWIQYKEGFGHLSP, SEQ ID NO: 2) consisting of lysine at position 232 to proline at position 246 of the fibrinogen ⁇ chain (SEQ ID NO: 20). Also became clear.
- Example 1 From the results of computer simulation shown in Reference Example 5 for producing the antibody of the present invention , the reason why the antibody 102-10 cannot bind to fibrinogen is that the epitope of the antibody is a region hidden in the molecule. is assumed. In addition, fibrinogen is cleaved by thrombin to produce fibrin monomers, which are further polymerized and cross-linked to change to insoluble fibrin. This structural change is expected to allow the 102-10 antibody to bind to insoluble fibrin by exposing the regions hidden within the fibrinogen molecule.
- the site consisting of amino acids 231 to 246 of the fibrinogen B ⁇ chain and the site consisting of amino acids 232 to 246 of the fibrinogen ⁇ chain are used to produce an antibody that binds to insoluble fibrin and does not bind to fibrinogen. Therefore, the production of antibodies using each of these sites as antigens was attempted by the following method.
- an antigen gene encoding a site consisting of amino acids 231 to 246 of the fibrinogen ⁇ chain or a site consisting of amino acids 232 to 246 of the fibrinogen ⁇ chain is inserted into the restriction enzyme site (NdeI-HindIII) of pET21b.
- NdeI-HindIII restriction enzyme site
- a plasmid DNA capable of expressing an antigen peptide fused with a histidine tag was prepared.
- These plasmid DNAs were then introduced into E. coli BL21 (DE3) (Novagen).
- the E. coli was inoculated into 200 mL of LB medium and cultured at 37 ° C. and 100 rpm.
- the culture was temporarily stopped and left on ice for 15 minutes. .
- IPTG isopropyl-1-thio- ⁇ -D-galactopyranoside
- the cells were cultured for 16 hours after addition of IPTG (antigen gene expression induction), centrifuged at 48,820 g for 15 minutes, and collected.
- the cells were suspended in 50 mM Tris-HCl (pH 8.5) containing 500 mM NaCl, and sonicated on ice. After crushing, the supernatant was collected by centrifugation at 48,820 g for 60 minutes. The collected supernatant was then passed through 1 mL Ni-NTA agarose (manufactured by Invitrogen) equilibrated with 50 mM Tris-HCl (pH 8.5) containing 500 mM NaCl, and the agarose was passed through 50 mM Tris- containing 5 mM imidazole and 500 mM NaCl. Washed with HCl (pH 8.5).
- the antigen peptide captured by Ni-NTA agarose was eluted with PBS (-) containing 100 mM imidazole.
- the obtained antigenic peptide was buffer-substituted with PBS (-) by ultrafiltration using Amicon Ultra-15 10K (Millipore).
- the purity of the antigenic peptide was confirmed by SDS-PAGE using SDS-page 10-20% (DRC).
- anti- ⁇ chain antibody The production of an antibody that binds to the site consisting of amino acids 232 to 246 of the fibrinogen ⁇ chain (hereinafter also referred to as “anti- ⁇ chain antibody”) was outsourced to ITM Corporation. Specifically, the antigen peptide derived from the ⁇ chain prepared above was injected into the ridge muscle of a mouse, and a monoclonal antibody was prepared using the mouse iliac lymph node (mouse iliac lymph node method, Sado Y. et al. Et al., Acta Histochem. Cytochem., 2006, 39, 89-94). An antibody that binds to a site consisting of amino acids at positions 231 to 246 of the fibrinogen B ⁇ chain (hereinafter also referred to as “anti- ⁇ chain antibody”) was prepared by the following method.
- the histidine tag of the ⁇ -chain-derived antigen peptide prepared above was replaced with a 4M-tag according to a conventional method. Then, an antigen peptide (immunogen) fused with 4M-tag was administered to 6 mice.
- an emulsion in which FCA and an immunogen were mixed was prepared so that the immunogen was 50 ug / mouse and administered to the abdominal cavity.
- Booster immunization was performed every 2 to 3 weeks, and 50 ug of immunogen was mixed with 200 ul of Sigma adjuvant system and administered to the abdominal cavity of mice.
- the hybridoma culture supernatant obtained by culturing was evaluated by an ELISA method using a plate on which an immunogen was immobilized, and subjected to primary screening.
- the hybridoma determined to be positive in the primary screening was evaluated by an ELISA method using a plate on which an immunogen, fibrinogen or fibrin was immobilized, and a secondary screening was performed.
- the hybridoma determined to be positive in the secondary screening was selected as a hybridoma that produces an antibody that binds to fibrin and does not bind to fibrinogen, and was further monocloned by limiting dilution.
- Example 2 The anti- ⁇ chain antibody and anti- ⁇ chain antibody prepared in Example 1 were compared with the 102-10 antibody by the ELISA method shown below.
- the hybridomas established in Example 1 Fib-0355 and Fib-03435 were selected, and the antibodies produced by these hybridomas were subjected to the ELISA method shown below as anti- ⁇ chain antibodies.
- 13-30 and 34-105 were selected from the hybridomas established in Example 1, and the antibodies produced by these hybridomas were subjected to the ELISA method shown below as anti- ⁇ chain antibodies.
- ⁇ ELISA method 100 ⁇ L of each antibody solution was added at 10 ⁇ g / mL to the fibrin plate and the fibrinogen plate, and allowed to stand at room temperature for 1 hour. After washing with TBS-T, ethyl anti-Human IgG-HRP (x1000 dilution) or ethyl anti-mouse IgG-HRP (x10000 dilution) was added as a secondary antibody. After washing with TBS-T, the color was compared with OPD for 10 minutes, and an absorption wavelength of 492 nm was measured. The obtained results are shown in FIG.
- the anti- ⁇ chain antibody and the anti- ⁇ chain antibody prepared this time did not bind to fibrinogen, and showed high affinity for insoluble fibrin.
- the affinity (absorbance at 492 nm) of these antibodies for insoluble fibrin was found to be much higher than that of the 102-10 antibody. That is, the affinity of the Fib-0355, Fib-3435, 13-30, and 34-105 antibodies to insoluble fibrin was 12 times, 9 times, 6 times, and 16 times that of the 102-10 antibody, respectively. It was.
- the ratio of that of insoluble fibrin to its affinity for fibrinogen is 13 times 18 for the Fib-0355, Fib-3435, 13-30, 34-105 and 102-10 antibodies.
- the anti- ⁇ chain antibody and the anti- ⁇ chain antibody prepared this time showed higher specificity for insoluble fibrin than the 102-10 antibody.
- variable region and complementarity determining region (CDR) sequences were determined by the following method. Were determined.
- RNA iso Plus manufactured by TAKARA
- RNeasy mini kit manufactured by QIAGEN
- cDNA was synthesized by a reverse transcription reaction using a high capacity cDNA reverse transcription kit (Applied Biosystems) and an attached random primer.
- PCR was performed using the synthesized cDNA as a template and mixed primers, and the antibody variable region was cloned.
- PCR product was developed by agarose gel electrophoresis, and bands presumed to be the target VH and VL regions from the size were cut out.
- cDNA was extracted from the gel using a QIA quick gel extraction kit (manufactured by Qiagen). The extracted cDNA was inserted into the pGEM-T Easy vector system (Promega) by TA cloning to obtain a plurality of clones containing the target sequence. E. coli JM109 or DH5 ⁇ is used as the host E.
- plasmid DNA was extracted from the obtained white colony using a QIA prep spin miniprep kit (manufactured by QIAGEN).
- the obtained candidate sequences were subjected to NCBI IgBLAST (http://www.ncbi.nlm.nih.gov/igblast/) to select antibody sequences and myeloma-derived sequences. Furthermore, the sequence information of the obtained variable region was compared with the sequence information recorded in the database on the web (IMGT: http: //www.imgt.org), and the CDR region was determined. The obtained results are shown in FIGS. In addition, the amino acid sequences of the variable regions and CDRs of the 34-105 antibody and the Fib-0355 antibody thus identified are also shown in the sequence listing with the following SEQ ID NOs.
- Amino acid sequence of the L chain (light chain) variable region of the 34-105 antibody SEQ ID NO: 3 Amino acid sequence of the light chain CDRs 1-3 of the 34-105 antibody: SEQ ID NOs: 4-6 Amino acid sequence of the heavy chain variable region of the 34-105 antibody: SEQ ID NO: 7 Amino acid sequence of the heavy chain CDRs 1-3 of the 34-105 antibody: SEQ ID NOs: 8-10.
- Amino acid sequence of L chain (light chain) variable region of Fib-0355 antibody SEQ ID NO: 11 Amino acid sequence of the light chain CDRs 1-3 of the Fib-0355 antibody: SEQ ID NOs: 12-14 Amino acid sequence of the heavy chain variable region of Fib-0355 antibody: SEQ ID NO: 15 The amino acid sequence of the heavy chain CDRs 1-3 of the Fib-0355 antibody: SEQ ID NOs: 16-18.
- the present invention it is possible to provide an antibody that does not bind to fibrinogen and has high affinity and specificity for insoluble fibrin.
- an antibody that does not bind to fibrinogen and has high affinity and specificity for insoluble fibrin.
- the presence of insoluble fibrin and thrombus can be detected with high sensitivity, reliability, and ease, and as a result, a thrombus-related disease can be determined.
- the present invention it becomes possible to deliver an appropriate compound or molecule to a site where a thrombus exists, for example, a tumor. Therefore, the present invention is useful in the medical diagnosis field and the pharmaceutical field.
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Abstract
Description
<1> 不溶性フィブリンと結合し且つフィブリノーゲンと結合しない抗体であって、配列番号:1に示される部位又は配列番号:2に示される部位に結合する抗体。
<2> 配列番号:4~6に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む軽鎖可変領域と、
配列番号:8~10に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む重鎖可変領域と
を保持する、<1>に記載の抗体。
<3> 配列番号:12~14に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む軽鎖可変領域と、
配列番号:16~18に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む重鎖可変領域と
を保持する、<1>に記載の抗体。
<4> 配列番号:3に記載のアミノ酸配列又は該アミノ酸配列において1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む軽鎖可変領域と、
配列番号:7に記載のアミノ酸配列又は該アミノ酸配列において1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む重鎖可変領域と
を保持する、<1>に記載の抗体。
<5> 配列番号:11に記載のアミノ酸配列又は該アミノ酸配列において1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む軽鎖可変領域と、
配列番号:15に記載のアミノ酸配列又は該アミノ酸配列において1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む重鎖可変領域と
を保持する、<1>に記載の抗体。
<6> <1>~<5>のいずれか1に記載の抗体を含む、免疫学的測定用試薬。
<7> <1>~<5>のいずれか1に記載の抗体を含む、血栓関連疾患の判定用試薬。
<8> <1>~<5>のいずれか1に記載の抗体を含む、血栓可視化剤。
<9> (a)<1>~<5>のいずれか1に記載の抗体とサンプルとを接触させるステップ、(b)該抗体がサンプル中の不溶性フィブリンと結合したか否かを検出するステップを含む、サンプル中の不溶性フィブリンを検出するための方法。
<10> (a)<1>~<5>のいずれか1に記載の抗体と被験体に由来するサンプルとを接触させるステップ、(b)該抗体がサンプル中の不溶性フィブリンと結合したか否かを検出するステップを含む、被験体における血栓関連疾患を判定するための方法。
<11> <1>~<5>のいずれか1に記載の抗体と抗腫瘍性部分との複合体。
後述の実施例において示す通り、本発明者らは、フィブリノーゲンBβ鎖の第231~246位のアミノ酸からなる部位若しくはフィブリノーゲンβ鎖の第187~202位のアミノ酸からなる部位(共に配列番号:1に示される部位)、又はフィブリノーゲンγ鎖の第232~246位のアミノ酸からなる部位(配列番号:2に示される部位)をエピトープとする抗体は、不溶性フィブリンに結合できるが、フィブリノーゲンには結合できないことを明らかにした。
軽鎖CDR1~CDR3のアミノ酸配列(配列番号:4~6に記載のアミノ酸配列)又は該アミノ酸配列の少なくともいずれかにおいて1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む軽鎖可変領域と、
重鎖CDR1~CDR3のアミノ酸配列(配列番号:8~10に記載のアミノ酸配列)又は該アミノ酸配列の少なくともいずれかにおいて1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む重鎖可変領域と
を保持する抗体、
軽鎖CDR1~CDR3のアミノ酸配列(配列番号:12~14に記載のアミノ酸配列)又は該アミノ酸配列の少なくともいずれかにおいて1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む軽鎖可変領域と、
重鎖CDR1~CDR3のアミノ酸配列(配列番号:16~18に記載のアミノ酸配列)又は該アミノ酸配列の少なくともいずれかにおいて1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む重鎖可変領域と
を保持する抗体
が挙げられる。
配列番号:3に記載のアミノ酸配列又は該アミノ酸配列において1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む軽鎖可変領域と、
配列番号:7に記載のアミノ酸配列又は該アミノ酸配列において1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む重鎖可変領域と
を保持する抗体、
配列番号:11に記載のアミノ酸配列又は該アミノ酸配列において1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む軽鎖可変領域と、
配列番号:15に記載のアミノ酸配列又は該アミノ酸配列において1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む重鎖可変領域とを保持する抗体
が挙げられる。
後述の実施例に示す通り、本発明の抗体を用いて、サンプル中の不溶性フィブリンを検出することが可能である。この検出は、抗体を用いる測定方法、すなわち免疫学的測定方法であれば、任意の方法に基づいて実施することができる。例えば、不溶性フィブリンの検出は、免疫組織化学染色法及び免疫電顕法、並びに免疫アッセイ(酵素免疫アッセイ(ELISA、EIA)、蛍光免疫アッセイ、放射性免疫アッセイ(RIA)、免疫クロマト法及びウエスタンブロット法等)等を利用して実施することができる。
本発明の抗体は、後述の実施例に示す通り、ヒト不溶性フィブリンと特異的に反応するため、フィブリンに関連する疾患又は障害、例えば血栓関連疾患の判定用試薬において用いることができる。本発明において「血栓関連疾患」とは、その疾患又は障害の状態と血栓の存在との間に相関性がある疾患又は障害を意味する。そのような血栓関連疾患としては、限定されるものではないが、梗塞、例えば心筋梗塞、脳梗塞、脳出血、脳塞栓、脳血栓、くも膜下出血、肺梗塞等、並びに癌、例えば膵癌、胃癌、食道癌、結腸直腸癌、大腸癌、卵巣癌、乳癌及び肺癌が含まれる。不溶性フィブリンの存在を検出することによって、血栓関連疾患の有無の判定、血栓関連疾患の進行状況(悪化、良化)の判定、血栓関連疾患の位置の特定を行うことができる。
本発明の抗体は、被験体に投与した場合に、被験体内の不溶性フィブリンと結合する。従って、本発明の抗体を利用して、被験体における不溶性フィブリン、すなわち血栓を可視化することが可能である。また、本発明の抗体に化合物又は分子を結合させることにより、被験体における不溶性フィブリン、すなわち血栓部位に該化合物又は分子を送達することが可能である。
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と連結し、他の末端において抗腫瘍性部分と連結する。
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と連結し、他の末端において抗腫瘍性部分と連結する。
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、例えば特許文献7の参考例1に記載のように調製することができる。
(I)先ず、一方の端にカルボキシル基を有し、他の一方の端にBoc、Fmoc等で保護されたアミノ基を有するポリエチレングリコールとSN-38とを脱水縮合させ、SN-38の水酸基にポリエチレングリコールリンカーを導入する。
(II)一方の端にスクシンイミド基を有し、他の一方の端にマレイミド基を有するポリエチレングリコールと(I)の生成物とを混合し、スクシンイミド基と(I)の生成物のアミノ基とを反応させることにより、ポリエチレングリコールリンカーへマレイミド基を導入する。
(III)(II)の生成物と抗体とを混合し、(II)の生成物中のマレイミド基と抗体中のチオール基とを反応させ、(II)の生成物と抗体とを結合させることにより、本発明の複合体を得る。
以下に示す方法にて、102-10抗体のフィブリン特異性について解析した。
フィブリノーゲンをウェル上に固相化したプレート(フィブリノーゲンプレート)は、Maxi soapプレート(nunc社製)に、ヒト又はマウス由来のフィブリノーゲン(Sigma社製、PBSにて希釈したもの)を1μg/100μL/ウェルになるよう添加し、該プレートにシールをして、4℃にて一晩静置することにより作製した。
フィブリノーゲン10mg、0.02M CaCl2、2.5NIHU/mL トロンビン及び7mM L-システインを含有するTBSを、1.5mLチューブ中にて、37℃、1時間インキュベートすることにより、フィブリンクロットを作製した。
2μg/mL エラスターゼ(Elastase、Sigma社製)、
70μg/mL カリクレイン(Kallikrein、Sigma社製)、
10μg/mL カテプシンB(Cathepsin B、Sigma社製)、
210Units/mL カテプシンD(Cathepsin D、Sigma社製)、
200ng/mL MMP-9(Sigma社製)又は
0.1μM プラスミン(Plasmin、American diagnostica社製)を添加することにより、該フィブリンクロットを分解した。そして、各分解物をELISAプレートに固相化して、前記標識102-10抗体の反応性を解析した。得られた結果を図2に示す。
下記市販の抗フィブリン抗体2種をペルオキシダーゼにて標識した。
NYB-T2G1(Accurate Chemical and Scientific社製)、
MH-1(American Tissue and Cell Culture社製)
そして、これら標識抗体及び前記標識102-10抗体について、フィブリン、フィブリノーゲン、D-ダイマー(積水メディカル社製)を各1μg固相化したELISAプレートを用い、各抗体の親和性を解析した。得られた結果を図3に示す。
以下に示す方法にて、102-10抗体による免疫染色を行い、組織サンプルにおけるフィブリン形成の検出を試みた。
各パラフィン包埋組織サンプルを脱パラフィン処理し、120℃で10分間、10mM クエン酸バッファー(pH6)にて抗原賦活した。5%スキムミルク/PBSにてブロッキング処理を施した後、10μg/mLの102-10抗体と共に、室温で1~2時間インキュベートした。次いで、ぺルオキシダーゼ標識した抗ヒトIgG二次抗体(MBL社製)と共に60分間インキュベートした。そして、DAB(Dako社製)にて染色した後に、ヘマトキシリン(武藤化学社製)にて核を染色した。得られた結果を図4~6に示す。
以下に示す方法にて、血栓が生じる疾患のモデル動物を用い、102-10抗体のフィブリン検出能を評価した。
化学発癌モデルは、メスのFVB/Nマウスを剃毛し、アセトンで希釈したDMBA(250μg/mL、Sigma社製)を1回塗布し、1週後からアセトンで希釈したPMA(25μg/mL、Sigma社製)を毎週塗布し、32週まで行うことにより、調製した。
102-10抗体とp-イソチシアネートベンジル-デスフェリオキサミンB(p-isothiocyanatobenzyl-desferrioxamine B、DF、Macrocyclics社製)とを、DFと抗体の比(1:1~1:3)にて結合させ、Sephadex G-50(GE)にて精製した。また、89Zr-シュウ酸塩(oxalate)はサイクロトロンで作製した。そして、DFを結合した102-10抗体(100μg/20μL PBS)と5.0-5.6M Bqの89Zr-oxalate(3.7-5.6 GBq/mL,pH7-9)とを1時間室温でインキュベートし、Sephadex G-50で精製した。このようにして得られたPETプローブ(図8 参照)は、放射化学的収率は73-96%を達成し、純度は96-98%で、50mMDTPAを用いた薄層クロマトグラフィーによる特異活性は37-44kBq/μgであった。
化学発癌モデルについては、前記PETプローブを用い、PET/CTを行った。すなわち、未標識換算で100μg/マウスの抗体投与量にて、3.7MBqの前記PETプローブを化学発癌モデルマウスに尾静脈から投与した。PETのデータは、小動物用PETシステム「Inveon」(small-animal PET system)を用い、麻酔下で10~20分間撮影することにより得た。体温はランプや恒温パットで37℃になるよう保った。画像は、減衰補正なしで3D 最大事後確率法(3D maximum a posteriori;18 iterations with 16 subsets,β=0.2resolution)を用いて処理した。PETスキャンの後、CTイメージは90kVp,200μAのX線光源を用いて、小動物用PETシステム「R_mCT2」(理学社製)にて撮影した。得られた結果を図9に示す。
以下に示す方法にて、102-10抗体によるフィブリンクロット中のフィブリン分子の検出を試みた。また、102-10抗体と、非還元フィブリノーゲン又は還元フィブリノーゲンとの親和性の有無についても解析した。
参考例1に記載の方法と同様の方法にて作製したフィブリンクロットを、OCTcompoundを用いて凍結し、6μm厚の凍結切片を調製した。これら凍結切片を風乾させた後、PBSで洗浄し、5%スキムミルク/PBSでブロッキングした。次いで、Alexa Folur 647 タンパク質ラベリングキット(invitrogen社製)で標識した102-10抗体を添加し、1時間インキュベートした後、Fluoromount G(Southern Biotec社製)で封入した。そして、このようにして調製したサンプルを蛍光顕微鏡にて観察した。得られた結果を図11に示す。
1μgのヒト及びマウス由来のフィブリノーゲンを、5% 2-メルカプトエタノール(2-ME)含有サンプルバッファー(Bio-Rad社製)又は不含サンプルバッファー(Bio-Rad社製)で各々希釈した。そして、2-ME含有サンプルバッファーで希釈したサンプルのみを96℃で5分間熱処理した。次いで、これらサンプルを4-20% TGXゲル(Bio-Rad社製)にアプライし、200Vの定電圧で30分間かけ泳動した。泳動後のゲルを、トランスブロットターボミニPVDF(Bio-Rad社製)に2.5A、25V、7分の条件で転写した。転写後のメンブレンをスナップi.d.(Millipore社製)に移し、0.3%スキムミルク/0.1%PBS-Tでブロッキングした後に、1μg/mLのペルオキシダーゼ標識した102-10抗体と共にインキュベートし、抗原抗体反応を行った。次いで、PBS-Tにて洗浄した後、ECLプライム(GE社製)にて化学発光を生じさせ、ペルオキシダーゼ標識した102-10抗体が結合した抗原を検出した。得られた結果を図13に示す。
102-10抗体のエピトープの同定を以下に示す方法にて試みた。
参考例4に記載の方法にて、還元・熱処理したフィブリノーゲンをSDS-PAGEにて展開した。次いで、イージーステイン・リバース及びアトプレップMF(共にAtto社製)を用い、ゲルからフィブリノーゲンBβ鎖を抽出した。抽出したフィブリノーゲンBβ鎖を、リシルエンドペプチダーゼにて切断した。そして、ウェスタンブロッティングにより、切断されたフィブリノーゲンBβ鎖ペプチドの中から、102-10抗体に結合する最小分子量のペプチド断片を抽出した。次いで、このペプチド断片についてアミノ酸シークエンスを行った。
前記86アミノ酸からなる領域をさらに5分割して、合成ペプチドを作製した。これらペプチドを用いて、競争阻害実験を行った。すなわち、1μg/mLの102-10抗体に対し、0.01~100μMで段階希釈した前記合成ペプチドを加え、室温で30分インキュベートした。抗体とペプチドとの混合溶液を100μLずつフィブリンプレートに添加し、室温で30分インキュベートした。その後、TBS-Tで洗浄し、1step slow TMB(Thermo社製)100μLを添加して5分間比色を行った。停止反応は2規定のH2SO4を100μL加えることによって行った。また、吸光度の測定は、SPECTRA MAX190(日本モレキュラーデバイス社製)で450nmの吸光波長を測定することにより得た。得られた結果の一部を図15に示す。なお、図15に記載のFib-1、Fib-3、No.1及びNo.5の配列は以下の通りである。
Fib-1:NIPVVSGKECEEIIRKGGETS(フィブリノーゲンBβ鎖の第179~252位、配列番号:21)
Fib-3:CNIPVVSGKE(フィブリノーゲンBβ鎖の第231~240位、配列番号:22)
No.1:HQLYIDETVNSNIPTNLRVLRSILENLRSK(フィブリノーゲンBβ鎖の第179~208位、配列番号:23)
No.5:CNIPVVSGKECEEIIR(フィブリノーゲンBβ鎖の第231~246位、配列番号:1)
また、Fib-1及びFib-3については、キャリアタンパク質(KLH)をコンジュゲートしたもの(Fib-1 KLH及びFib-3 KLH)も用意し、競争阻害実験に供した。
本発明の抗体の作製
参考例5に示したコンピューターシュミレーションの結果から、102-10抗体がフィブリノーゲンに結合できないのは、当該抗体のエピトープは分子内に隠れている領域であることが原因であると想定される。また、フィブリノーゲンがトロンビンによって切り出されてフィブリンモノマーを生成し、さらにそれらが重合、架橋化することによって、不溶性フィブリンに変化する。この構造変化によって、フィブリノーゲン分子内に隠れていた前記領域が暴露されることにより、102-10抗体が不溶性フィブリンに結合できるようになると想定される。
先ず、フィブリノーゲンβ鎖の第231~246位のアミノ酸からなる部位又はフィブリノーゲンγ鎖の第232~246位のアミノ酸からなる部位をコードする抗原遺伝子を、pET21bの制限酵素サイト(NdeI-HindIII)に挿入することにより、ヒスチジンタグを融合させた抗原ペプチドを発現させることのできるプラスミドDNAを調製した。そして、これらプラスミドDNAを、大腸菌 BL21(DE3)(Novagen社製)に各々導入した。
フィブリノーゲンγ鎖の第232~246位のアミノ酸からなる部位に結合する抗体(以下、「抗γ鎖抗体」とも称する)の作製は、ITM株式会社に委託した。すなわち、前記にて調製したγ鎖由来の抗原ペプチドをマウスの尾根部筋肉内に注射し、該マウスの腸骨リンパ節を用い、モノクローナル抗体を作製した(マウス腸骨リンパ節法、Sado Y.ら、Acta Histochem. Cytochem.、2006年、39巻、89~94ページ 参照)。フィブリノーゲンBβ鎖の第231~246位のアミノ酸からなる部位に結合する抗体(以下、「抗β鎖抗体」とも称する)は以下の方法にて作製した。
実施例1にて作製した抗β鎖抗体及び抗γ鎖抗体と、102-10抗体とを、以下に示すELISA法にて比較した。なお、実施例1にて樹立したハイブリドーマのうち、Fib-0355及びFib-03435を選択し、これらハイブリドーマが産生する抗体を、抗β鎖抗体として以下に示すELISA法に供した。また、実施例1にて樹立したハイブリドーマのうち、13-30及び34-105を選択し、これらハイブリドーマが産生する抗体を、抗γ鎖抗体として以下に示すELISA法に供した。
各抗体溶液を10μg/mLで100μLずつ、前記フィブリンプレートと前記フィブリノゲンプレートとに添加し、1時間室温で静置した。TBS-Tにて洗浄した後、二次抗体としてbethyl anti-Human IgG-HRP(×1000希釈)又はbethyl anti-mouse IgG-HRP(×10000希釈)を添加した。そして、TBS-Tにて洗浄した後、OPDで10分間比色し、492nmの吸収波長を測定した。得られた結果を図16に示す。
前述のFib-0355が産生する抗体(Fib-0355抗体)及び34-105が産生する抗体(34-105抗体)について、以下に示す方法にて可変領域及び相補性決定領域(CDR)の配列を決定した。
34-105抗体のL鎖(軽鎖)可変領域のアミノ酸配列:配列番号:3
34-105抗体のL鎖CDR1~3のアミノ酸配列:配列番号:4~6
34-105抗体のH鎖(重鎖)可変領域のアミノ酸配列:配列番号:7
34-105抗体のH鎖CDR1~3のアミノ酸配列:配列番号:8~10。
Fib-0355抗体のL鎖(軽鎖)可変領域のアミノ酸配列:配列番号:11
Fib-0355抗体のL鎖CDR1~3のアミノ酸配列:配列番号:12~14
Fib-0355抗体のH鎖(重鎖)可変領域のアミノ酸配列:配列番号:15
Fib-0355抗体のH鎖CDR1~3のアミノ酸配列:配列番号:16~18。
<223> 軽鎖可変領域(34-105)
配列番号:4
<223> 軽鎖CDR1(34-105)
配列番号:5
<223> 軽鎖CDR2(34-105)
配列番号:6
<223> 軽鎖CDR3(34-105)
配列番号:7
<223> 重鎖可変領域(34-105)
配列番号:8
<223> 重鎖CDR1(34-105)
配列番号:9
<223> 重鎖CDR2(34-105)
配列番号:10
<223> 重鎖CDR3(34-105)
配列番号:11
<223> 軽鎖可変領域(Fib-0355)
配列番号:12
<223> 軽鎖CDR1(Fib-0355)
配列番号:13
<223> 軽鎖CDR2(Fib-0355)
配列番号:14
<223> 軽鎖CDR3(Fib-0355)
配列番号:15
<223> 重鎖可変領域(Fib-0355)
配列番号:16
<223> 重鎖CDR1(Fib-0355)
配列番号:17
<223> 重鎖CDR2(Fib-0355)
配列番号:18
<223> 重鎖CDR3(Fib-0355)
Claims (11)
- 不溶性フィブリンと結合し且つフィブリノーゲンと結合しない抗体であって、配列番号:1に示される部位又は配列番号:2に示される部位に結合する抗体。
- 配列番号:4~6に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む軽鎖可変領域と、
配列番号:8~10に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む重鎖可変領域と
を保持する、請求項1に記載の抗体。 - 配列番号:12~14に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む軽鎖可変領域と、
配列番号:16~18に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む重鎖可変領域と
を保持する、請求項1に記載の抗体。 - 配列番号:3に記載のアミノ酸配列又は該アミノ酸配列において1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む軽鎖可変領域と、
配列番号:7に記載のアミノ酸配列又は該アミノ酸配列において1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む重鎖可変領域と
を保持する、請求項1に記載の抗体。 - 配列番号:11に記載のアミノ酸配列又は該アミノ酸配列において1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む軽鎖可変領域と、
配列番号:15に記載のアミノ酸配列又は該アミノ酸配列において1若しくは複数のアミノ酸が置換、欠失、付加及び/又は挿入されているアミノ酸配列を含む重鎖可変領域と
を保持する、請求項1に記載の抗体。 - 請求項1~5のいずれか1項に記載の抗体を含む、免疫学的測定用試薬。
- 請求項1~5のいずれか1項に記載の抗体を含む、血栓関連疾患の判定用試薬。
- 請求項1~5のいずれか1項に記載の抗体を含む、血栓可視化剤。
- (a)請求項1~5のいずれか1項に記載の抗体とサンプルとを接触させるステップ、
(b)該抗体がサンプル中の不溶性フィブリンと結合したか否かを検出するステップ
を含む、サンプル中の不溶性フィブリンを検出するための方法。 - (a)請求項1~5のいずれか1項に記載の抗体と被験体に由来するサンプルとを接触させるステップ、
(b)該抗体がサンプル中の不溶性フィブリンと結合したか否かを検出するステップ
を含む、被験体における血栓関連疾患を判定するための方法。 - 請求項1~5のいずれか1項に記載の抗体と抗腫瘍性部分との複合体。
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EP2963059B1 (en) | 2018-04-04 |
JPWO2014133093A1 (ja) | 2017-02-02 |
US20160011217A1 (en) | 2016-01-14 |
ES2673583T3 (es) | 2018-06-22 |
JP2018193386A (ja) | 2018-12-06 |
EP3339327A2 (en) | 2018-06-27 |
EP2963059A4 (en) | 2017-03-22 |
EP3339327A3 (en) | 2018-09-19 |
US9429584B2 (en) | 2016-08-30 |
EP2963059A1 (en) | 2016-01-06 |
JP7012360B2 (ja) | 2022-02-14 |
JP6490574B2 (ja) | 2019-03-27 |
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