JP2012115243A - Anti-mt1-mmp single-stranded antibody and radioactive molecule imaging probe using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new single-stranded antibody usable for diagnosis and examination of cancer, and to provide a radioactive molecule imaging probe using the antibody.SOLUTION: There is provided a single-stranded antibody having bonding activity to MT1-MMP, which single-stranded antibody includes an H chain variable region and an L chain variable region having specific amino acid sequences. A polynucleotide encoding the amino acid sequence of the single-stranded antibody, a vector comprising the polynucleotide and a method for producing the single-stranded antibody by using a transformant transformed with the vector are provided. Further, a use method of the single-stranded antibody as a molecular imaging probe agent for cancer is provided.

Description

  The present invention relates to a novel single-chain antibody used for cancer diagnosis and testing, and a radiomolecular imaging probe using the same.

  Degradation of ECM and basement membrane is an essential step in cancer invasion and metastasis, and MMP is an important group of enzymes that control this process. MMPs are broadly classified into secretory and membrane-bound types based on their structures. Among these, MT1-MMP, which is membrane-bound, is (1) expressed early in tumor formation and its expression is limited to tumor tissue. (2) It has degradation activity against a wide range of ECM components such as type I, II and type III collagen, laminin, fibronectin, and (3) regulates cell adhesion molecules such as integrin and CD44 on the cell surface. (4) MMP-2 -13 has the property of activating -13 on the cell surface, and the expression of MT1-MMP has been shown to correlate with the malignancy of cancer, and the MT1-MMP mutant was expressed. It has been shown that proliferation and invasiveness are markedly suppressed in cancer cells. In addition, MT1-MMP has been shown to be expressed in most human cancer tissues, and development of cancer diagnosis and treatment methods targeting MT1-MMP has become an urgent task. So far, development of molecular imaging probes targeting MMP (particularly secreted MMP) has been actively promoted, but no effective MT1-MMP imaging probe has been reported yet.

  Radioactive probes targeting MT1-MMP have been reported using full-length IgG antibodies (see Non-Patent Documents 1 and 2), but the tumor blood ratio as an index of imaging is 1. It is about 5 and is not sufficient. In addition, since 99mTc having a half-life of 6 hours is used as the radionuclide, the radioactivity is remarkably attenuated 48 hours after administration, and cannot be used for practical imaging. A report using a two-step administration method (pre-targeting method) (see Non-Patent Document 3) has succeeded in SPECT imaging of tumor-bearing mice, but streptavidin used as a linker pair has a problem of immunogenicity. There is a low possibility of clinical application.

  As an imaging probe targeting cancer, 18F-labeled fluorodeoxyglucose (FDG) that accumulates according to the sugar metabolism of cells is known, but has a problem that it is not specific to cancer lesions. Yes.

Development of a radiolabeled probe for detecting membrane type-1 matrix metalloproteinase on malignant tumors.Temma T, Sano K, Kuge Y, Kamihashi J, Takai N, Ogawa Y, Saji H. Biol Pharm Bull. 2009 Jul; 32 (7): 1272-7. Imaging with radiolabelled anti-membrane type 1 matrix metalloproteinase (MT1-MMP) antibody: potentials for characterizing atherosclerotic plaques.Kuge Y, Takai N, Ogawa Y, Temma T, Zhao Y, Nishigori K, Ishino S, Kamihashi J, Kiyono Y, Shiomi M, Saji H. Eur J Nucl Med Mol Imaging. 2010 Nov; 37 (11): 2093-104. Radioimmunodetection of membrane type-1 matrix metalloproteinase relevant to tumor malignancy with a pre-targeting method.Sano K, Temma T, Kuge Y, Kudo T, Kamihashi J, Zhao S, Saji H. Biol Pharm Bull. 2010; 33 (9) : 1589-95.

  The problem to be solved by the present invention is to provide a diagnostic probe for cancer that is specific to cancer lesions, exhibits optimal pharmacokinetics as diagnostic imaging, and is suitable for clinical application without immunogenicity problems. there were.

  In view of the above problems, the present inventors have made extensive studies and succeeded in obtaining single-chain antibodies specific for MT1-MMP, and those radiolabeled are useful as cancer-specific imaging agents. As a result, the present invention has been completed.

  ADVANTAGE OF THE INVENTION According to this invention, the probe for the diagnostic imaging of a cancer suitable for a clinical application which is specific to a cancer lesion, shows the optimal pharmacokinetics as an imaging diagnosis, and does not have an immunogenicity problem is provided.

Accordingly, the present invention provides the following:
(1) A single-chain antibody having binding activity to MT1-MMP, an H-chain variable region having the amino acid sequence represented by SEQ ID NO: 2, and an L-chain variable region having the amino acid sequence represented by SEQ ID NO: 4. A single chain antibody comprising:
(2) A single-chain antibody having binding activity to MT1-MMP, the H-chain variable region having the amino acid sequence represented by SEQ ID NO: 6, and the L-chain variable region having the amino acid sequence represented by SEQ ID NO: 8 A single chain antibody comprising:
(3) A single-chain antibody having binding activity to MT1-MMP, an H-chain variable region having the amino acid sequence represented by SEQ ID NO: 10, and an L-chain variable region having the amino acid sequence represented by SEQ ID NO: 12 A single chain antibody comprising:
(4) a polynucleotide encoding the amino acid sequence of the single-chain antibody according to any one of (1) to (3);
(5) A vector comprising the polynucleotide according to (4);
(6) A transformant transformed with the vector according to (5);
(7) A method for producing a single-chain antibody according to any one of (1) to (3), wherein a transformant transformed with the vector according to (5) is cultured in an appropriate medium, Recovering the single chain antibody from the transformant or medium;
(8) Cells deposited with the Independent Administrative Institution Product Evaluation Technology Infrastructure and assigned the receipt number NITE AP-1008, and deposited with the Independent Administrative Institution Product Evaluation Technology Infrastructure and received the receipt number NITE AP-1009 Or a cell deposited with an Independent Administrative Institution Product Evaluation Technology Foundation and given the receipt number NITE AP-1010 in an appropriate medium, and recovering a single chain antibody from the medium (1 ) To (3), a method for producing a single-chain antibody;
(9) The single chain antibody according to any one of (1) to (3), which is labeled;
(10) A molecular imaging probe agent for cancer, comprising the radiolabeled single-chain antibody according to any one of (1) to (3);
(11) A diagnostic imaging agent or kit for cancer comprising the molecular imaging probe agent according to (10);
(12) A method for examining the presence of cancer in a subject, the method comprising contacting a single-chain antibody according to any one of (1) to (3) with a sample obtained from the subject;
(13) A reagent or kit comprising the single-chain antibody according to any one of (1) to (3) for immunological measurement or detection of MT1-MMP;
(14) A method for immunologically measuring or detecting MT1-MMP, the method comprising contacting the single-chain antibody according to any one of (1) to (3) with a sample;
(15) The method according to (12) or (14), wherein the single chain antibody is labeled, or the kit according to (13).

FIG. 1 shows the results of an antigen-immobilized ELISA of a single chain antibody of the present invention. FIG. 2 shows the results of Cell ELISA of the single chain antibody of the present invention. FIG. 3 shows an inhibition curve by indirect competitive ELISA of the single chain antibody of the present invention. FIG. 4 shows the results of Western blotting of the single chain antibody of the present invention. Lane 1 is MMP98-1 (1 μg / ml), Lane 2 is MMPK9E-1 (1 μg / ml), and Lane 3 is commercially available antibody IgG (10 μg / ml). FIG. 5 shows the results of immunostaining of tumors (left panel) and autoradiography (right panel) with single chain antibody MMPK9E-1. FIG. 6 shows an inhibition curve of the single-chain antibody of the present invention by flow immunoassay.

  The present invention is described in detail below. In the present specification, amino acids are represented by one-letter code or three-letter code known to those skilled in the art. Unless otherwise noted, other terms in this specification are also construed in the usual meaning described in biology textbooks and dictionaries. Moreover, unless otherwise indicated in this specification, MT1-MMP means MT1-MMP protein.

  In the first aspect, the present invention provides a novel single chain antibody having binding activity to MT1-MMP. As described above, MT1-MMP is expressed in most human cancer tissues, and its expression correlates with the malignancy of cancer. Therefore, by examining the expression of MT1-MMP using the novel single chain antibody of the present invention, the presence and malignancy of cancer in the body of the subject can be known.

  In one specific example, the single-chain antibody of the present invention has a binding activity to MT1-MMP, an H chain variable region having the amino acid sequence represented by SEQ ID NO: 2, and an amino acid sequence represented by SEQ ID NO: 4 A light chain variable region having In this specification, the antibody of this specific example is referred to as “antibody MMP98-1.”

  In a further embodiment, the single-chain antibody of the present invention has a binding activity to MT1-MMP, an H chain variable region having the amino acid sequence represented by SEQ ID NO: 6, and an amino acid sequence represented by SEQ ID NO: 8. Having a light chain variable region. In this specification, the antibody of this specific example is referred to as “antibody MMPK9E-1.”

  In a further embodiment, the single-chain antibody of the present invention has a binding activity to MT1-MMP, an H chain variable region having the amino acid sequence represented by SEQ ID NO: 10, and an amino acid sequence represented by SEQ ID NO: 12. Having a light chain variable region. In this specification, the antibody of this specific example is referred to as “antibody MMPK9I-1.”

All of the above single-chain antibodies of the present invention have a binding activity to MT1-MMP, and a sequence in which Cys is added to the C-terminal of PQPRTTSRPSVPDKPKN, which is a partial sequence of MT1-MMP:
PQPRTTSRPSVVPDKKNC (SEQ ID NO: 13)
A KD value of 50 nM or less, preferably 40 nM or less, more preferably 30 nM or less is shown for a peptide consisting of

  In a further aspect, the present invention also provides a mutant single chain antibody having the same properties as the single chain antibody of the present invention described above. Specific examples of these mutant single chain antibodies include the following.

A heavy chain variable region having an amino acid sequence having binding activity to MT1-MMP and having one to several amino acids substituted, added or deleted in the amino acid sequence represented by SEQ ID NO: 2, and SEQ ID NO: A single chain antibody comprising an L chain variable region having an amino acid sequence in which one to several amino acids are substituted, added or deleted in the amino acid sequence represented by 4;
A heavy chain variable region having an amino acid sequence having binding activity to MT1-MMP and having one to several amino acids substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 6, and SEQ ID NO: A single chain antibody comprising an L chain variable region having an amino acid sequence in which one to several amino acids are substituted, added or deleted in the amino acid sequence represented by 8;
A heavy chain variable region having an amino acid sequence having binding activity to MT1-MMP and having one to several amino acids substituted, added or deleted in the amino acid sequence represented by SEQ ID NO: 10, and SEQ ID NO: A single-chain antibody comprising an L chain variable region having an amino acid sequence in which one to several amino acids are substituted, added or deleted in the amino acid sequence shown in FIG. 12; and SEQ ID NO: 2, SEQ ID NO: 6 or An H chain variable region having any one of the amino acid sequences represented by column number 10 and an L chain variable region having any one of the amino acid sequences represented by SEQ ID NO: 4, SEQ ID NO: 8 or column number: 12; A single chain antibody consisting of any combination.
These single chain antibodies exhibit a KD value of 50 nM or less, preferably 40 nM or less, more preferably 30 nM or less with respect to the peptide consisting of the amino acid sequence represented by SEQ ID NO: 13.

From the viewpoint of diagnostic imaging of cancer or measurement or detection of MT1-MMP, the above single-chain antibody is preferably labeled in a detectable manner. Antibody labeling methods are known to those skilled in the art, and can be appropriately selected to label the antibody. Examples of the antibody labeling method include a fluorescence labeling method using FITC, an enzyme labeling method using peroxidase, a radioactive labeling method using ^99m Tc or ¹¹¹ In, a biotin labeling method, and the like. The single-chain antibody of the present invention has a molecular size of about one fifth that of IgG and has improved pharmacokinetics. Therefore, as a radionuclide used for radiolabeling, ¹¹¹ In having a half-life of about 3 days is easy to use when SPECT is used. In the present specification, the single chain antibody of the present invention includes a labeled single chain antibody of the present invention.

Further, when radiolabeling is performed, it is preferable to introduce a plurality of lysine residues for introducing a bifunctional chelate at the C-terminus of the antibody so that the target recognition function is not impaired by the radiolabeling. Therefore, the single-chain antibody of the present invention labeled with ¹¹¹ In and having a plurality of lysine residues introduced at the C-terminus can obtain a high tumor blood radioactivity ratio necessary for imaging from the early stage of administration. Therefore, it is particularly preferable.

  The single chain antibody of the present invention can be obtained by a method known to those skilled in the art, for example, a phage display method or a hybridoma method. These methods can be further combined with known gene manipulation techniques. For details of these methods, see the Examples. However, it goes without saying that the method for obtaining the single-chain antibody of the present invention is not limited to the above method.

  In a further aspect, the present invention provides a polynucleotide encoding the amino acid sequence of the single chain antibody or mutated single chain antibody of the present invention described above. Furthermore, in another aspect, the present invention provides a vector incorporating the polynucleotide, and a cell transformed with the vector.

  Such a polynucleotide includes a polynucleotide encoding a linker sequence, a His tag sequence, a V5 tag sequence, and the like (not limited to these sequences) in addition to the polynucleotide encoding the H chain variable region and the L chain variable region. May be.

  A polynucleotide encoding the amino acid sequence of the single-chain antibody of the present invention is incorporated into an expression vector or cloning vector, introduced into an appropriate cell for transformation, and the transformed cell is cultured in an appropriate medium to produce the produced single unit. By recovering the chain antibody from the medium, the single chain antibody of the present invention, that is, the single chain antibody MMP98-1, the single chain antibody MMPK9E-1, and the single chain antibody MMPK9I-1 can be obtained. Means and methods for selection of cell types, cell transformation methods, cell culture methods, and recovery and purification of single-chain antibodies from culture media are known to those skilled in the art. A method can be appropriately selected and used. For example, introduction of a polynucleotide into cells includes a method of introducing a vector using PEG and Ca ions, an electroporation method, a method of using a gene gun, and the like.

  The above mutant single chain antibodies can also be obtained by the same means / method using polynucleotides encoding their amino acid sequences.

  A single-chain antibody MMP98-1, a single-chain antibody MMPK9E-1, and a polynucleotide encoding the single-chain antibody MMPK9I-1 were incorporated into a vector, and these were used to transform insect cells, whereby a single-chain antibody MMP98- 1 cells (named MMP98-0 in the applicant), cells producing the single chain antibody MMPK9E-1 (named MMPK9E-0 in the applicant) and cells producing the single chain antibody MMPK9I-1 (applicant) And named MMPK9I-0). Cells producing these single-chain antibody MMP98-1, single-chain antibody MMPK9E-1 and single-chain antibody MMPK9I-1 were each deposited with the National Institute of Technology and Evaluation and received on December 2, 2010. The numbers NITE AP-1008, NITE AP-1009 and NITE AP-1010 have been assigned. By culturing these cells in an appropriate medium and recovering the single chain antibody from the medium, the single chain antibody of the present invention, that is, the single chain antibody MMP98-1, the single chain antibody MMPK9E-1, and the single chain antibody MMPK9I −1 can be obtained.

In a further aspect, the present invention provides a molecular imaging probe for cancer comprising a radiolabeled single chain antibody of the present invention. That is, cancer can be image-diagnosed using the radiolabeled single-chain antibody of the present invention. As the radiolabel, γ-ray emitting nuclides such as ¹¹¹ In, ^99m Tc, ⁶⁷ Ga, ²⁰¹ Tl, ¹²³ I, and ¹³³ Xe can be used for SPECT, but are not limited to these nuclides. For PET, positron emitting nuclides such as ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸ F, ⁶² Cu, ⁶⁸ Ga, and ⁷⁶ Br can be used for labeling, but are not limited to these nuclides.

^Since the single-chain antibody of the present invention labeled with ¹¹¹ In and having a plurality of lysine residues introduced at the C-terminus can obtain a high tumor blood radioactivity ratio necessary for imaging from early administration, This is particularly preferable for diagnostic imaging of cancer.

The molecular imaging probe agent for cancer of the present invention may contain a commonly used excipient or carrier in addition to the radiolabeled single-chain antibody of the present invention. These excipients and carriers are also known, and those skilled in the art can appropriately select and use them according to the purpose. The dosage form of the molecular imaging probe of the present invention may be any dosage form, but is preferably an injection or an infusion. The cancer molecular imaging probe agent of the present invention is generally used after labeling the single-chain antibody of the present invention with a radionuclide before use. The method for labeling the single-chain antibody of the present invention with a radionuclide can be performed using a known method. See the examples herein for specific examples of labeling with ¹¹¹ In. Using the molecular imaging probe for cancer of the present invention, the presence of cancer in the subject, its site, and the malignancy of the cancer can be examined.

  Explaining the use of the molecular imaging probe agent for cancer of the present invention, the molecular imaging probe agent for cancer of the present invention is injected into the subject (any of local administration and systemic administration), administered by means such as infusion, By measuring radiation with SPECT or PET after an appropriate time has elapsed, for example, 1 to 24 hours after administration, the presence of cancer in the subject, its site, and the malignancy of the cancer can be examined. In the case of cancer diagnosis, it is desirable to compare with normal subjects. In addition, when looking at the progress and prognosis of cancer in a subject, it is desirable to perform an examination over time. These diagnosis and examination methods and procedures are basically the same as normal cancer diagnosis and examination, as well as normal diagnostic imaging methods and procedures.

  In a further aspect, the present invention provides a diagnostic imaging agent or kit for cancer comprising the molecular imaging probe agent as an essential component. Usually, an instruction manual is attached to the kit.

  In a further aspect, the present invention is a method for determining the presence or malignancy of a cancer in a subject comprising administering a single chain antibody of the present invention to the subject and binding to the MT1-MMP in the subject. Provided is a method characterized by examining the amount of chain antibody. In this method, the single chain antibody of the present invention is preferably labeled with a radionuclide.

  In a further aspect, the present invention relates to a method for examining the presence or malignancy of cancer in a subject, wherein the single-chain antibody according to any one of (1) to (3) is used in a sample obtained from the subject. Is contacted and the presence or amount of MT1-MMP in the sample is measured. In this method, the single chain antibody of the present invention is preferably labeled. Such labels include, but are not limited to, enzyme labels, fluorescent labels, radioactive labels and the like. Here, the contact means that MT1-MMP in the sample and the single chain antibody of the present invention can be bound. For example, the sample solution and the single chain antibody of the present invention are mixed and mixed at room temperature or in the animal. Incubation may be performed at a temperature near body temperature for an appropriate time.

  In another aspect, the present invention includes the single-chain antibody according to any one of (1) to (3) for immunologically measuring, detecting or capturing MT1-MMP. A reagent or kit is provided. Using the reagent or kit, MT1-MMP in a sample can be captured, or the presence or amount thereof can be examined. In such reagents and kits for the purpose of measurement and detection, the single-chain antibody of the present invention is preferably labeled. Such labels include, but are not limited to, enzyme labels, fluorescent labels, radioactive labels and the like.

  In another aspect, the present invention provides a method for immunologically measuring, detecting or capturing MT1-MMP, wherein the single-chain antibody according to any one of (1) to (3) is used as a sample. Provided is a method characterized by contacting. In such measurement and detection methods, the single chain antibody of the present invention is preferably labeled. Such labels include, but are not limited to, enzyme labels, fluorescent labels, radioactive labels and the like.

  EXAMPLES The present invention will be described more specifically and in detail below with reference to examples, but the examples should not be construed as limiting the present invention.

(1-1) Preparation of Peptide Conjugate A hinge region peptide having an amino acid sequence contained in the MT1-MMP hinge region was synthesized and bound to a carrier protein using a linker Sulfo-EMCS (manufactured by PIERCE), and peptide conjugate A gate was synthesized.

(1-2) Immunization of mice Mice were immunized using the obtained conjugate as an immunogen.
The peptide conjugate and the adjuvant RASR-700 (RIBI) were mixed in a 1: 1 volume and sufficiently emulsified, and the resulting emulsion was abdominal cavity of BALB / c mice (7-8 weeks old, female). The mice were immunized by administration of 200 μL. Booster immunization was performed at intervals of about 2 to 3 weeks, and blood was collected from the tail vein one week after the booster immunization, and the antibody titer against the immunogen was observed by measuring the antibody titer in the blood by ELISA.

(1-3) Production of hybridoma Final immunization was performed by further administering the immunogen into the tail vein of mice in which high antibody production against the immunogen was observed in the blood. Three days after the final immunization, the spleen is removed from the immunized mouse, spleen cells are prepared, and myeloma cells (P3-X63-Ag8.653 or Sp2 / O) in the logarithmic growth phase and spleen cells are 1: 5. And cell fusion was performed by the polyethylene glycol (PEG) method. The cells after fusion are suspended in RPMI 1640 medium containing 10% FCS supplemented with HAT (hypoxanthine, aminopterin and thymidine), and cultured in 96 wells so that the number of spleen cells is 1-2 × 10 ⁵ cells / well. Plates were seeded and cultured at 37 ° C., 5% CO ₂ .

(1-4) Screening and cloning of antibody-producing hybridomas Seven to ten days after cell fusion, antibody titers were measured by ELISA using the culture supernatant of wells in which clone growth was observed.

(1-5) Screening of antibody-producing hybridoma (measurement of antibody titer)
To each well of a microtiter plate (manufactured by CORNING), 50 μL of a 0.25 to 1 μg / mL solution of each immunogen in PBS (−) was added and allowed to stand at room temperature for 1 hour to be immobilized. In addition, in order to exclude antibody producing clones against the carrier protein in the peptide conjugate, a well immobilized with BSA or KLH was used as a control. Each well was washed three times with 300 μL of PBS (−) (washing solution) containing 0.05% Tween 20 and then 1 g of Block Ace powder (Yuki-Ink, UK-B80) was dissolved in 100 mL of purified water. (Blocking reagent) 300 microliters was added to the well, and it left still at room temperature for 2 hours, and blocked. The wells were washed three times with 300 μL of the washing solution, 50 μL of the culture supernatant containing the primary antibody was added to each well and mixed, and then allowed to stand for 30 to 60 minutes at room temperature for reaction. Next, after washing each well three times with 300 μL of washing solution, a solution in which 0.4 g of block ace powder was dissolved in 100 mL of purified water in order to detect the primary antibody bound to each immobilized immunogen. 50 μL of horseradish peroxidase-labeled anti-mouse IgG (γ chain recognition) (manufactured by KPL) prepared to a 3000-fold dilution with (secondary antibody diluent) was added as a secondary antibody solution and reacted at room temperature for 1 hour. After the wells were washed three times with 300 μL of the washing solution in the same manner, 3,3 ′, 5,5′-tetramethylbenzidine (TMB) solution (manufactured by KPL) was added to each well, and a color reaction was performed at room temperature. It was. After 5 to 15 minutes, 50 μL of 1M phosphoric acid was added to stop the reaction, and the absorbance at 450 nm (control: OD650) was immediately measured with a microplate reader (Multiscan MS, manufactured by Labstems). The difference in absorbance between the well in which each immunogen was immobilized and the control well was defined as the binding activity of the antibody to the immunogen.

(1-6) Selection of specific antibody-producing clones by indirect competition method According to (1-5) above, Block Ace prepared by immobilizing each immunogen in each well of a microtiter plate, washing, and then diluting 4 times with purified water 300 μL of the solution was added to the well and allowed to stand at room temperature for 2 hours for blocking. The wells were washed 3 times with 300 μL of washing solution.
Next, a hinge region peptide solution prepared with PBS (−) was prepared, and 25 μL of each was added to each well of the immunogen-immobilized plate. Next, 25 μL of the hybridoma culture supernatant was added to the well and mixed, and then allowed to stand for 30 to 60 minutes at room temperature to carry out the reaction.
Each well was washed 3 times with 300 μL of the washing solution, and then horseradish peroxidase-labeled anti-mouse IgG (γ) prepared by diluting 3000 times with a secondary antibody diluent to detect the primary antibody bound to the immobilized immunogen. 50 μL of chain recognition) was added as a secondary antibody solution, and the mixture was reacted at room temperature for 1 hour.
After the wells were washed three times with 300 μL of the washing solution in the same manner, a TMB solution was added to each well and a color reaction was performed at room temperature. After 5 to 15 minutes, 50 μL of 1M phosphoric acid was added to stop the reaction, and the absorbance at 450 nm (control: OD650) was immediately measured with a microplate reader (Multiscan MS, manufactured by Labsystems). The ratio (B / B0) of the well absorbance value (B) in the presence of the hinge region peptide to the absorbance value of the control well (control OD; B0) was calculated, and the reactivity of the antibody to the coexisting substance was judged. The hybridoma 66C and hybridoma 19B in wells with high binding activity to the hinge region peptide and confirmed to have high antibody production continuously were selected and subjected to cloning by limiting dilution.

(1-7) Purification of mRNA and cDNA synthesis The hybridoma 66C and hybridoma 19B selected in the above (1-6) were grown in RPMI 1640 medium containing 10% FCS under 5% CO ₂ aeration. Poly (A) ⁺ RNA was purified from about 1 × 10 ⁷ cells in the logarithmic growth phase using a QuickPrep Micro mRNA Purification Kit (manufactured by GE Healthcare). Subsequently, first strand cDNA was synthesized according to a conventional method using Superscript III reverse transcriptase (Invitrogen). Using the obtained cDNA as a template, P C R was performed using Mouse Ig-Primer Set (manufactured by Novagen) and Taq DNA polymerase (manufactured by Applied Biosystems) under the conditions recommended in the attached document of Ig-Primer Set. The antibody gene variable region gene DNA was amplified.

(1-8) Determination of base sequence and analysis of amino acid sequence The antibody gene variable region gene DNA amplified in (1-7) above was sequenced using BigDye Terminator Cycle Sequencing Ready Reaction Kit v3.1 (Applied Biosystems). Reaction was performed. Subsequently, the sequence was analyzed by ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Analysis software DNAsis (Hitachi Soft Engineering) was used for the analysis of the base sequence and the estimation and analysis of the amino acid sequence. In addition, the ImmunoGeneTics database (http://imgt.cines.fr) was used to identify the variable distribution region. As a result, the base sequence of the cDNA encoding the H chain and L chain variable regions of the obtained antibody gene and its deduced amino acid sequence were as follows.

Nucleotide sequence encoding heavy chain variable region of antibody (antibody K9E) produced by hybridoma 66C: SEQ ID NO: 5

CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGCTATACCTTCACAAGCTATGCATTGCACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGAAATACACCAACACTGGAGAGCCAACATATGGTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATTAACAACCTCAAAAATGAGGACATGGCTACATATTTCTGTGCAAGAGGGGGACTTAGTTCCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

Amino acid sequence of heavy chain variable region of antibody (antibody K9E) produced by hybridoma 66C: SEQ ID NO: 6

QIQLVQSGPELKKPGETVKISCKASGYTFTSYALHWVKQAPGKGLKWMGWKYTNTGEPTYGDDFKGRFAFSLETSASTAYLQINNLKNEDMATYFCARGGLSSYAMDYWGQGTSVTVSS

Nucleotide sequence encoding the light chain variable region of the antibody produced by hybridoma 66C (antibody K9E): SEQ ID NO: 7

GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCACTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATCTTCTGGTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG

Amino acid sequence of L chain variable region of antibody (antibody K9E) produced by hybridoma 66C: SEQ ID NO: 8

DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGTGSGTDFTLTISSVQAEDLAVYYCKQSYNLLVTFGAGTKLELKR

Nucleotide sequence encoding the heavy chain variable region of the antibody (antibody K9I) produced by the hybridoma 19B: SEQ ID NO: 9

CAGGTTCAACTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGTCCTGCAAGGCTTCGGGCTACAGATTTACTGACTATGAAATGCACTGGGTGAGGCAGACACCTGTGCATGGCCTGGAATGGATTGGAGCTATTGATCCTGAAACTGGTGGTACTGCCTACAATCAGAAGTTCAAGGGCAAGGCCACACTGACTGAAGACAAATCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTACAACTATGATTACGACAGCTCTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

Amino acid sequence of heavy chain variable region of antibody (antibody K9I) produced by hybridoma 19B: SEQ ID NO: 10

QVQLQQSGAELVRPGASVTLSCKASGYRFTDYEMHWVRQTPVHGLEWIGAIDPETGGTAYNQKFKGKATLTEDKSSSTAYMELRSLTSEDSAVYYCTTMITTALDYWGQGTSVTVSS

Nucleotide sequence encoding the L chain variable region of the antibody (antibody K9I) produced by hybridoma 19B: SEQ ID NO: 11

GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTGATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGGTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCACCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGG

Amino acid sequence of the L chain variable region of the antibody (antibody K9I) produced by hybridoma 19B: SEQ ID NO: 12

DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSDGNTYLEWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKITRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKR

(1-9) Preparation of Single-Chain Antibody Expression Vector Antibody IgG heavy and light chain variable region gene cDNAs were ligated with DNA encoding a linker sequence using PCR, and these were ligated to plasmid vector pMIB / V5-His. After insertion between restriction enzyme sites SfiI-NotI (manufactured by Invitrogen), cloning was performed according to a conventional method, and the one derived from hybridoma 66C was designated as single chain antibody expression vector pMIB-MMPK9E, and the one derived from hybridoma 19B was designated as a single chain antibody The expression vector was pMIB-MMPK9I.

(2-1) Preparation of single-chain antibody gene library An antibody gene library is prepared from cells producing antibodies in vivo such as spleen cells, and a recombinant antibody encoded by the antibody gene is displayed on the phage surface. As a method for obtaining a gene of a recombinant antibody having reactivity with a target antigen, a phage display method has been known for a long time. Using the MT1-MMP hinge region peptide-conjugated immunized mouse of (1-2), an antibody gene library was prepared, and an anti-MT1-MMP single chain antibody was screened by the phage display method. Library preparation and phage display were performed with reference to MaCafferty J., Hoogenboom HR, Chiswell DJ, Antibody Engineering, (Oxford University Press) and Kontermann R., Dubel S., Antibody Engineering, (Springer).

Total RNA was extracted from the mouse spleen cells immunized in (1-2) using Sepazole RNA I Super (manufactured by Nacalai), and mRNA was purified from this total RNA by mRNA Purification Kit (manufactured by GE Healthcare). First-strand cDNA was synthesized by reverse transcription reaction similar to the previous period (1-7) using the prepared mRNA. Using the first strand cDNA as a template, the variable region gene DNAs of the antibody IgG heavy chain and light chain were amplified by PCR using primers specific to the mouse antibody gene. As in (1-9) above, the variable region gene DNAs of the H chain and L chain were ligated with DNA encoding the linker sequence using PCR. These were inserted between the restriction enzyme sites SfiI-NotI of the phagemid vector pCANTAB5E, and then Escherichia coli TG1 strain was transformed according to a conventional method. The transformed E. coli was plated on 2 × YT agar medium containing 100 μg / ml ampicillin and cultured at 30 ° C. overnight. The grown cells were added with several ml of medium, scraped and collected with a large stick, and then phage rescued with M13KO7 phage. An Escherichia coli culture solution adjusted to OD600 = 0.3 with 2 × YT medium containing 100 μg / ml ampicillin and 2% glucose, 35 ml of total culture was shaken at 37 ° C. for 1 hour, and then M13KO7 phage was added at a final concentration of 4 X10 ⁹ pfu / ml was added, and the mixture was further cultured with shaking at 37 ° C. for 1 hour. Thereafter, the cells were collected by centrifugation, resuspended in 40 ml of 2 × YT medium containing 100 μg / ml of ampicillin and 50 μg / ml of kanamycin, and cultured overnight at 37 ° C. to produce single-chain antibody-displayed phages in the medium. . Phages were collected by polyethylene glycol precipitation and used as a single chain antibody gene library. The culture supernatant after removing the bacterial cells was collected by centrifuging the culture solution after overnight culture, and 2.5 ml of a 20% polyethylene glycol solution containing 2.5 M NaCl was added to and mixed with 12.5 ml of the culture. After standing on ice for 1 hour, the mixture was centrifuged at 10000 G for 20 minutes under cooling. The supernatant was completely discarded and resuspended in 1 ml of 2 × YT medium to obtain a single-chain antibody-displayed phage solution.

(2-2) Screening by the phage display method In order to concentrate the highly reactive thing with respect to an antigen from the prepared single chain antibody display phage solution, the biopanning was performed. 100 μL / well of the prepared single-chain antibody-displayed phage solution was placed in a 96-well microtiter plate solid-phased with MT1-MMP hinge region peptide conjugate and blocked with Block Ace, and allowed to stand at room temperature for 1 hour. After the reaction, remove the solution, wash 3 times with 300 μL of washing solution, completely remove the washing solution, add 0.1 M glycine-hydrochloric acid buffer pH 2.2, leave it for 10 minutes, pipet it and collect it. And immediately neutralized with Tris solution pH 8.0.
Concentrated single chain antibody-displayed phages were amplified by reinfection and phage rescue. The collected solution was mixed with a culture solution of E. coli TG1 cultured in 10 mL of 2 × YT medium until OD600 = 0.3, and cultured at 37 ° C. for 1 hour to be reinfected. After reinfection, for phage rescue, each solution was added so that ampicillin had a final concentration of 100 μg / mL, glucose had a final concentration of 2%, and M13KO7 phage had a final concentration of 4 × 10 ⁹ pfu / mL. In culture. After culturing for 1 hour, the cells were collected by centrifugation, resuspended in 10 mL of 2 × YT medium containing 100 μg / mL of ampicillin and 50 μg / mL of kanamysi, cultured at 37 ° C. overnight, and single-chain antibody-displayed phage in the medium Was produced. The amplified phage was recovered again by polyethylene glycol precipitation. Concentration and reinfection by biopanning and amplification by phage rescue are repeated 3 to 5 times, and the phage after biopanning is infected with Escherichia coli TG1, which is plated on an agar plate medium and cultured at 30 ° C. overnight to form colonies. I let you.

(2-3) Reactivity evaluation of single-chain antibody-displayed phage after biopanning The reactivity of the single-chain antibody-displayed phage obtained by biopanning to the immobilized antigen was evaluated by ELISA.
50 μL of MT1-MMP hinge region peptide conjugate was added to the microtiter plate and allowed to react for 1 hour at room temperature. PBS (-) was used as a control. After washing with a washing solution, blocking was performed with Block Ace. After washing the wells of the microtiter plate, 50 μL of a single-chain antibody-displayed phage solution prepared with 0.1% Block Ace was added and allowed to react at room temperature for 1 hour. After washing the wells, peroxidase-labeled anti-M13 antibody (Amersham Biosciences) diluted 5000 times was added and reacted at room temperature for 1 hour. After washing the microplate and thoroughly removing the unreacted solution, add 50 μL of the substrate solution (TMB substrate, manufactured by KPL), react at room temperature for 5-15 minutes, and then add 50 μL of 1M phosphoric acid solution to react. Stopped. Absorbance OD450 (control: OD650) was measured with a plate reader (manufactured by Labsystems). As a result, it was found that the single-chain antibody-displayed phage of clone number 98 has reactivity with the immobilized antigen.

(2-4) Determination of base sequence, analysis of amino acid sequence and preparation of single-chain antibody expression vector The sequence of the single-chain antibody-displayed phage clone gene of clone number 98 that reacted with the immobilized antigen by ELISA was analyzed. The sequence was performed by the dye terminator method as in (1-8) above. As a result, it was confirmed to have the gene sequences of antibody H chain and L chain variable regions. The analysis results are shown below.

Nucleotide sequence encoding the heavy chain variable region of the expressed single chain antibody (single chain antibody MMP98): SEQ ID NO: 1

GAGGTTCAGCTTCAGCAGTCTGGACCTGAACTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATCCCGTCACAAAGTACGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACGTACACTGGAGAGCCAACATATACTGATGACTTCAAGGGACGATTTGCCTTCTCTTTGGAAACCTCTGCCAACACTGCCTATTTGCAGATCAACAACCTCAAAGATGAGGACACGGCTACATATTTCTGTGCAAACTCCCACGGTAAATACGTGGAATGGTTTACTTACTGGGGCCAAGGGACCACGGTCACCGTCTCTTCC

Amino acid sequence of the H chain variable region of the expressed single chain antibody (single chain antibody MMP98): SEQ ID NO: 2

EVQLQQSGPELKKPGETVKISCKASGYPVTKYGMNWVKQAPGKGLKWMGWINTYTGEPTYTDDFKGRFAFSLETSANTAYLQINNLKDEDTATYFCANSHGKYVEWFTYWGQGTTVTVSS

Nucleotide sequence encoding the light chain variable region of the expressed single chain antibody (single chain antibody MMP98): SEQ ID NO: 3

GACATTGAGCTCACCCAGTCTCCAACCACCATGGCTGCATCTCCCGGGGAGAAGATCACTATCACCTGCAGTGCCAGCTCAAGTATAAGTTCCAATTACTTGCATTGGTATCAGCAGAAGCCAGGATTCTCCCCTAAACTCTTGATTTATAGGACATCCAATCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATTGGCACCATGGAGGCTGAAGATGTTGCCACTTACTACTGCCAGCAGGGTAGTAGTATACCACTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG

Amino acid sequence of the L chain variable region of the expressed single chain antibody (single chain antibody MMP98): SEQ ID NO: 4

DIELTQSPTTMAASPGEKITITCSASSSISSNYLHWYQQKPGFSPKLLIYRTSNLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQGSSIPLTFGAGTKLELKR

  A single chain antibody gene was excised from the phagemid of clone no. 98 with restriction enzymes SfiI and NotI, inserted between the restriction enzyme sites SfiI-NotI of plasmid vector pMIB / V5-His (manufactured by Invitrogen), and cloned according to a conventional method. The chain antibody expression vector was pMIB-MMP98.

(3-1) Production of recombinant single-chain antibody-expressing cell line Using a gene introduction kit InsectSelect ^™ BSD System (manufactured by Invitrogen), a single-chain antibody expression vector was introduced into insect cells High Five (manufactured by Invitrogen), After culturing in a culture medium (without antibiotics) for 48 hours, the cells were diluted to 1/5 concentration in the culture medium (with antibiotics) and seeded in a culture dish. After standing for 5 or 6 hours, it was confirmed that the cells adhered to the bottom surface, and the medium was removed. The medium was changed to a selective medium (20 μg / ml blasticidin (Invitrogen)) and cultured for 4 days. By addition of the selective medium, cells that had not been transfected with the gene were killed. The culture supernatant was evaluated by the antigen-immobilized ELISA method, and positive clones were selected and then single-cloned by the limiting dilution method to establish a single-chain antibody stably expressing cell line. The established cell lines were each introduced with the single-chain antibody expression vector pMIB-MMP98, the single-chain antibody expression cell line MMP98-0, and the single-chain antibody expression vector pMIB-MMPK9E was introduced with the single-chain antibody expression cell line MMPK9E- 0, single-chain antibody expression vector pMIB-MMPK9I was introduced into single-chain antibody-expressing cell line MMPK9I-0, both deposited at the National Institute of Technology and Evaluation, each received on December 2, 2010 The numbers NITE AP-1008, NITE AP-1009, and NITE AP-1010 were assigned.

(4-1) Preparation of purified single-chain antibody The single-chain antibody-expressing cell line established in (3-1) above is cultured on about 1 L scale, and cultured when the cell concentration reaches 2 × 10 ⁶ cells / ml. The supernatant was collected. A single chain antibody was prepared from the collected culture supernatant by affinity purification using two types of metal chelate columns.
First, 1 ml of carrier Ni Sepharose 6Fast Flow (GE Healthcare) was packed in a PD10 column (GE Healthcare). After equilibrating the column with PBS (−), 1 L of the culture supernatant was applied. Washing was performed in the order of 10 ml of PBS (-), 10 ml of 20 mM imidazole, 0.3 mM sodium chloride, and 50 mM dihydrogen phosphate buffer (pH 8.3). Elution was performed with 3 ml of 500 mM imidazole, 0.3 mM sodium chloride, 50 mM dihydrogen phosphate buffer (pH 8.3) to obtain a single chain antibody fraction. The single chain antibody fraction was dialyzed against 0.3 mM sodium chloride and 50 mM dihydrogen phosphate buffer (pH 8.3).

  Subsequently, 5 ml of carrier TARON Metal Affinity Resin (Clontech) was packed in a PD10 column (GE Healthcare). After equilibrating the column with 0.3 mM sodium chloride and 50 mM dihydrogen phosphate buffer (pH 8.3), the antibody solution after dialysis was applied. Washing was performed with 50 ml of 10 mM imidazole, 0.3 mM sodium chloride, and 50 mM dihydrogen phosphate buffer (pH 8.3). Elution was performed with 20 ml of 500 mM imidazole, 0.3 mM sodium chloride, 50 mM dihydrogen phosphate buffer (pH 8.3) to obtain a single chain antibody fraction. The single chain antibody fraction was dialyzed with PBS (−) to obtain purified single chain antibodies of single chain antibody MMP98-1, single chain antibody MMPK9E-1, and single chain antibody MMPK9I-1.

Evaluation of purified single chain antibody by ELISA In order to evaluate the suitability of the obtained purified single chain antibody in an immunoassay, three types of ELISA were performed: an antigen-immobilized ELISA method, a Cell ELISA method, and an indirect competitive ELISA method. For comparison, a commercially available mouse anti-MT1-MMP monoclonal antibody was used.

(5-1) Antigen-immobilized ELISA
50 μl of MT1-MMP hinge region peptide conjugate solution adjusted to 0.5 μg / ml with PBS (−) was added to each well of a 96-well plate (manufactured by Costar) and allowed to stand at room temperature for 1 hour. The well was washed 3 times with 300 μl of a washing solution (PBS (−) containing 0.05% Tween 20), and then 250 μl of 1% Block Ace (manufactured by Snow Brand) was added, followed by blocking at room temperature for 2 hours. After washing the well with a washing solution, 50 μl of a single-chain antibody solution (0.1 μg / ml, prepared with PBS (−)) was added to the well and allowed to react at room temperature for 30 minutes. The wells were washed and 50 μl of anti-4 × histidine antibody (Qiagen) diluted 2000-fold with 0.1% Block Ace was added to the wells to which the single chain antibody was added. Add 50 μl of HRPO-labeled anti-mouse IgG (γ) antibody (manufactured by KPL) 3000 times diluted with 1% Block Ace (prepared with MilliQ water), react at room temperature for 1 hour, and then re-well Was washed. To the well to which the single chain antibody was added, 50 μl of HRPO-labeled anti-mouse IgG (γ) antibody (manufactured by KPL) diluted 3000-fold with 0.1% Block Ace was added and reacted at room temperature for 1 hour. To wells to which commercially available antibodies were added, 50 μl of TMB Microwell Peroxidase Substrate (manufactured by KPL) was added as a substrate and reacted at room temperature for 10 minutes, and then 50 μl of 1M phosphoric acid was added to stop the reaction. The absorbance (measurement wavelength: 450 nm, target wavelength: 650 nm) of each well was measured with a plate reader. The well to which the single chain antibody was added was washed, TMB was added in the same manner as the commercially available antibody, and the absorbance was measured.

The results are shown in Table 1 and FIG. All single-chain antibodies were confirmed to be highly reactive with the immobilized antigen of the MT1-MMP hinge region peptide conjugate as compared with the commercially available antibody.

(5-2) Cell ELISA
Human breast cancer cells, MDA-MB-231, were seeded in a 96-well plate at 1 × 10 ⁴ cells / well and cultured overnight. After washing 3 times with PBS (−) containing 300 μl% FBS, 50 μl of antibody solution (0.1 μg / ml, prepared with PBS (−)) was added to the well and allowed to react at room temperature for 30 minutes. . Furthermore, 50 μl of anti-4 × histidine antibody (Qiagen) diluted 2000-fold with 0.1% Block Ace was added to the wells to which the wells were washed and the single chain antibody was added. 50 μl of HRPO-labeled anti-mouse IgG (γ) antibody (manufactured by KPL) diluted 3000 times with 0.1% Block Ace was added and reacted at room temperature for 1 hour, and then the wells were washed again. To the well to which the single chain antibody was added, 50 μl of HRPO-labeled anti-mouse IgG (γ) antibody (manufactured by KPL) diluted 3000-fold with 0.1% Block Ace was added and reacted at room temperature for 1 hour. To the well to which the commercially available antibody was added, 50 μl of TMB Microwell Peroxidase Substrate (manufactured by KPL) was added as a substrate and reacted at room temperature for 10 minutes, and then 50 μl of 1M phosphoric acid was added to stop the reaction. The absorbance (measurement wavelength: 450 nm, target wavelength: 650 nm) of each well was measured with a plate reader. The well to which the single chain antibody was added was washed, TMB was added in the same manner as the commercially available antibody, and the absorbance was measured.

The results are shown in Table 2 and FIG. 2. All single-chain antibodies were confirmed to be highly reactive against human breast cancer cells and MDA-MB-231 surface antigen as compared with commercially available antibodies.

(5-3) Indirect competition ELISA
50 μl of MT1-MMP hinge region peptide conjugate (0.5 μg / ml, prepared with PBS (−)) was added to each well of a 96-well plate (manufactured by Costar) and allowed to stand at room temperature for 1 hour. The wells were washed 3 times with 300 μl of a washing solution (PBS (−) containing 0.05% Tween 20) and then 250 μl of 1% Block Ace (manufactured by Snow Brand) was added, followed by blocking at room temperature for 2 hours. After washing the wells with a washing solution, MT1-MMP peptide (from 400 μg / ml to 5 times, diluted in 9-step diluted PBS (−)) 25 μl and antibody (0.2 μg / ml, prepared in PBS (−)) 25 μl was added and allowed to react for 30 minutes at room temperature. Further, the wells were washed, and 50 μl of anti-4 × histidine antibody (Qiagen) diluted 2000-fold with 0.1% Block Ace was added to the wells to which the single chain antibody was added. 50 μl of HRPO-labeled anti-mouse IgG (γ) antibody (manufactured by KPL) diluted 3000 times with 0.1% Block Ace was added, reacted at room temperature for 1 hour, and then the wells were washed again. To the well to which the single chain antibody was added, 50 μl of HRPO-labeled anti-mouse IgG (γ) antibody (manufactured by KPL) diluted 3000-fold with 0.1% Block Ace was added and reacted at room temperature for 1 hour. To the well to which the commercially available antibody was added, 50 μl of TMB Microwell Peroxidase Substrate (manufactured by KPL) was added as a substrate and reacted at room temperature for 10 minutes, and then 50 μl of 1M phosphoric acid was added to stop the reaction. The absorbance (measurement wavelength: 450 nm, target wavelength: 650 nm) of each well was measured with a plate reader. The well to which the single chain antibody was added was washed, TMB was added in the same manner as the commercially available antibody, and the absorbance was measured.

The results are shown in Table 3 and FIG. The single-chain antibody MMP98-1 and the single-chain antibody MMPK9I-1 were confirmed to have higher reactivity with the antigen peptide compared to the commercially available antibody, and the single-chain antibody MMPK9E-1 was almost equivalent to the commercially available antibody.

Western Blotting To examine whether the reactivity of the single chain antibody prepared in Example 4 is specific to MT1-MMP, Western blotting was performed using MT1-MMP-expressing cells. The Western blotting method was based on the basic experimental method for proteins and enzymes. Cellulate of human breast cancer cell MDA-MB-231 was prepared with PBS (−) to a protein concentration of 2 mg / ml, mixed with 20 μl of cellulase diluted solution and 20 μl of β-mercaptoethanol-containing SDS buffer, and heated at 96 ° C. for 5 minutes. Then it was converted to SDS. The SDS-modified sample was applied to acrylamide gel (concentrated gel: 4%, separation gel 7.5%) at 10 μl / lane, and electrophoresed at 100 V using Miniprotean II (BIO-RAD). Next, the gel was immersed in a transfer buffer (Tris, glycine, methanol) and conditioned, and then transferred to a membrane at 100 V, 250 mA for 45 minutes using a mini-transblotting module (manufactured by BIO-RAD). After the transfer was completed, the membrane was washed with deionized water by shaking and immersed in 1% Block Ace for blocking. Next, shaking washing was performed 3 times with (0.05% Tween20-containing PBS (-)). Each antibody (0.2 μg / ml, prepared with PBS (−)) was dropped on the membrane and reacted at room temperature for 1 hour. After washing with shaking three times, a membrane added with a single chain antibody was added dropwise with an anti-4 × histidine antibody (Qiagen) diluted 1000-fold with 0.1% Block Ace and allowed to react at room temperature for 1 hour. . HRPO-labeled anti-mouse IgG (γ) antibody diluted 10,000-fold with 0.1% Block Ace was added dropwise to the membrane to which the target commercially available antibody was added. After reacting at room temperature for 1 hour, the membrane was washed and ECL was washed. After adding Plus (manufactured by GE Healthcare) dropwise and reacting at room temperature for 5 minutes, chemiluminescence was detected. The membrane on which the single-chain antibody was dropped was washed, and HRPO-labeled anti-mouse IgG (γ) antibody diluted 10,000 times with 0.1% Block Ace was dropped and reacted at room temperature for 1 hour, then the membrane was washed and ECL After adding Plus (manufactured by GE Healthcare) dropwise and reacting at room temperature for 5 minutes, chemiluminescence was detected.

  The results are shown in FIG. A band was confirmed in the vicinity of a molecular weight of 66 kD in human breast cancer cell MDA-MB-231 for each single-chain antibody and commercially available antibody, indicating that the single-chain antibody was specifically reacting with MT1-MMP. Moreover, it was suggested that the single chain antibody MMP98-1 and the single chain antibody MMPK9E-1 have higher reactivity compared to the commercially available antibody.

Evaluation of binding ability of single-chain antibody of the present invention to mouse macrophage (RAW264.7) by FACS (7-1) Experimental method Each single-chain antibody is concentrated with amicon (3KDa) so as to be 100 μg / 100 μl in 20 mM Na ₂ HPO ₃ , added with 0.77 μl of Alexa Fluor 488 succinimidyl ester (10 mg / ml in DMSO), and incubated at room temperature for 15 minutes. did.
2. After purification on a PD-10 column, absorbance at 280 nm and 488 nm was measured.
3. Concentration was performed to about 100 μl using amicon (3KDa).
4). Antibody concentration was calculated by BCA quantification.
5. A cell solution was prepared so that mouse macrophages (RAW264.7) were 2 × 10 ⁶ cells / sample, and washed with PBS.
6). 100 μl of 10 μg / ml of fluorescently labeled antibody was added and incubated at 37 ° C. for 30 or 60 minutes.
7). The cells were washed, suspended in 1 ml of PBS (−), and measured by FACS.

The amount of fluorescence was measured by measuring absorbance at 488 nm (A488), and the concentration of antibody could be calculated by measuring absorbance at 280 nm (A280), and the amount of fluorescent labeling for each antibody was compared with the value of A488 / A280. Since each antibody had a different labeling amount, it was corrected by dividing by the value of A ₄₈₈ / A ₂₈₀ to determine the antibody binding amount. Since single-chain antibodies are directly fluorescently labeled on the primary antibody, the amount of label in each single-chain antibody is considered to be different, so correction using absorbance was performed.

(7-2) Results Table 4 shows the results. It was found that single-chain MMP98-1 and single-chain antibody MMPK9E-1 have strong binding to cells, and single-chain antibody MMPK9I-1 has slightly weak binding to cells.

Analysis of the binding of the single-chain antibody of the present invention to MT1-MMP by surface plasmon resonance (SPR) (8-1) Experimental method As a peptide to be immobilized, immunization during antibody production or panning by phage display method The peptide-BSA complex used was used. The protein is MT1-MMP 1-350aa. Partial protein (commercially available) was used.
Procedure 1. MT1-MMP protein or peptide was diluted to 10 μg / ml in pH 4.0 sodium acetate buffer and immobilized on a sensor chip.
Each (single chain) antibody 267, 88, 29, 9.6, 3.2 (1250, 412, 136, 45, 14.8) nM was allowed to flow on the sensor chip immobilized in 2.1.
The value obtained by immobilizing and binding 3.1 lanes with BSA alone was regarded as non-specific binding and reduced as background.
4). Analysis was performed from the obtained binding / dissociation curves, and a KD value was calculated.
The MT1-MMP partial peptide used was Peptide 3: PQPRTTSRPSVPDKPKN-Cys (SEQ ID NO: 13)
Met.

(8-2) Experimental results In surface plasmon analysis, the single chain antibody MMP98-1, the single chain antibody MMPK9E-1, and the single chain antibody MMPK9I-1 all showed binding ability to peptide 3. The calculation of the KD value was examined by varying the concentration of these single chain antibodies. The single chain antibody MMP98-1 was also examined by preparing a DTPA (diethylenetriaminepentaacetic acid) form. These single-chain antibodies have KD values on the order of 10 ⁻⁸ M, and were found to bind strongly to MT1-MMP proteins and peptides. The results are shown in Table 5.

Biodistribution of single-chain antibody of the present invention (9-1) Experimental method FM3A mouse breast cancer cells as a tumor-bearing mouse were suspended in PBS (−) (5 × 10 ⁶ cells / 100 μL PBS (−) / mouse), and C3H / He A mouse (female, 5 weeks old) was transplanted subcutaneously into the right lower limb and used after 2 weeks.
Procedure 1. Single chain antibody buffer exchanged in 50 mM NaHCO ₃ to p-SCN-Bn-DTPA 20 eq. In DMF. And incubated for 1 hour under shaking at room temperature.
2. After the reaction, the mixture was centrifuged three times with Amicon (10 KDa) (7500 × g, 20 minutes) to remove unreacted DTPA.
3. The concentrated single chain antibody concentration was measured by BCA quantification.
4). ¹¹¹ InCl ₃ was diluted with Na acetate solution to 0.5 mCi / ml and incubated for 5 minutes under shaking at room temperature.
5. Four solutions were added to the antibody solution to 1 μCi per 1 μg of single chain antibody, and incubated for 1 hour under shaking at room temperature.
6). Purification was performed with a PD-10 column, and only a single chain antibody fraction was collected and concentrated with Amicon (10 KDa).
7. Diluted with saline to give 0.5 μCi / 100 μl saline.
8). ¹¹¹ In-labeled anti-MT1-MMP single chain antibody (0.5 μCi, 0.5 μg / 100 μL sale) was administered from the tail vein of the mouse and sacrificed after 3 and 24 hours. Each organ was removed, and its weight and radioactivity were determined. It was measured. For the single-chain antibody MMP98-1, measurements were performed for 15 minutes, 1 hour, 3 hours, 6 hours, and 24 hours.

(9-2) Experimental Results The biodistribution of the single-chain antibody MMP98-1 considered promising from the FACS and SPR results was examined at time points of 15 minutes, 1 hour, 3 hours, 6 hours, and 24 hours. The results are shown in Table 6. Accumulation in the kidney was very high. Accumulation in the tumor peaked for 3 hours. A good T / B (tumor / blood) ratio was obtained with good escape from blood.

Other single chain antibodies were also evaluated for the 3-hour and 24-hour groups, which are the peak of tumor accumulation. The results are shown in Tables 7 and 8.

In Example 8, single-chain antibodies (MMP98-1, MMPK9E-1, MMPK9I-1) having affinity for peptide 3 showed high tumor accumulation. Table 9 shows the results of examining the tumor / blood (T / B) ratio of these three single-chain antibodies at 3 hours and 24 hours. All single chain antibodies showed a high T / B ratio at 24 hours, and the T / B ratios of the single chain antibody MMPK9E-1 and the single chain antibody MMPK9I-1 were particularly high. In particular, the single-chain antibody MMPK9E-1 is less accumulated in the kidney than others, and thus was considered to have the most suitable properties for imaging among the single-chain antibodies evaluated this time.

Tumor autoradiography and immunostaining of the single-chain antibody of the present invention (10-1) Experimental method FM3A mouse breast cancer cells as a tumor-bearing mouse were suspended in PBS (−) (5 × 10 ⁶ cells / 100 μL PBS (−). / Mouse) and C3H / He mice (female, 5 weeks old) were transplanted subcutaneously into the right lower limb and used after 2 weeks.
Autoradiography procedure ¹¹¹ In-labeled single chain antibody C (15 μCi / 1 μg) was administered to tumor-bearing mice from the tail vein.
2. 24 hours after administration, transcardial reflux was performed with physiological saline, and then PLP fixative (37.5 mM phosphate buffer (pH 7.4) containing 75 mM L (+)-lysine hydrochloride, 4% paraformaldehyde). And fixed at reflux.
3. The excised tumor was made into a tumor section with a cryostat.
4). The tissue section was exposed to an imaging plate for 1 week, and an autoradiogram was obtained using an image analyzer.

Immunostaining procedure Adjacent to the specimen subjected to autoradiography, a section was prepared using a cryostat.
2. After hydrophilization with alcohol, it was reacted with 10% aqueous hydrogen peroxide at room temperature for 30 minutes.
3. TBS-BSA (Tris 0.1M, NaCl 0.15M (pH 7.6), 1% BSA, 0.05% Triton) was reacted at room temperature for 30 minutes.
4. The mixture was reacted with the primary antibody (MT1-MMP F-84 antibody) at 4 ° C. overnight.
5. Reacted with ENVISON polymer reagent (K4000) for 30 minutes.
6). After reacting with DAB for 15 minutes, nuclear staining was performed with hematoxylin.
7). After dehydration and permeation with xylene, it was encapsulated with an encapsulant.

(10-2) Experimental Results FIG. 5 shows the results of tumor autoradiography and immunostaining with the single-chain antibody MMPK9E-1. Both staining patterns were confirmed to match.

Immunoassay of MT1-MMP by flow immunoassay using the antibody according to the present invention JIS K using the antibody IgG and MMPK9I produced by the hybridoma 19B which isolated the gene of the single-chain antibody MMPK9I-1 in Example 1. In 0464, the application to a flow type immunoassay described as an immunoassay for PCB was examined. As an apparatus for performing the flow type immunoassay, for example, a flow type immunosensor DXS-610 (manufactured by Kyoto Electronics Industry Co., Ltd.) can be used. The MT1-MMP hinge region peptide conjugate prepared in (1-1) above is physically adsorbed on Gantz Kasei polystyrene beads, Gantz Pearl GS-100S, and blocked with a Block Ace (Snow Brand) solution. A binding carrier was obtained. Anti-MT1-MMP antibody MMPK9I and Cy ^™ 5-conjugated AffiniPure F (ab ′) ₂ Fragment Goat Anti-Mouse IgG (H + L) (Jackson ImmunoResearch) in 0.1 w / v% BSA-containing PBS- I let you. Subsequently, this solution was reacted with a solution of MT1-MMP hinge region peptide having a final concentration of 0.0 to 100 ng / mL prepared in PBS (-) containing 0.1 w / v% BSA, and then transferred to an antibody-binding carrier. It was applied at a flow rate of 0.75 mL / min. Subsequently, PBS (−) containing 0.1 w / v% BSA was passed at a flow rate of 0.75 mL / min, and after removing non-specific binding to the carrier, the fluorescence intensity was measured. A sufficient amount of fluorescence was obtained in an antibody solution to which no MT1-MMP hinge region peptide was added. Using this as the control (B0) value, the ratio (B / B0) of the fluorescence amount (B) decreased by the addition of the MT1-MMP hinge region peptide to the B0 value was plotted against the MT1-MMP hinge region peptide concentration. Dependent reaction was confirmed. The inhibition curve is shown in FIG. IC50 calculated | required from the inhibition curve was about 4.74 ng / ml, and the CV value of the 3 times measurement was stable at less than 3.5% at the maximum. From the above, it was suggested that this antibody is useful for immunoassay by flow immunoassay.

  The present invention can be used in the fields of cancer diagnostics and research.

The single chain antibody MMP98-producing cells were assigned the independent evaluation corporation product evaluation technology base organization receipt number NITE AP-1008.
The single chain antibody MMPK9E-producing cells were given the independent evaluation corporation product evaluation technology base mechanism receipt number NITE AP-1009.
The single chain antibody MMPK9I-producing cells were given the independent evaluation corporation product evaluation technology base mechanism receipt number NITE AP-1010.

SEQ ID NO: 1 is a nucleotide sequence encoding the heavy chain variable region of single chain antibody MMP98.
SEQ ID NO: 2 is the amino acid sequence of the heavy chain variable region of the single chain antibody MMP98.
SEQ ID NO: 3 is a nucleotide sequence encoding the L chain variable region of single chain antibody MMP98.
SEQ ID NO: 4 is the amino acid sequence of the L chain variable region of the single chain antibody MMP98.
SEQ ID NO: 5 is a nucleotide sequence encoding the heavy chain variable region of single chain antibody MMPK9E.
SEQ ID NO: 6 is the amino acid sequence of the heavy chain variable region of single chain antibody MMPK9E.
SEQ ID NO: 7 is a nucleotide sequence encoding the light chain variable region of single chain antibody MMPK9E.
SEQ ID NO: 8 is the amino acid sequence of the L chain variable region of single chain antibody MMPK9E.
SEQ ID NO: 9 is a nucleotide sequence encoding the heavy chain variable region of single chain antibody MMPK9I.
SEQ ID NO: 10 is the amino acid sequence of the heavy chain variable region of single-chain antibody MMPK9I.
SEQ ID NO: 11 is a nucleotide sequence encoding the light chain variable region of single chain antibody MMPK9I.
SEQ ID NO: 12 is the amino acid sequence of the L chain variable region of single chain antibody MMPK9I.
SEQ ID NO: 13 is the amino acid sequence of a peptide with a cysteine added to the C-terminus of the partial amino acid sequence of mouse MT1-MMP protein.

Claims (11)

  A single-chain antibody having binding activity to MT1-MMP, the antibody comprising a single chain antibody comprising an H-chain variable region having the amino acid sequence represented by SEQ ID NO: 2 and an L-chain variable region having the amino acid sequence represented by SEQ ID NO: 4. Chain antibody.   A single-chain antibody having binding activity to MT1-MMP, comprising a single-chain antibody having an H-chain variable region having the amino acid sequence represented by SEQ ID NO: 6 and an L-chain variable region having the amino acid sequence represented by SEQ ID NO: 8 Chain antibody.   A single-chain antibody having binding activity to MT1-MMP, comprising a single-chain antibody having an H-chain variable region having the amino acid sequence represented by SEQ ID NO: 10 and an L-chain variable region having the amino acid sequence represented by SEQ ID NO: 12 Chain antibody.   The polynucleotide which codes the amino acid sequence of the single chain antibody of any one of Claims 1-3.   A vector comprising the polynucleotide according to claim 4.   A transformant transformed with the vector according to claim 5.   It is a manufacturing method of the single chain antibody of any one of Claims 1-3, Comprising: The transformant transformed with the vector of Claim 5 is culture | cultivated in a suitable culture medium, This transformant Or recovering the single chain antibody from the medium.   A cell deposited with the National Institute of Technology and Evaluation and received the receipt number NITE AP-1008, a cell deposited with the National Institute of Technology and Evaluation and granted the receipt number NITE AP-1009, or independent A cell deposited with an administrative corporation Product Evaluation Technology Infrastructure and given a receipt number NITE AP-1010 is cultured in an appropriate medium, and a single chain antibody is recovered from the medium. A method for producing a single-chain antibody according to any one of the above.   The single-chain antibody according to any one of claims 1 to 3, which is labeled.   A molecular imaging probe agent for cancer comprising the radiolabeled single-chain antibody according to any one of claims 1 to 3.   A diagnostic imaging agent or kit for cancer comprising the molecular imaging probe agent according to claim 10.