WO2015137441A1 - Anticorps anti-protéine myristoylée ou fragment de liaison à l'antigène de celui-ci, trousse de détection d'une protéine myristoylée, médicament, gène et vecteur - Google Patents

Anticorps anti-protéine myristoylée ou fragment de liaison à l'antigène de celui-ci, trousse de détection d'une protéine myristoylée, médicament, gène et vecteur Download PDF

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WO2015137441A1
WO2015137441A1 PCT/JP2015/057287 JP2015057287W WO2015137441A1 WO 2015137441 A1 WO2015137441 A1 WO 2015137441A1 JP 2015057287 W JP2015057287 W JP 2015057287W WO 2015137441 A1 WO2015137441 A1 WO 2015137441A1
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seq
amino acid
acid sequence
sequence shown
cdr
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PCT/JP2015/057287
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Japanese (ja)
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林　宣宏
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国立大学法人東京工業大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to an anti-myristoylated protein antibody or an antigen-binding fragment thereof, a myristoylated protein detection kit, a medicine, a gene, and a vector.
  • Non-Patent Document 1 Since it was discovered that myristic acid was bound to the N-terminus during primary sequencing of cAMP-dependent protein kinase catalytic subunit (see Non-Patent Document 1), more than 100 ⁇ myristoylated proteins have been found so far. It has been reported that several percent of the total protein has undergone myristoylation from the genomic sequence (see Non-Patent Document 2).
  • Myristoylation is a type of post-translational modification in which myristic acid, a saturated fatty acid having 14 carbon atoms, is added to the N-terminus of protein.
  • This modification is N-myristoyl that is universally present in all eukaryotes It is performed simultaneously with translation by a transferase (NMT: N-myristoytransferase, see Non-Patent Document 3).
  • Myristoylation occurs when NMT adds myristic acid via an amide bond to the ⁇ -amino group of glycine exposed by the removal of methionine at the N-terminus by methionine aminopeptidase with the peptide chain bound to ribosome during translation ( (Refer nonpatent literature 4.).
  • Non-Patent Document 5 There is no consensus sequence for myristoylation, but it is essential that the first from the N-terminus is glycine, the second is an uncharged amino acid (except for proline and aromatic amines), and the fifth is a charge. There is a tendency that amino acids having no amino acid, particularly serine, are present, and the sixth is an amino acid other than proline (see Non-Patent Document 5).
  • Nef which is an HIV gene product
  • Src kinase which is an oncogene product
  • myristoylation is involved in the functional expression of proteins (see Non-Patent Documents 6 and 7).
  • N-terminal glycine is mutated so that it cannot be myristoylated cannot bind to the cell membrane, and as a result, cancer metastasis activity may be lost.
  • the present invention has been made in view of the above circumstances, and provides an anti-myristoylated protein antibody or an antigen-binding fragment thereof, a myristoylated protein detection kit, a medicine, a gene, and a vector that can be recognized only by interaction.
  • the present inventors have used an antibody library technology capable of cloning an antibody only by an intermolecular interaction between the antibody and the antigen, to thereby obtain an anti-myristoylated protein antibody or an antigen thereof.
  • the binding fragment was found and the present invention was completed.
  • the present invention provides an anti-myristoylated protein antibody or antigen-binding fragment thereof, myristoylated protein detection kit, drug, gene, and vector having the following characteristics.
  • (a1) CDR-L1 comprising the amino acid sequence represented by SEQ ID NO: 1 or the amino acid sequence represented by SEQ ID NO: 1, wherein one to several amino acids are deleted, substituted or added
  • (B1) CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2 in which one to several amino acids are deleted, substituted or added
  • (C1) CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 3 or the amino acid sequence shown in SEQ ID NO: 3, wherein one to several amino acids are deleted, substituted or added
  • a light chain variable domain comprising: and / or (D1) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 4 or the amino acid sequence shown in SEQ ID NO: 4 in which one to several amino acids are deleted, substituted or added
  • (E1) CDR-H2 comprising the amino acid sequence represented by SEQ ID NO: 5 or the amino acid sequence represented by SEQ ID NO: 5, wherein 1
  • (a2) CDR-L1 comprising the amino acid sequence represented by SEQ ID NO: 8 or the amino acid sequence represented by SEQ ID NO: 8, wherein one to several amino acids are deleted, substituted or added
  • (B2) CDR-L2 comprising the amino acid sequence represented by SEQ ID NO: 9 or the amino acid sequence represented by SEQ ID NO: 9, wherein 1 to several amino acids are deleted, substituted or added
  • (C2) CDR-L3 comprising the amino acid sequence represented by SEQ ID NO: 10 or the amino acid sequence represented by SEQ ID NO: 10, wherein one to several amino acids are deleted, substituted or added
  • a light chain variable domain comprising: and / or (D2) CDR-H1 comprising the amino acid sequence represented by SEQ ID NO: 11 or the amino acid sequence represented by SEQ ID NO: 11, wherein one to several amino acids are deleted, substituted or added
  • (E2) CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence shown in SEQ ID NO: 12 in which one
  • CDR-L1 including the amino acid sequence shown in SEQ ID NO: 8
  • CDR-L2 including the amino acid sequence shown in SEQ ID NO: 9
  • CDR-L3 including the amino acid sequence shown in SEQ ID NO: 10.
  • Light chain variable domain, and / or A heavy chain variable comprising CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 11, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 12, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 13.
  • the anti-myristoylated protein antibody or antigen-binding fragment thereof according to (5) having a domain.
  • CDR-L1 including the amino acid sequence shown in SEQ ID NO: 15, CDR-L2 including the amino acid sequence shown in SEQ ID NO: 16, and CDR-L3 including the amino acid sequence shown in SEQ ID NO: 17
  • the anti-myristoylated protein antibody or antigen-binding fragment thereof according to (9) having a domain.
  • the amino acid sequence shown in SEQ ID NO: 28 the amino acid sequence shown in SEQ ID NO: 29, the amino acid sequence shown in SEQ ID NO: 30, the amino acid sequence shown in SEQ ID NO: 25, and the amino acid shown in SEQ ID NO: 26
  • the anti-myristoylated protein antibody or antigen-binding fragment thereof according to (13) or (14) above which has the sequence and the amino acid sequence shown in SEQ ID NO: 27 in this order.
  • a myristoylated protein detection kit comprising the anti-myristoylated protein antibody or antigen-binding fragment thereof according to any one of (1) to (16).
  • a medicament comprising the anti-myristoylated protein antibody or antigen-binding fragment thereof according to any one of (1) to (16) as an active ingredient.
  • a gene comprising a DNA encoding the anti-myristoylated protein antibody or antigen-binding fragment thereof according to any one of (1) to (16).
  • (21) (g2) DNA comprising the base sequence represented by SEQ ID NO: 23, (H2) DNA encoding a protein consisting of a base sequence in which 1 to several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 23 and having the ability to recognize myristoylated protein, (I2) DNA encoding a protein consisting of a base sequence having 80% or more identity with the base sequence shown in SEQ ID NO: 23 and having the ability to recognize myristoylated protein, or (J2) A protein comprising a base sequence capable of hybridizing under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence represented by SEQ ID NO: 23, and having a myristoylated protein recognition ability A gene characterized by comprising a DNA encoding.
  • (22) (g3) DNA comprising the base sequence represented by SEQ ID NO: 24, (H3) a DNA encoding a protein consisting of a base sequence in which one to several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 24, and having a myristoylated protein recognition ability; (I3) DNA encoding a protein consisting of a base sequence having 80% or more identity with the base sequence shown in SEQ ID NO: 24 and having the ability to recognize myristoylated protein, or (J3) a protein comprising a base sequence capable of hybridizing under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence represented by SEQ ID NO: 24, and having a myristoylated protein recognition ability A gene characterized by comprising a DNA encoding.
  • an anti-myristoylated protein antibody or antigen-binding fragment thereof that can be recognized only by interaction can be provided.
  • the anti-myristoylated protein antibody of the present embodiment or an antigen-binding fragment thereof (hereinafter also referred to as the antibody of the present embodiment) (A1) CDR-L1 comprising the amino acid sequence represented by SEQ ID NO: 1 or the amino acid sequence represented by SEQ ID NO: 1, wherein one to several amino acids are deleted, substituted or added; (B1) CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2 in which one to several amino acids are deleted, substituted or added; (C1) CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 3 or the amino acid sequence shown in SEQ ID NO: 3, wherein one to several amino acids are deleted, substituted or added; A light chain variable domain comprising: and / or (D1) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 4 or the amino acid sequence shown in S
  • the anti-myristoylated protein antibody or antigen-binding fragment thereof of this embodiment specifically recognizes the myristoyl group of the myristoylated protein. Therefore, as long as it retains the myristoyl group-specific recognition ability, one to several amino acids are deleted, substituted or added in the amino acid sequences shown in SEQ ID NOs: 1 to 6 of (a1) to (f1). It may be.
  • the number of amino acids that may be deleted, substituted, or added is preferably 1 to 5, more preferably 1 to 2.
  • the anti-myristoylated protein antibody of the present embodiment or the antigen-binding fragment thereof flexibly combines the heavy chain variable domain and the light chain variable domain, which are the minimum units necessary for recognizing the antigen. It is preferably a single variable domain fragment linked by a simple peptide linker. That is, it is preferably an scFv antibody.
  • the anti-myristoylated protein antibody or antigen-binding fragment thereof of the present embodiment includes an amino acid sequence represented by SEQ ID NO: 4, an amino acid sequence represented by SEQ ID NO: 5, and an amino acid sequence represented by SEQ ID NO: 6.
  • It preferably has the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 2, and the amino acid sequence shown in SEQ ID NO: 3 in this order, and has the amino acid sequence shown in SEQ ID NO: 7. Is more preferable.
  • the method for producing the anti-myristoylated protein antibody or antigen-binding fragment thereof of the present embodiment is not particularly limited, and a commonly used animal immunization method or a method for producing an antibody using a hybridoma may be used.
  • the growth rate of animal cells is slow and time consuming, antigens that are harmful to animals (lethal) cannot be used, only epitopes (antigen binding sites of antibodies) and specific antibodies with limited binding power can be obtained. Since there are problems such as difficulty in obtaining an antibody gene, it is preferable to use a method for producing a monoclonal antibody using a phage antibody library that can clone an antibody gene only by the intermolecular interaction between the antibody and the antigen.
  • An antibody library is obtained by cloning all antibody genes from a single solid animal or a plurality of solid animals, and converting all the variable domains thereof into recombinant forms (scFv antibodies), each with one filamentous phage (for example, This is a technique for producing a set of phages that display all antibodies without omission by fusion expression with the outer protein of M13) (see FIG. 1).
  • the target anti-myristoylated protein antibody is selected by panning with a set of antibodies displayed on phage using such an antibody library. By repeating the panning cycle several times, an anti-myristoylated protein antibody having a high affinity for the myristoyl group as an antigen can be obtained from the antibody library.
  • myristoylated protein that is a target protein (antigen) is immobilized on a plate, reacted with a phage antibody, unbound phage is washed, and the bound phage antibody is eluted to infect Escherichia coli (for example, DH12S). And repeat the operation. Thereby, it is possible to select an antibody only by whether the antibody expressed to the antigen molecule binds or not. Further, as shown in the Examples, myristoylated protein may be immobilized on beads. From the two viewpoints of (1) increase in the absolute number of antigen molecules to be presented and (2) exposure of appropriate antigen sites, bead screening using beads is preferred.
  • Non-immune libraries and immune libraries are prepared by cloning all antibody genes prepared in vivo by the animal from which they are derived. At this time, an animal that has not been immunized is used in the non-immunized library, and an animal that has been immunized with the target antigen is used in the immune library. In the former, any antigen can be used, and various antibodies with different epitopes and binding powers can be obtained. In the latter case, antibodies with high specificity and strong binding force are often obtained.
  • a synthetic library is a library in which CDR region genes having diversity among antibody variable regions are substituted with random amino acids.
  • An antibody library does not necessarily require immunity, and all antibodies possessed by animals are candidates, so that they may exist in large amounts in vivo, such as toxic substances and antibodies of the present invention, and cause an immune reaction. Antibodies against difficult molecules can also be obtained.
  • affinity maturation antibody affinity maturation
  • affinity maturation that produces antibodies with higher affinity by repeated stimulation with antigen is performed, but by introducing mutations and panning repeatedly, This phenomenon can be reproduced in vitro.
  • antibody genes can be obtained together with antibody molecules, one of the merits is that continuous antibody production is possible and antibody molecules can be modified.
  • an antibody expression vector for phage display is the vector shown in FIG. 3 (provided by Fujita Health University, Kurosawa Laboratory).
  • the antibody is first expressed as a single chain antibody (scFv) in a form fused with cp3, which is a part of the phage outer membrane.
  • scFv single chain antibody
  • this vector is recovered from the phage bound to the antigen via the antibody, and the cp3 gene is removed by SalI treatment to obtain an scFv antibody fused with proteinA. Therefore, the obtained scFv antibody can be easily purified on a column on which IgG to which protein A binds is immobilized.
  • the antibody of this embodiment is (A2) the amino acid sequence shown in SEQ ID NO: 8, or CDR-L1 comprising an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 8, (B2) CDR-L2 comprising the amino acid sequence represented by SEQ ID NO: 9 or the amino acid sequence represented by SEQ ID NO: 9, wherein 1 to several amino acids are deleted, substituted or added; (C2) CDR-L3 comprising the amino acid sequence represented by SEQ ID NO: 10 or the amino acid sequence represented by SEQ ID NO: 10, wherein one to several amino acids are deleted, substituted or added; A light chain variable domain comprising: and / or (D2) CDR-H1 comprising the amino acid sequence represented by SEQ ID NO: 11 or the amino acid sequence represented by SEQ ID NO: 11, wherein one to several amino acids are deleted, substituted or added; (E2) CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 12 or
  • a light chain variable domain comprising -L3, and / or A heavy chain variable comprising CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 11, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 12, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 13.
  • it includes a domain.
  • one to several amino acids may be deleted, substituted or added in the amino acid sequences represented by SEQ ID NOs: 8 to 13 of (a2) to (f2).
  • the antibody of the present embodiment is preferably an scFv antibody.
  • the amino acid sequence shown in SEQ ID NO: 11, the amino acid sequence shown in SEQ ID NO: 12, the amino acid sequence shown in SEQ ID NO: 13, and SEQ ID NO: 8 It is more preferable to have the amino acid sequence shown in SEQ ID NO: 9, the amino acid sequence shown in SEQ ID NO: 9, and the amino acid sequence shown in SEQ ID NO: 10 in this order, and it is particularly preferable to have the amino acid sequence shown in SEQ ID NO: 14. .
  • the antibody of this embodiment is (A3) the amino acid sequence shown in SEQ ID NO: 15, or CDR-L1 comprising an amino acid sequence in which 1 to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 15, (B3) an amino acid sequence represented by SEQ ID NO: 16, or CDR-L2 comprising an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 16, (C3) CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 17 or the amino acid sequence shown in SEQ ID NO: 17 in which one to several amino acids are deleted, substituted or added; A light chain variable domain comprising: and / or (D3) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 18 or the amino acid sequence shown in SEQ ID NO: 18 in which one to several amino acids are deleted, substituted or added; (E3) the amino acid sequence shown in SEQ ID NO: 19 or CDR-H2 comprising
  • a light chain variable domain comprising -L3, and / or A heavy chain variable comprising CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 18, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 19, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 20.
  • it includes a domain.
  • one to several amino acids may be deleted, substituted, or added in the amino acid sequences shown in SEQ ID NOs: 15 to 20 in the above (a3) to (f3).
  • the antibody of this embodiment is preferably an scFv antibody, and the amino acid sequence shown in SEQ ID NO: 18, the amino acid sequence shown in SEQ ID NO: 19, the amino acid sequence shown in SEQ ID NO: 20, and SEQ ID NO: 15 It is more preferable to have the amino acid sequence shown in SEQ ID NO: 16, the amino acid sequence shown in SEQ ID NO: 16, and the amino acid sequence shown in SEQ ID NO: 17 in this order, and it is particularly preferable to have the amino acid sequence shown in SEQ ID NO: 21 .
  • the antibody of this embodiment is (A4) the amino acid sequence shown in SEQ ID NO: 25, or CDR-L1 comprising an amino acid sequence in which 1 to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 25; (B4) the amino acid sequence shown in SEQ ID NO: 26, or CDR-L2 comprising an amino acid sequence in which one to several amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 26; (C4) the amino acid sequence shown in SEQ ID NO: 27, or CDR-L3 comprising an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 27; A light chain variable domain comprising: and / or (D4) the amino acid sequence shown in SEQ ID NO: 28, or CDR-H1 comprising an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 28; (E4) the amino acid sequence shown in SEQ ID NO: 25
  • a light chain variable domain comprising -L3, and / or A heavy chain variable comprising CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 18, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 19, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 20.
  • it includes a domain.
  • one to several amino acids may be deleted, substituted or added in the amino acid sequences shown in SEQ ID NOs: 25 to 30 in the above (a4) to (f4).
  • the antibody of the present embodiment is preferably an scFv antibody.
  • the amino acid sequence shown in SEQ ID NO: 28, the amino acid sequence shown in SEQ ID NO: 29, the amino acid sequence shown in SEQ ID NO: 30, and SEQ ID NO: 25 It is more preferable to have the amino acid sequence shown in SEQ ID NO: 26, the amino acid sequence shown in SEQ ID NO: 27, and the amino acid sequence shown in SEQ ID NO: 27 in this order, and it is particularly preferable to have the amino acid sequence shown in SEQ ID NO: 31. .
  • the gene of the present invention comprises DNA encoding the above-described anti-myristoylated protein antibody of the present invention or an antigen-binding fragment thereof.
  • the gene is not particularly limited as long as it includes DNA encoding the heavy chain variable domain and the light chain variable domain of the antibody of the present invention, and the gene including DNA encoding the heavy chain variable domain, and the light chain It may be composed of two genes including a gene encoding a variable domain. From the viewpoint of easy handling, a base sequence having high homology (base sequence identity) with the base sequence of the DNA encoding the scFv antibody described above is preferable.
  • the gene of this embodiment is composed of the DNA encoding the anti-myristoylated protein antibody or antigen-binding fragment thereof of the first embodiment described above, specifically, any one of the following bases (g1) to (j1): DNA consisting of sequences.
  • the number of bases that may be deleted, substituted or added is preferably from 1 to 30, more preferably from 1 to 15, particularly preferably from 1 to 10, and most preferably from 1 to 5 .
  • stringent conditions includes, for example, a method described in Molecular Cloning-A LABORATORY MANUAL 2nd EDITION (Sambrook et al., Cold Spring Harbor Laboratory Press). For example, 5 ⁇ SSC (composition of 20 ⁇ SSC: 3M sodium chloride, 0.3M citric acid solution, pH 7.0), 0.1 wt% N-lauroyl sarcosine, 0.02 wt% SDS, 2 wt% The hybridization can be performed by incubation for several hours to overnight at 55 to 70 ° C. in a hybridization buffer consisting of a blocking reagent for nucleic acid hybridization and 50% formamide.
  • the washing buffer used for washing after incubation is preferably a 0.1 ⁇ SSC solution containing 0.1 wt% SDS, more preferably a 0.1 ⁇ SSC solution containing 0.1 wt% SDS.
  • the gene of this embodiment consists of DNA encoding the anti-myristoylated protein antibody or antigen-binding fragment thereof of the second embodiment described above, and specifically, any one of the following bases (g2) to (j2): DNA consisting of sequences.
  • the gene of this embodiment consists of DNA encoding the anti-myristoylated protein antibody or antigen-binding fragment thereof of the third embodiment described above, specifically, any one of the following bases (g3) to (j3): DNA consisting of sequences.
  • the gene of this embodiment is composed of the DNA encoding the anti-myristoylated protein antibody or antigen-binding fragment thereof of the fourth embodiment described above, specifically, any one of the following bases (g4) to (j4): DNA consisting of sequences.
  • (G4) DNA consisting of the base sequence represented by SEQ ID NO: 32
  • (H4) a DNA encoding a protein consisting of a base sequence in which 1 to several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 32, and having a myristoylated protein recognition ability
  • (I4) DNA encoding a protein consisting of a base sequence having 80% or more identity with the base sequence shown in SEQ ID NO: 32 and having the ability to recognize myristoylated protein
  • (J4) a protein comprising a base sequence capable of hybridizing under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence represented by SEQ ID NO: 32 and having the ability to recognize myristoylated proteins
  • the recombinant vector of the present invention is obtained by inserting the above-described gene of the present invention into an expression vector. Specifically, a DNA encoding an anti-myristoylated protein antibody or an antigen-binding fragment thereof is added to the expression vector. , Inserted through a restriction enzyme site.
  • the DNA inserted into the recombinant vector of the present invention includes DNA consisting of the base sequence shown in SEQ ID NO: 22, DNA consisting of the base sequence shown in SEQ ID NO: 23, DNA consisting of the base sequence shown in SEQ ID NO: 24, Or the DNA which consists of a base sequence shown by sequence number 32 is preferable.
  • the expression vector used in the present invention includes a cell-based vector that allows a host cell to express an anti-myristoylated protein antibody or an antigen-binding fragment thereof; a protein translation system comprising a component having the ability to synthesize proteins extracted from a suitable cell.
  • a cell-free vector that expresses myristoylated protein antibody or antigen-binding fragment thereof can be mentioned.
  • the cell system vector a known expression vector suitable for the host cell is used.
  • Escherichia coli examples include ColE-type plasmids typified by pBR322 derivatives, pACYC-type plasmids having a p15A origin, pSC-type plasmids, and Bac-type F factor-derived mini-F plasmids.
  • Other examples include expression vectors having tryptophan promoters such as trc and tac, lac promoter, T7 promoter, T5 promoter, T3 promoter, SP6 promoter, arabinose-inducible promoter, cold shock promoter, tetracycline-inducible promoter, and the like.
  • Introduction of a recombinant vector into which a gene is incorporated into a host can be performed using a conventionally known method.
  • a competent cell method using microbial cells treated with calcium, an electroporation method, and the like can be mentioned.
  • a phage vector may be used to infect and introduce into a microbial cell.
  • the cell-free vector examples include an expression vector having a T7 promoter and an expression vector having a T3 promoter mentioned in the cell-based vector; a vector for wheat cell-free protein synthesis such as a pEU-based plasmid having an SP6 promoter or a T7 promoter. It is done.
  • RNA synthesis using a cell-free vector In protein synthesis using a cell-free vector, first, using a transcription system, the DNA of an anti-myristoylated protein antibody or antigen-binding fragment thereof is transcribed to synthesize mRNA.
  • transcription systems include conventionally known ones that are transcribed with RNA polymerase.
  • the RNA polymerase include T7 RNA polymerase.
  • a cell-free protein synthesis system which is a translation system, mRNA is translated and a protein is synthesized. This system contains elements necessary for translation, such as ribosomes, translation initiation factors, translation elongation factors, dissociation factors, and aminoacyl-tRNA synthetases.
  • Examples of such protein translation systems include Escherichia coli extract, rabbit reticulocyte extract, and wheat germ extract. Furthermore, there is a reconstituted cell-free protein synthesis system in which the elements necessary for the translation are composed solely of independently purified factors.
  • An anti-myristoylated protein antibody or an antigen-binding fragment thereof can be purified from a protein synthesized using a cell-based vector or a cell-free vector. Examples of the purification method include salting out methods and methods using various chromatographies. If the expression vector is designed to express a tag sequence such as a histidine tag at the N-terminus or C-terminus of the target protein, use an affinity column that uses a substance having an affinity for this tag, such as nickel or cobalt. A purification method is mentioned. In addition, the purity of the purified anti-myristoylated protein antibody or an antigen-binding fragment thereof can be increased by purifying it in combination as appropriate, such as ion exchange chromatography or gel filtration chromatography.
  • the myristoylated protein detection kit of the present invention is not particularly limited as long as it has the above-described anti-myristoylated protein antibody of the present invention or an antigen-binding fragment thereof.
  • the myristoylated protein detection kit of the present invention includes an ELISA kit for colorimetric determination by the sandwich method. Specifically, a 96-well plate coated with the anti-myristoylated protein antibody of the present invention or an antigen-binding fragment thereof, a secondary antibody labeled with an enzyme such as HRP (Horseadish peroxidase), and a chromogenic substrate of the enzyme And including.
  • HRP Hexeadish peroxidase
  • the medicament of the present invention contains an anti-myristoylated protein antibody or an antigen-binding fragment thereof as an active ingredient.
  • protein myristoylation has been reported in proteins involved in intracellular signal transduction systems such as oncogene product Src kinase and HIV gene products such as Nef.
  • the anti-myristoylated protein antibody of the present invention or an antigen-binding fragment thereof functions as a neutralizing antibody
  • the medicament of the present invention containing this as an active ingredient is expected to have a therapeutic effect on various diseases.
  • target diseases for which the therapeutic effect of the medicament of the present invention is expected include human solid cancer.
  • human solid cancer examples include brain cancer, head and neck cancer, esophageal cancer, thyroid cancer, small cell cancer, non-small cell cancer, breast cancer, stomach cancer, gallbladder / bile duct cancer, and liver.
  • Cancer pancreatic cancer, colon cancer, rectal cancer, ovarian cancer, choriocarcinoma cancer, endometrial cancer, cervical cancer, renal pelvis / ureter cancer, bladder cancer, prostate cancer, penis Cancer, testicular cancer, fetal cancer, Wilms cancer, skin cancer, malignant melanoma, neuroblastoma, osteosarcoma, Ewing's tumor, soft tissue sarcoma, etc.
  • neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease; diabetes and the like.
  • Examples of formulation in the medicament of the present invention include those used orally as tablets, capsules, elixirs, and microcapsules with sugar coating as necessary. Or what is used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injection of suspension. Furthermore, a pharmacologically acceptable carrier or medium, specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative And those formulated by mixing in a unit dosage form generally required for pharmaceutical practice, in appropriate combination with agents, binders and the like.
  • Additives that can be mixed into tablets and capsules include, for example, binders such as gelatin, corn starch, gum tragacanth and gum arabic, excipients such as crystalline cellulose, swelling such as corn starch, gelatin and alginic acid Agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red mono oil or cherry.
  • the above material can further contain a liquid carrier such as fats and oils.
  • Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.
  • Aqueous solutions for injection include, for example, isotonic solutions containing physiological saline, glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride.
  • Suitable solubilizers such as Alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 (TM), HCO-50 may be used in combination.
  • oily liquid examples include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent.
  • oily liquid examples include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent.
  • buffer for example, phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, stabilizer, for example, benzyl alcohol, phenol, antioxidant.
  • the prepared injection solution is usually filled into a suitable ampoule.
  • intraarterial injection, intravenous injection, subcutaneous injection, etc., as well as intranasal, transbronchial, intramuscular, percutaneous, or oral administration to patients are performed by methods known to those skilled in the art. sell.
  • the dose varies depending on the weight and age of the patient, the administration method, etc., but those skilled in the art can appropriately select an appropriate dose.
  • the dose and administration method vary depending on the weight, age, symptoms, etc. of the patient, but can be appropriately selected by those skilled in the art.
  • the dose of the medicament of the present invention varies depending on the symptoms, but in the case of oral administration, generally for an adult (with a body weight of 60 kg), about 0.1 to 100 mg per day, preferably about 1.0 to It is believed that 50 mg, more preferably about 1.0 to 20 mg.
  • the single dose When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, and administration method. For example, in the form of an injection, it is usually 1 for an adult (with a body weight of 60 kg). It may be convenient to administer about 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection.
  • One aspect of the present invention also provides the anti-myristoylated protein antibody or antigen-binding fragment thereof for therapy.
  • One aspect of the invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the anti-myristoylated protein antibody or antigen-binding fragment thereof.
  • One aspect of the present invention also provides use of the anti-myristoylated protein antibody or antigen-binding fragment thereof for the manufacture of a therapeutic agent.
  • One aspect of the present invention also provides a therapeutic method comprising administering an effective amount of the anti-myristoylated protein antibody or antigen-binding fragment thereof to a patient in need of treatment.
  • NAP22 neural axial membrane protein 22 kDa: myristoylated protein specifically expressed in the brain
  • E. coli BL21 (DE3) strain having a plasmid (pET14b_NAP22) in which the rat-derived NAP22 gene is inserted into pET14b vector is used.
  • the BL21 (DE3) strain transformed with pET14b_NAP22 was cultured overnight at 37 ° C. on an LB plate (hereinafter referred to as “Amp.”) containing 100 ⁇ g / mL ampicillin (hereinafter referred to as “Amp.”).
  • LBA medium LB medium
  • plasmid extraction 5 mL of LB medium (hereinafter referred to as LBA medium) containing 100 ⁇ g / mL Amp.
  • glycerol stock preparation 5 mL of LB medium (hereinafter referred to as LBA medium) containing 100 ⁇ g / mL Amp.
  • LBA medium LB medium
  • the collected cells before and after induction were suspended in 60 ⁇ L cell lytic B (SIGMA), and the culture supernatant was collected at 12,000 rpm, 10 min, 4 ° C. Subsequently, the supernatant was boiled at 95 ° C. for 15 min, and the resulting precipitate was removed by centrifugation at 12,000 rpm, 15 min, 4 ° C. This was again used as a sample before and after induction, and the expression level was confirmed by SDS-PAGE using 12.5% acrylamide gel. The results are shown in FIG. As shown in FIG. 4, the most highly expressed clone2 was used as an expression strain of NAP22, and mass expression was performed.
  • SIGMA cell lytic B
  • NAP22 Hydrophobic chromatography was used for the purification of NAP22. According to the above procedure, NAP22 was expressed in 200 mL LBA medium, and the cells were collected at 10,000 rpm, 15 min, 4 ° C. After induction of 200 mL of this culture solution, the cells were suspended in 12 mL of TEG solution (10 mM Tris-HCl, 0.2 mM EGTA, pH 7.5). Using BRANSON SONIFIER 250, Duty Cycle: 50%, Output control: power 4, 5 min sonication ⁇ 2.5 min interval was repeated 3 times on ice to disrupt the cells. The supernatant was collected by centrifugation at 12,000 rpm, 30 min, 4 ° C.
  • This soluble fraction was used as a sample before purification, and subjected to FPLC using AKTAPlime (GE Healthcare).
  • AKTAPlime GE Healthcare
  • a HiPrep Phenyl FF (high sub) 16/10 column GE healthcare
  • 5 mL of pre-purification sample was injected into this column equilibrated beforehand with 40% saturated ammonium sulfate TEG solution, and 40% saturated ammonium sulfate TEG solution was allowed to flow at a flow rate of 1 mL / min for 10 minutes for washing.
  • a 0% saturated ammonium sulfate TEG solution was prepared as an elution buffer, and the protein was eluted by gradually decreasing the ammonium sulfate ratio from 40% to 0% at a flow rate of 1 ml / min and 30 min.
  • SDS-PAGE was performed on a 12.5% acrylamide gel for the fraction considered to contain protein from the absorbance value. The results are shown in FIG. In FIG. 5, the band indicated by the arrow indicates NAP22.
  • the fraction of fractions 110 to 120 in which a single band was confirmed was considered to be usable as a protein solution, and the fraction in this section was recovered as purified NAP22.
  • myrNAP22 was constructed by constructing a co-expression strain of NAP22 and N-myristoy transferase (hereinafter abbreviated as NMT). 3) The pBB131 vector in which the yeast N-myristoy transferase cDNA was incorporated was introduced into the NAP22 expression strain that was determined to have the highest expression level by the above expression confirmation.
  • NMT N-myristoy transferase
  • the NAP22 expression strain which was determined to have the highest expression level by confirming the expression, was made competent cells.
  • a glycerol stock of a NAP22 expression strain was generated and cultured overnight at 37 ° C. on an LBA plate.
  • the cells were suspended in 20 mL of an ice-cooled transformation buffer, incubated for 10 min on ice, and then centrifuged at 4 ° C., 3,000 rpm, 10 min, and the supernatant was removed.
  • the cells were suspended again with 5 mL transformation buffer, DMSO with a final concentration of 7% was added, and the mixture was allowed to stand for 10 min on ice. Thereafter, 100 ⁇ L each was dispensed, snap-frozen with liquid nitrogen, and stored at ⁇ 80 ° C.
  • the transformation buffer was prepared by adjusting the composition of Table 2 to pH 6.7 to 6.8 with KOH, adding 5.45 g of MnCl 2 .4H 2 O, making up to 500 mL with milliQ, and sterilizing by filter.
  • a pBB131 vector (pBB131_yNMT) in which yeast N-myristol transfer cDNA was incorporated was introduced into this competent cell by the heat shock method.
  • 1 ⁇ L of pBB131_yNMT and 20 ⁇ L of NAP22 expression strain competent cell were mixed. The mixture was allowed to stand for 30 min on ice, then incubated at 42 ° C. for 45 sec, and immediately left for 2 min on ice. Four times the amount of LB medium was added and mixed, followed by shaking culture at 37 ° C. for 1 hour.
  • LBAK plate LBA plate containing 50 ⁇ g / mL kanamycin
  • 20 ⁇ L of non-transformed NAP22-expressing strain competent cells were diluted with 80 ⁇ L of LB medium, then each 50 ⁇ L was plated on LBAK and LBA plates, and cultured at 37 ° C. overnight.
  • Five grown colonies were picked up and cultured overnight at 37 ° C. in 2 mL of LBA medium containing 50 ⁇ g / mL kanamycin (hereinafter referred to as “LBAK medium”). From each preculture, 1) plasmid extraction, 2) glycerol stock preparation, and 3) expression confirmation were performed.
  • BRANSON SONIFIER 250 Duty Cycle: 50%, Output control: power3, 30 min sonication ⁇ 1 min interval was repeated twice. This was centrifuged at 12,000 rpm, 10 min, 4 ° C., and the culture supernatant was collected. This was used as a sample after induction. Pre-induction cells were collected from 2 mL of the pre-induction culture by centrifugation at 12,000 rpm, 5 min, 4 ° C. After adding 240 ⁇ L cell lytic B and disrupting the cells by pipetting, the culture supernatant was collected at 2,000 rpm, 5 min, 4 ° C. to obtain a sample before induction.
  • myrNAP22 was expressed in large quantities on a 200 mL scale.
  • a glycerol stock of myrNAP22 expression strain was raised, inoculated into 3 mL of LBAK medium, and cultured at 37 ° C. overnight. This preculture was added to 200 mL of LBAK and cultured at 30 ° C. for 4 hours. Expression was induced with a final concentration of 1 mM IPTG, and then the culture was continued for 8 hours. After centrifuging at 12,000 rpm for 15 min at 4 ° C.
  • RNA extraction preparation of a rabbit immune library [Total RNA extraction] Since the myristoyl group has a low molecular weight and is contained in the living body, even if the myristoyl group is immunized as it is, it does not exhibit antigenicity. Therefore, a myristoyl group was conjugated to KLH (Keyhole limpet hemocyanin, 450 kDa-13000 kDa). The myristoyl group does not bind unless the N-terminal of the protein to be bound is glycine, and KLH does not bind unless the molecule to be bound is a peptide or protein. Combined. The linker peptide was designed to have a sequence that does not exist naturally among myristoylated proteins.
  • myristol-GKRKC-KLH (abbreviated as myrKLH)
  • RNAlater RNA Stabilization Reagent QIAGEN
  • concentration of total RNA extracted by absorbance was estimated.
  • a solution having the composition shown in Table 5 was mixed and incubated at 65 ° C. for 5 minutes and on ice for 2 minutes.
  • the solution having the composition shown in Table 4 was further mixed with the solution having the composition shown in Table 5 and incubated at 42 ° C. for 2 minutes.
  • 6 ⁇ L of reverse transcriptase was further mixed with this solution, and incubated at 42 ° C. for 50 minutes, 70 ° C. for 15 minutes, and on ice for 5 minutes.
  • Ribonuclease H (manufactured by Invitrogen) was added to this solution and incubated at 37 ° C. for 20 minutes.
  • the synthesized cDNA solution was purified using QIAquick purification Kit (manufactured by QIAGEN).
  • each antibody gene was amplified by PCR using primers specific to each antibody gene. At that time, the optimum number of cycles was estimated.
  • Each specific primer is a mixture of sequences common to the V gene group (V primer) and the J gene group (J primer) at both ends of the variable region.
  • the number of cycles in amplification of each gene was distributed from 24 cycles to 36 cycles, PCR reaction was performed, and amplification products were confirmed by agarose electrophoresis.
  • the composition of the reaction solution in the PCR reaction is shown in Table 6, and the reaction conditions for the PCR reaction are shown in Table 7.
  • Each PCR reaction solution was treated with phenol / chloroform and precipitated with ethanol, and then purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN). Each reaction solution was mixed with 1/5 equivalent of 6 ⁇ Loading Dye (TaKaRa) and electrophoresed on a 2% agarose gel to confirm that the target band was present.
  • the purified antibody gene light chain (k chain, ⁇ chain) and the antibody display vector for phage display shown in FIG. 3 were treated with restriction enzymes with NcoI and AscI. 10 ⁇ L of 10 ⁇ buffer, 1.2 ⁇ L each of NcoI and AscI were added to 4 ⁇ g of each antibody gene, and incubated at 37 ° C. for 2 hours. 18 ⁇ L of 10 ⁇ buffer and 9 ⁇ L each of NcoI and AscI were added to 30 ⁇ g of vector, and incubated at 37 ° C. for 2 hours.
  • the antibody gene after the restriction enzyme treatment is purified using QIA PCR purification Kit (manufactured by QIAGEN), and the vector after the restriction enzyme treatment is purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN) and agarose electrophoresis is performed. Confirmed cutting.
  • the restriction enzyme-treated sample was ligated at 15 ° C. for 3 hours with the following reaction composition.
  • the k chain and ⁇ chain ligation products were confirmed by agarose electrophoresis, and ethanol precipitation was performed.
  • 5 ⁇ L of each sample and 100 ⁇ L of DH12S were mixed, transformed by electroporation (25 ⁇ F, 2.5 kV, 200 ⁇ ), cultured in 2 ⁇ YT medium 10 mL at 37 ° C. for 1 hour, and a part (100 ⁇ L, 10 ⁇ L). 1 ⁇ L) was plated on LBGA plates and incubated overnight at 37 ° C. Further, 190 mL of 2 ⁇ YTGA was added to the 10 mL culture solution and cultured overnight at 30 ° C. The composition of the medium used is shown below.
  • 2 ⁇ YT triptone 16 g, yeast extract 5 g, NaCl 10 g (1 L) 2 ⁇ YTGA: 2 ⁇ YT + Amp final 20 ⁇ g / mL, Glucose final 1%
  • LB plate triptone 10g, yeast extract 5g, NaCl 10g, agar 15g (1L) LBGA plate: LB plate + Amp final 20 ⁇ g / mL, Glucose final 1%
  • Plasmids were extracted from 200 mL of each culture solution using Maxi prep (manufactured by QIAGEN), and the target bands were confirmed by agarose electrophoresis, which were designated as rabbit immune k chain and ⁇ chain library plasmids, respectively.
  • the repertoire was estimated from the number of colonies on the plate, 10 colonies were picked up, and the base sequence of plasmid DNA extracted from the selected colonies was analyzed.
  • the composition of the solution used in the sequence reaction is shown in Table 11, and the conditions for the sequence reaction are shown in Table 12.
  • the purified heavy chain gene (H chain) and light chain (k chain, ⁇ chain) library was treated with SfiI at 50 ° C. for 2 hours. Specifically, 10 ⁇ L of 10 ⁇ buffer and 6 ⁇ L of SfiI were added to 4 ⁇ g heavy chain gene, incubated at 50 ° C. for 2 hours, and cleavage was confirmed by agarose electrophoresis. In addition, 20 ⁇ L of 10 ⁇ buffer and 10 ⁇ L of SfiI were added to 5 ⁇ g of a light chain library in which the light chain k chain gene and the light chain ⁇ chain gene were mixed at 9: 1, and incubated at 50 ° C.
  • the heavy chain ligation product was confirmed by electrophoresis and ethanol precipitation was performed.
  • 19 ⁇ L of the sample and 100 ⁇ L of DH12S were mixed, transformed by electroporation, added to 1000 mL of 2 ⁇ YT medium, cultured at 37 ° C. for 1 hour, and a part (100 ⁇ L, 10 ⁇ L, 1 ⁇ L) was plated on an LBGA plate. Incubated overnight at 37 ° C. Further, Glucosecofinal 1% and Ampicileine final 20 ⁇ g / ml were added to the culture solution, followed by incubation at 30 ° C. overnight.
  • the number of repertoires was estimated from the number of colonies on the plate, 10 colonies were picked up, and the base sequence of plasmid DNA extracted from the selected colonies was analyzed.
  • the composition of the solution used for the sequence reaction is the same as in Table 11, and the sequence reaction conditions are the same as in Table 12.
  • LBAK plate overnight at 1/10 ⁇ L, 1/100 ⁇ L, and 1/1000 ⁇ L, respectively.
  • the bacterial cells were removed from the 200 mL liquid medium by centrifugation at 10,000 rpm, 4 ° C., and 10 min, and the culture supernatant containing the phage antibody was recovered.
  • 4 mL of 20% polyethylene glycol (hereinafter abbreviated as “PEG”) was added, vigorously mixed by vortexing, and then allowed to stand for 10 min for Room Temperature (hereinafter abbreviated as “RT”).
  • PEG polyethylene glycol
  • 50 ⁇ L of each post-infection bacterial solution was plated on an LBA plate and cultured at 37 ° C. overnight.
  • the composition of the medium and reagents used is shown below.
  • a glycerol stock was prepared using 140 ⁇ L of the above preculture. The manufacturing method was the same as described above.
  • Antigen beads using myrNAP22 prepared for screening were prepared. MyrNAP22 was biotinylated for immobilization on beads via streptavidin.
  • Biotinylation of myrNAP22 Biotinylation was performed using Biotin Labeling kit-NH 2 (DOJINDO). Based on the result of SDS-PAGE, the concentration of myrNAP22 was estimated, and a sample solution containing about 50 ⁇ g of myrNAP22 was mixed with 200 ⁇ L of 1/10 ⁇ WS buffer diluted with PBS. Using Amicon Ultra4, 10,000 MWCO previously moistened with pure water, centrifugation was performed at 5,000 g, RT, 15 min to remove and concentrate impurities. After discarding the filtrate and washing again in the same manner, about 20 ⁇ L of the sample was collected and transferred to a 1.5 mL Eppendorf tube.
  • DOJINDO Biotin Labeling kit-NH 2
  • Bio-myrNAP22 Biotinylated myrNAP22
  • phage antibody 500 ⁇ L of the above culture solution was transferred to 5 mL 2 ⁇ YT medium, and 50 ⁇ L of M13KO7 helper phage was added thereto and infected at 37 ° C. for 1 hour. Thereafter, 2 ⁇ YTA was added to a total volume of 40 mL, and a final concentration of 50 ⁇ M kanamycin was added, followed by shaking culture at 30 ° C. overnight. After completion of the cultivation, the supernatant was collected by centrifugation at 10,000 rpm, 4 ° C., and 15 minutes. 5 mL of 20% PEG was added and mixed vigorously with vortex.
  • Monoclonal antibodies were prepared from the plate culture at each screening. The colonies grown in the plate culture at each screening stage were picked up, and the expression of each cp3 form monoclonal antibody and the determination of the amino acid sequence of a part of the antibody genes were attempted.
  • [Cp3 form expression] A portion of the phage-infected E. coli was plated. Colonies were picked up from each plate, and each was cultured in 2 mL of 2 ⁇ YTA medium at 37 ° C. overnight. 50 ⁇ L of each preculture was subcultured to 1 mL of 2 ⁇ YTA medium (hereinafter abbreviated as 2 ⁇ YTAI) containing IPTG having a final concentration of 1 mM, and further cultured at 30 ° C. overnight. In addition, 70 ⁇ L of each preculture solution was mixed with 30 ⁇ L of 50% glycerol to obtain each monoclonal antibody glycerol stock.
  • 2 ⁇ YTAI 2 ⁇ YTA medium
  • the supernatant was collected by centrifugation at 12,000 rpm, 4 ° C., 5 min, and used as each cp3 form monoclonal antibody solution. Subsequently, concentration by ammonium sulfate precipitation was performed. An equal amount of saturated ammonium sulfate was added to the sample, vigorously mixed using vortex, and then incubated at RT for 5 min. Centrifugation was performed at 12,000 rpm, 4 ° C., 7 min, and the resulting precipitate was suspended in 0.05% NaN 3 in PBS of 1/10 volume of the sample. The mixture was centrifuged again at 12,000 rpm, 4 ° C., 7 min to remove insoluble matters, and this was used as the final cp3 form monoclonal antibody solution.
  • the plate was washed 3 times with PBS, and 100 ⁇ L of 1/2000 ⁇ ⁇ -cp3 antibody in PBST0.05 was added as a primary antibody. After reacting for 1 hour with shaking at RT, the antibody solution was discarded and washed 3 times with PBS. Subsequently, 100 ⁇ L of 1/4000 ⁇ ⁇ -IgG (H + L chain) (Rabbit) pAb-HRP (MBL) in PBST0.05 was added as a secondary antibody and allowed to react for 1 hour with shaking at RT. It was. After discarding the antibody solution and washing with PBS three times, a substrate solution using an OPD tablet was prepared for detection, and 200 ⁇ L was added to each well.
  • the concentration of the plasmid was estimated based on the results of agarose electrophoresis.
  • a PCR reaction solution was prepared with the composition shown in Table 16, and PCR was performed under the reaction conditions shown in Table 17 to amplify the antibody gene. After the reaction, 5 ⁇ L of 125 mM EDTA and 60 ⁇ L of 100% EtOH were added and tapped. The mixture was incubated at -20 ° C for 20 min and centrifuged at 12,000 rpm for 20 min at 4 ° C to remove the supernatant. 60 ⁇ L of 70% EtOH was added, centrifuged at 12,000 rpm, 10 min, 4 ° C., and the supernatant was removed again. The lid of the Eppendorf tube was opened and dried at room temperature for 5 minutes, and 20 ⁇ L of formamide was added and pipetted. Heating block was performed at 95 ° C. for 3 min and analyzed.
  • the SPR method was used to quantify the binding force of the antibody to the antigen.
  • BIAcore-X GE Healthcare
  • NAP22 and myrNAP22 purified as described above were used as ligands.
  • the concentration was calculated using SDS-PAGE and ultraviolet absorption method for NAP22, and using bradford method for myrNAP22 in addition to SDS-PAGE.
  • the unpurified cp3 form monoclonal antibody of the above three clones having a high color intensity ratio (myrNAP / NAP) was used.
  • the concentration of cp3 form was estimated from the results of SYPRO Ruby staining by SDS-PAGE using 12.5% acrylamide gel.
  • a protein was expressed in E. coli transformed with a vector (pTZ19R_original) before cloning of the antibody gene, and the lysate was used.
  • CM5 to be bound by the amino coupling method was used as a sensor chip for binding the ligand.
  • HBS 10 mM HEPES 150 mM NaCl pH 7.5
  • the optimum pH of the ligand dilution buffer was examined. 10 mM sodium acetate pH 4.0, 4.5, 5.0
  • the ligand solution was diluted with each immobilization buffer and flowed at 10 ⁇ L / min. The buffer with the largest amount of immobilization was set to the optimum pH.
  • TBS-T0.05 0.05% Tween20 in TBS
  • Skim-milk in TBS-T 1 hour at room temperature.
  • the above three clones diluted 5-fold with 5% skim milk in TBS-T0.05 were reacted as primary antibodies.
  • 1/2000 ⁇ ⁇ -cp3 antibody in TBS-T0.05 capable of recognizing cp3 of a monoclonal antibody was used.
  • this secondary antibody is a rabbit-derived IgG type, it can be detected by using 1/4000 ⁇ ⁇ -IgG (H + L chain) (Rabbit) pAb-HRP (MBL) in TBS-T0.05 as the tertiary antibody. Met.
  • Western blotting using 1/100 ⁇ ⁇ -NAP22 (M-65): sc-66995 (SANTA CRUZ BIOTECHNOLOGY) in TBS-T0.05 was also performed.
  • 1/4000 ⁇ ⁇ -IgG (H + L chain) (Rabbit) pAb-HRP (MBL) in TBS-T0.05 was used as the secondary antibody.
  • the clone 119 was noted as a clone having a high color intensity ratio (myrNAP / NAP).
  • the results of ELISA for clone 119 are shown in Table 21.
  • the supernatant was recovered, and the precipitate obtained by ammonium sulfate precipitation was dissolved in 40 mL of PBS to obtain an antibody solution.
  • 50 ⁇ L of IgG Sepharose 6 Fast Flow (manufactured by GE Healthcare) was filled in the column, and the antibody solution (40 mL) obtained previously was allowed to react through the column to obtain antibody beads.
  • the antibody beads were eluted with SDS elution buffer (2% SDS, 100 mM Tris-HCl pH 7.5, 10% glycerol, 0.5 mM EDTA, 100 mM DTT, BPB) to check the expression of bound clone 119 and to the beads.
  • lane 1 shows a clone 119 bound to the beads
  • lane 2 shows a control clone (an antibody clone randomly cloned from the library) bound to the beads used as a control.
  • a band was confirmed at a position of 40 kDa, and the bands of the two types of antibodies had the same intensity, so it was confirmed that equal amounts of clone 119 and control clone could be bound to each bead.
  • the signal intensity of lane 1 was 61% of the signal intensity of lane 3.
  • the signal intensity of Lane 2 was 90% of the signal intensity of Lane 3. That is, it was confirmed that about 30% to 40% of myrNAP22 antigen can be immunoprecipitated using clone 119.
  • the reason why a variety of antibodies with high specificity were obtained in this example is that (1) increase in the absolute number of antigen molecules to be presented by using bead screening (2) exposure of appropriate antigen sites There are two points.
  • the beads used in this example can bind to 1,000 pmol of molecule per mg. Considering that the amount of protein that can be immobilized on one well in a plate screening is about 10 pmol, the number of antigen molecules to be presented has a difference of several tens of times or more.
  • plate screening has a major problem that a portion where the antibody cannot react at all is formed due to adsorption to the plate.
  • the myristoyl group used as an antigen in this example is so hydrophobic that it can anchor the protein to the membrane.
  • Anti-myristoylated protein antibodies or antigen-binding fragments thereof, myristoylated protein detection kits, drugs, genes, and vectors that can be recognized only by interaction can be provided.

Abstract

L'invention concerne un anticorps anti-protéine myristoylée, ou un fragment de liaison à l'antigène de celui-ci, caractérisé en ce qu'il a un domaine variable de la chaîne légère consistant en CDR-L1 comprenant la séquence d'acides aminés représentée par Séquence N° 1, CDR-L2 comprenant la séquence d'acides aminés représentée par Séquence N° 2, et CDR-L3 comprenant la séquence d'acides aminés représentée par Séquence N° 3, et/ou un domaine variable de la chaîne lourde consistant en CDR-H1 comprenant la séquence d'acides aminés représentée par Séquence N° 4, CDR-H2 comprenant la séquence d'acides aminés représentée par Séquence N° 5, et CDR-H3 comprenant la séquence d'acides aminés représentée par Séquence N° 6.
PCT/JP2015/057287 2014-03-14 2015-03-12 Anticorps anti-protéine myristoylée ou fragment de liaison à l'antigène de celui-ci, trousse de détection d'une protéine myristoylée, médicament, gène et vecteur WO2015137441A1 (fr)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
WO2018025886A1 (fr) * 2016-08-02 2018-02-08 Ｓｐｉｂｅｒ株式会社 Procédé de production d'une protéine recombinante

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