WO2017138471A1 - Anti-dog cd70 monoclonal antibody - Google Patents

Anti-dog cd70 monoclonal antibody Download PDF

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Publication number
WO2017138471A1
WO2017138471A1 PCT/JP2017/004147 JP2017004147W WO2017138471A1 WO 2017138471 A1 WO2017138471 A1 WO 2017138471A1 JP 2017004147 W JP2017004147 W JP 2017004147W WO 2017138471 A1 WO2017138471 A1 WO 2017138471A1
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canine
variable region
chain variable
monoclonal antibody
seq
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PCT/JP2017/004147
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French (fr)
Japanese (ja)
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拓也 水野
友紀 長谷川
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日本全薬工業株式会社
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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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to an anti-canine CD70 antibody used for treatment and diagnosis of canine lymphoma and a method for using the same.
  • the present invention aims to identify a CD70 molecule as a new diagnostic and disease monitoring marker for lymphoma in dogs, and to provide a therapeutic method and a diagnostic method using the same.
  • lymphoma which is a relatively large blood system tumor in dogs.
  • the present inventors have identified molecules that are specifically expressed in canine lymphomas and found that CD70, a member of the tumor necrosis factor (TNF) superfamily, is specifically expressed.
  • CD70 a member of the tumor necrosis factor (TNF) superfamily
  • the present inventors have prepared a monoclonal antibody against CD70, and by using the monoclonal antibody, by measuring the expression of CD70 in canine lymphocytes, it is possible to diagnose whether or not a dog suffers from lymphoma. I found it.
  • an anti-CD70 antibody can be used for the treatment of canine lymphoma and have completed the present invention.
  • an anti-canine CD70 monoclonal antibody or canine CD70 that binds to canine CD70 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 4 Its functional fragment that binds to.
  • a heavy chain variable region comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 and having binding activity to canine CD70, and an amino acid sequence represented by SEQ ID NO: 4
  • An anti-canine CD70 monoclonal antibody that binds to canine CD70 or a functional fragment thereof that binds to canine CD70, comprising a light chain variable region having an amino acid sequence having 90% or more sequence identity and binding activity to canine CD70 .
  • Binds to canine CD70 which includes a heavy chain variable region encoded by DNA consisting of the base sequence represented by SEQ ID NO: 1 and a light chain variable region encoded by DNA consisting of the base sequence represented by SEQ ID NO: 3.
  • An anti-canine CD70 monoclonal antibody or a functional fragment thereof that binds to canine CD70 [4] A heavy chain variable region having binding activity to canine CD70 encoded by a base sequence having 90% or more sequence identity with the base sequence represented by SEQ ID NO: 1, and a base sequence represented by SEQ ID NO: 3 A canine CD70 monoclonal antibody that binds to canine CD70 or a functional fragment thereof that binds to canine CD70, comprising a light chain variable region having binding activity to canine CD70 encoded by a nucleotide sequence having 90% or more sequence identity.
  • the anti-canine CD70 monoclonal antibody that binds to the canine CD70 of any one of [1] to [4], wherein the heavy chain constant region and the light chain constant region are constant regions of a canine IgG antibody, or that that binds to a canine CD70 Functional fragment.
  • the functional fragment is selected from the group consisting of Fab, Fab ′, F (ab ′) 2 , disulfide bond Fv (dsFv), dimerization V region (diabody), single chain Fv (scFv) and CDR
  • Fab fragment antigen
  • Fab ′ fragment antigen binding domain
  • F (ab ′) 2 fragment antigen binding domain
  • dsFv disulfide bond Fv
  • dimerization V region dimerization V region
  • scFv single chain Fv
  • CDR An anti-canine CD70 monoclonal antibody that binds to canine CD70 according to any one of [1] to [5], or a functional fragment that binds to canine CD70.
  • a polypeptide that is a light chain variable region of an anti-canine CD70 monoclonal antibody comprising the amino acid sequence represented by SEQ ID NO: 4.
  • a polynucleotide encoding the heavy chain variable region of an anti-canine CD70 monoclonal antibody comprising the DNA sequence represented by SEQ ID NO: 1.
  • a polynucleotide encoding the light chain variable region of an anti-canine CD70 monoclonal antibody comprising the DNA sequence represented by SEQ ID NO: 3.
  • a cell comprising the vector according to [11].
  • a DNA encoding the heavy chain variable region of the anti-canine CD70 monoclonal antibody consisting of the DNA sequence represented by SEQ ID NO: 1 is ligated with the DNA encoding the heavy chain constant region of the antibody, and represented by SEQ ID NO: 3.
  • a method for producing an anti-canine CD70 monoclonal antibody comprising transforming a host cell or host animal and producing an antibody by the host cell or host animal.
  • CD70 expressed in canine peripheral blood lymphocytes is measured.
  • a method for diagnosing canine lymphoma, wherein CD70 is expressed in lymphocytes and it is judged that the patient is affected by lymphoma is performed using a flow cytometer.
  • a detection reagent for canine lymphoma comprising the anti-canine CD70 monoclonal antibody that binds to canine CD70 according to any one of [1] to [6] or a functional fragment thereof that binds to canine CD70.
  • the canine lymphoma can be diagnosed by the anti-canine CD70 antibody of the present invention.
  • the anti-canine CD70 antibody of the present invention is also effective for the treatment of canine lymphoma.
  • CD70 in canine lymphoma. It is a figure which shows the base sequence of the gene which codes canine CD70 and CD27. It is a figure which shows the binding property of anti-canine CD70 monoclonal antibody and NRK / cCD70. It is a figure which shows the binding property of cCD27-hIg and NRK / cCD70. It is a figure which shows that an anti-canine CD70 monoclonal antibody does not have the binding inhibitory effect of canine CD70 and canine CD27. It is a figure which shows the result of the expression analysis of CD70 in 9 canine lymphoma tumor cell lines.
  • the present invention is an anti-canine CD70 monoclonal antibody that specifically binds to canine CD70 including the heavy chain variable region and light chain ( ⁇ chain) variable region of monoclonal antibody 4F2-12 that specifically binds to canine CD70.
  • the present invention also includes polypeptides that are the heavy chain variable region or light chain ( ⁇ chain) variable region of the 4F2-12 antibody, and polynucleotides encoding these variable regions.
  • the antibody of the present invention includes a functional fragment of an antibody or a modified product thereof.
  • a functional fragment of an antibody is a fragment of an antibody that can specifically bind to an antigen.
  • Functional fragments include Fab, F (ab ′) 2, Fv, a single chain Fv in which Fab / c having one Fc and a complete Fc, H chain or L chain Fv are linked by an appropriate linker ( scFv) or CDR.
  • Polynucleotide includes both DNA and RNA.
  • the base sequence of the DNA encoding the heavy chain variable region of the 4F2-12 antibody consists of the base sequence represented by SEQ ID NO: 1, and the amino acid sequence of the heavy chain variable region consists of the amino acid sequence represented by SEQ ID NO: 2. Further, the base sequence of the DNA encoding the light chain variable region of the 4F2-12 antibody is composed of the base sequence represented by SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is composed of the amino acid sequence represented by SEQ ID NO: 4. .
  • the heavy chain variable region includes not only the heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 2, but also one or several, for example, 1 to 10, preferably 1 to 5, more preferably in the amino acid sequence.
  • the light chain variable region includes not only the light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 4, but also 1 or several, for example, 1 to 10, preferably 1 to 5, more preferably 1 in the amino acid sequence.
  • a light chain variable basin comprising a protein having an amino acid sequence in which two, more preferably one amino acid has been deleted, substituted, or added, and having the activity of the light chain variable region of an antibody, ie, the binding activity to canine CD70.
  • an amino acid sequence of SEQ ID NO: 2 or 4 and BLAST Basic Local Alignment Search Tool at the (E.g., National Center for Biological Information (Basic National Alignment Search Tool of the National Center for Biological Information)) (for example, default or default parameters), at least 85%, preferably 90% or more, More preferred are those having a sequence identity of 95% or more, particularly preferably 97% or more.
  • a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2 or 4 is substantially the same as a protein having the amino acid sequence of SEQ ID NO: 2 or 4. is there.
  • the base sequence consisting of the sequence represented by SEQ ID NO: 1 or 3 above and BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information), etc.)
  • sequence identity is at least 85% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 97% or more when calculated using default or default parameters.
  • DNA that can hybridize under stringent conditions with a DNA consisting of a sequence complementary to the DNA consisting of the sequence represented by SEQ ID NO: 1 or 3, wherein the heavy chain variable region or light chain of the antibody.
  • DNA encoding a protein having the activity of the chain variable region, that is, the binding activity to canine CD70 is also included in the DNA encoding the heavy chain variable region or the light chain variable region of the present invention. That is, after hybridization at 68 ° C.
  • a 0.1 to 2 fold concentration SSC solution (1 fold concentration SSC is 150 mM NaCl, 15 mM) This is a condition that can be identified by washing at 68 ° C. using sodium citrate.
  • hybridization buffer [50% formamide, 4 ⁇ SSC, 50 mM HEPES (pH 7.0), 10 ⁇ Denhardt , s) solution, 100 ⁇ g / ml salmon sperm DNA]
  • the heavy chain variable region or the light chain variable region of the anti-canine CD70 monoclonal antibody that specifically binds to the canine CD70 of the present invention can be obtained by a known method using canine CD70 as an immunogen, using mouse or rat cells. It can be obtained by obtaining an anti-canine CD70 antibody-producing hybridoma, isolating a DNA encoding a heavy chain variable region or a DNA encoding a light chain variable region from the hybridoma, and expressing the DNA.
  • the anti-canine CD70 monoclonal antibody that specifically binds to canine CD70 containing the heavy chain variable region and the light chain variable region includes the heavy chain variable region and the heavy chain constant region, and the light chain variable region and the light chain. It consists of a chain constant region.
  • the heavy chain constant region is composed of three domains C H 1, C H 2 and C H 3.
  • the heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is most preferably an IgG1 or IgG4 constant region.
  • the light chain constant region is comprised of one domain C L.
  • the light chain constant region is a kappa or lambda constant region.
  • the antibody-encoding DNA is obtained by linking the DNA encoding the heavy chain variable region and the DNA encoding the heavy chain constant region, and further linking the DNA encoding the light chain variable region and the DNA encoding the light chain constant region.
  • the species derived from the variable region may be different from the species derived from the constant region
  • the anti-canine CD70 antibody of the present invention includes a chimeric antibody in which the species derived from the variable region and the species derived from the constant region are different.
  • the heavy chain variable region encoded by the DNA represented by SEQ ID NO: 1 and the light chain variable region encoded by the DNA represented by SEQ ID NO: 3 are derived from rat.
  • a chimeric antibody comprising a rat-derived variable region and a dog-derived constant region can be prepared by ligation with a region-encoding DNA.
  • DNA encoding a heavy chain variable region or DNA encoding a light chain variable region is inserted into an expression vector, and a host cell for expression is transformed using the vector. It can be transformed and cultivated by culturing host cells.
  • the anti-canine CD70 monoclonal antibody of the present invention inserts the DNA encoding the heavy chain and the DNA encoding the light chain into an expression vector, transforms a host cell using the vector, and cultures the host cell. Can be produced. At this time, the DNA encoding the heavy chain and the DNA encoding the light chain may be inserted into the same expression vector, and the host cell may be transformed using the vector, or the DNA encoding the heavy chain and the light chain may be lightly mixed. The DNA encoding the strands may be inserted into separate vectors and the two cells may be used to transform host cells.
  • DNA encoding the heavy chain variable region and the light chain variable region may be inserted into a vector into which DNA encoding the heavy chain constant region and the light chain constant region of a specific isotype has been inserted in advance.
  • the vector may also contain DNA encoding a signal peptide that promotes antibody secretion from the host cell.
  • the DNA encoding the signal peptide and the DNA encoding the antibody are linked in frame. After the antibody is produced, the signal peptide can be removed to obtain the antibody as a mature protein.
  • a DNA obtained by ligating DNA and DNA encoding a light chain constant region may be operably linked to elements such as a promoter, an enhancer, and a polyadenylation signal.
  • functionally connected means that elements are connected so as to perform their functions.
  • cytomegalovirus CMV
  • SV40 simian virus 40
  • adenovirus-derived promoter and enhancer can be used.
  • the vector for inserting the gene of the present invention is not particularly limited as long as it can be replicated in a host such as an animal cell, a bacterium, or a yeast, and examples thereof include a plasmid and a phage.
  • a well-known thing can be used as a vector used for construction of an expression vector.
  • Flexi registered trademark
  • pUC19, pTV118 N Teakara Shuzo
  • pUEX2 Amersham
  • pGEX-4T pKK233-2
  • pMAM-neo Clontech
  • the expression vector can be introduced into a host cell by a known method to transform the host cell. For example, electroporation method, calcium phosphate precipitation method, DEAE-dextran transfection method and the like.
  • prokaryotic cells such as Escherichia coli and Bacillus subtilis and eukaryotic cells such as yeast and animal cells can be used, but eukaryotic cells are preferably used.
  • Animal cells include human fetal kidney cell line HEK293 cells, Chinese hamster ovary (CHO) cells, lepidopteran insect cells such as silkworms, Sf 21 cells, Sf 9 cells and TN5 cells, monkey COS cells, mice Examples include fibroblasts, and examples of yeast include Saccharomyces cerevisiae.
  • the variable region or antibody of the present invention can also be produced using an animal body such as a silkworm worm. Production using a silkworm body can be performed by a known method.
  • the purification of the expressed and produced antibody may be carried out using the separation and purification methods used for ordinary proteins.
  • antibodies can be separated and purified by appropriately selecting and combining affinity chromatography, other chromatography, filters, ultrafiltration, salting out, dialysis, etc. (Antibodies A Laboratory Manual. Ed Harlow, David Lane , Cold Spring Harbor Laboratory, 1988).
  • affinity tag sequence include a polyhistidine sequence comprising 2 to 12, preferably 4 or more, more preferably 4 to 7, more preferably 5 or 6 histidines.
  • the synthetic protein can be purified by using nickel chelate column chromatography using nickel as a ligand. It can also be purified by affinity chromatography using a column in which an antibody against polyhistidine is immobilized as a ligand.
  • HAT tags and HN tags composed of sequences containing histidine can also be used.
  • other affinity tags include V5 tag, Xpress tag, AU1 tag, T7 tag, VSV-G tag, DDDDK tag, S tag, CruzTag09, CruzTag22, CruzTag41, Glu-Glu tag, Ha.11 tag, KT3 tag, etc. There is.
  • the present invention also includes anti-canine CD70 antibodies to which these tags are bound.
  • the canine CD70 monoclonal antibody of the present invention can be used for both detection and diagnosis of canine lymphoma and treatment of canine lymphoma.
  • the canine CD70 monoclonal antibody elicits an immune response when administered into the body of a dog. What you do not want is desirable.
  • Examples of such an antibody include a chimeric antibody containing a canine constant region as an antibody constant region, and a canine antibody in which all variable regions except the constant region and the hypervariable region are replaced with a canine sequence.
  • the anti-canine CD70 monoclonal antibody of the present invention can be used for diagnosis of canine lymphoma. That is, it is possible to diagnose whether or not a dog suffers from lymphoma by immunologically detecting CD70 expressed on the lymphocyte surface of a dog using the anti-canine CD70 monoclonal antibody of the present invention. If CD70 is expressed in the lymphocytes of a canine subject, the canine subject can be determined to be suffering from a lymphoma. Detection of canine CD70 in lymphocytes may be performed by detecting the presence or absence of expression of CD70 antigen on the surface of lymphocytes in the peripheral blood of a canine subject.
  • the expression of the CD70 antigen on the surface of lymphocytes can be determined by, for example, microscopic observation as to whether or not the cells are stained with an anti-CD70 antibody labeled with a chromogenic enzyme, a fluorescent compound, or the like.
  • cells can be immunostained using these antibodies to determine the presence or absence of surface antigens, or can be determined using magnetic beads to which the antibodies are bound. It can also be determined whether there is a surface antigen using a FACS or flow cytometer.
  • FACSAria manufactured by Becton Dickinson
  • FACS vantage manufactured by Becton Dickinson
  • FACS Calibur manufactured by Becton Dickinson
  • these surface antigens are negative, it means that they are not sorted as positive cells when analyzed using FACS as described above, or that expression is not observed when expression is examined by RT-PCR, Even if it is expressed to such an extent that it cannot be detected by these techniques, it is considered negative in the present invention.
  • the measurement may be performed simultaneously with cells known to be positive for the marker, and may be negative if the expression level is hardly detected or significantly lower than those positive cells.
  • the CD70 expression level of lymphocytes collected from healthy dogs in advance was measured, a cut-off value was determined from the measured value, and the CD70 expression level of lymphocytes in the dog subject was compared with the cut-off value. Can also be diagnosed.
  • the present invention is also intended to provide a kit that enables detection of canine CD70 in lymphocytes in canine blood, and the kit contains at least an anti-canine CD70 monoclonal antibody.
  • the canine lymphomas targeted by the detection method of the present invention include Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, large B-cell lymphoma, mantle cell lymphoma, MALT lymphoma, follicular lymphoma, and the like.
  • the anti-CD70 monoclonal antibody of the present invention can also be used for the treatment of canine lymphoma.
  • the dosage form of the preparation containing the antibody of the present invention is not limited, and it is administered orally, parenterally, transmucosally (for example, sublingual or buccal administration), topical, transdermal, rectal, inhalation (for example, nasal or deep lung inhalation), etc. can do.
  • Parenteral administration includes intravenous, subcutaneous, intramuscular injection and the like.
  • Topical or transdermal formulations are used in the form of powders, emulsions, suspensions, sprays, creams, ointments, lotions and pastes, or in the form of medicated salves, patches or films.
  • the amount of the antibody of the present invention required for treatment will vary depending on the nature of the condition being treated, the age and condition of the subject, and can ultimately be determined by the attending veterinarian.
  • the antibody may be administered at 0.05 to 2 mg of antibody per day.
  • the predetermined dose may be given as a single dose, or may be given at appropriate intervals of 2, 3, 4 or more divided doses per day.
  • the present invention also includes a therapeutic agent for canine lymphoma comprising an anti-canine CD70 monoclonal antibody as an active ingredient, and further includes a method for treating canine lymphoma comprising administering an anti-canine CD70 monoclonal antibody to a dog.
  • therapeutic agents targeting canine CD70 as a molecular target are also included in the scope of the present invention.
  • the tumor cells may be brought into contact with the anti-CD70 antibody so that the growth of CD70-expressing tumor cells is inhibited.
  • Example 1 CD70 Expression Analysis in Canine Lymphoma Tumor cells were collected from 3 cases of canine T-cell lymphomas who visited the Yamaguchi University Animal Medical Center and stored at ⁇ 80 ° C. until use. On the other hand, as a control, peripheral blood was collected from each of three healthy beagles, and after isolating peripheral blood mononuclear cells, T cells were separated by magnetic bead method using anti-canine CD3 antibody (AbD Serotec), and normal T lymphocytes were isolated. A sphere.
  • CD70 As a result of cDNA microarray, the expression level of CD70 selected from among genes whose expression was increased was examined in canine lymphoma by Realtime PCR. 10 canine lymphoma tumor cell lines (GL-1, CL-1, Ema, UL-1, Nody-1, CLBL-1. CLGL90, 17-71, CLL1390, 3132) and 38 canine lymphomas In the case, there was a sample in which increased expression of CD70 was observed as compared with unstimulated lymphocytes, stimulated T lymphocytes and stimulated B lymphocytes derived from healthy dogs.
  • Example 2 Cloning of the gene of the CD70 molecule of canine CD70 and determination of the nucleotide sequence cDNA prepared using the superscript III cDNA synthesis kit using RNA extracted from lungs and ConA-activated lymphocytes collected from healthy dogs as a template, Using YTM946 (ggagggctgacgtttcatc (SEQ ID NO: 5)) and YTM947 (aagagaaaaacacattctcccaat (SEQ ID NO: 6)), YTM1277 (agctcccctgagtgagaacc (SEQ ID NO: 7)) and YTM1278 (tgggatagaggctgaggttt (SEQ ID NO: 8)) as primers, KOD Plus neo Gene cloning of CD70 molecule and canine CD27 molecule was performed, and the nucleotide sequence was determined (FIG.
  • YTM946 ggagggctga
  • Example 3 Production of a cell line (NRK / cCD70) in which a canine CD70 molecule was overexpressed in a rat kidney cell line NRK and production of an anti-canine CD70 monoclonal antibody
  • a canine CD70 molecule was transformed into a pMx-IP vector (Institute of Medical Science, University of Tokyo) Incorporated into Drat Toshio Kitamura) and retrovirus produced after gene transfer with pCVSVG into PLAT-gp cells (distributed by Dr. Toshio Kitamura, Institute of Medical Science, The University of Tokyo) By infecting, canine CD70 overexpressing cells (NRK / cCD70) were obtained.
  • a rat monoclonal antibody (4F2-12) against canine CD70 was produced by immunizing SD rats with NRK / cCD70. These antibodies were purified with a protein A column and confirmed to be rat IgG2a and kappa chains. In addition, NRK / cCD70 cells and 4F12-E6 antibody diluted to various concentrations are incubated, further reacted with a secondary antibody, and then the fluorescence intensity is measured by flow cytometry (Becton Dickinson, BD Accuri). Was confirmed to bind to NRK / cCD70 (FIG. 3).
  • Example 4 Production of Fusion Protein cCD27-hIg of Canine CD70 Ligand (CD27) and Human Ig Fc Region
  • CD27 Canine CD70 Ligand
  • Human Ig Fc Region The extracellular region of canine CD27, which is a ligand for CD70, was amplified by PCR, and pFUSE-hIgF2-Fc (Invivogen) vector And transferred to HEK293T cells, and the culture supernatant was collected.
  • a fusion protein cCD27-hIg of the Fc region of human IgG2 was prepared from these culture supernatants using Protein A beads. Using these fusion proteins, binding to NRK / cCD70 was confirmed in the same manner as in Example 3.
  • Example 5 Assay for Properties of Anti-Canine CD70 Monoclonal Antibody (4F2-12) Further, whether the anti-canine CD70 antibody (4F2-12) obtained in Example 3 can inhibit the binding between cCD27-hIg and NRK / cCD70, The examination was performed in the same manner as in Example 3.
  • Example 6 Determination of base sequence of variable region of antibody heavy chain and light chain The base sequence of the variable region of heavy chain and light chain of each antibody obtained in Example 3 was determined. The base sequence was determined by the following method.
  • RACE PCR was performed using PrimeSTAR® GXL DNA polymerase (TaKaRa) and primers specific to the rat IgG constant region.
  • the obtained PCR product was cloned into pGEM easy T-vector (Promega), and then the gene sequence of the randomly picked clone was analyzed.
  • H-RT1 and L-RT1 described below were used as primers.
  • H-PCR-N1, H-PCR-N2 and L-PCR were used as primers.
  • Table 1 shows the primer sequences.
  • Example 7 Expression analysis of CD70 in tumor cells collected from canine lymphoma cases by flow cytometry using anti-CD70 antibody
  • Nine canine lymphoma tumor cell lines (T cell types Ema, Nody-1, CLC, UL-1, CLGL90, CLK; B cell type CLBL-1, GL-1, 17-71), 2 canine lymphoma cases (B cell type), chronic lymphocytic leukemia (CLL) 1 tumor flow
  • B cell type B cell type
  • CLL chronic lymphocytic leukemia
  • Example 8 Analysis of CD70 Expression in Canine Peripheral Blood Lymphocytes by Flow Cytometry Using Anti-CD70 Antibody Lymphocytes isolated from the peripheral blood of 4 healthy beagle heads, and further treated with ConA stimulation (5 ⁇ g / ml) or LPS ( 5 ⁇ g / ml) and cells stimulated with CD40 ligand for 2 days were examined for canine CD70 expression by flow cytometry (Becton Dickinson, BD Accuri). No canine CD70 expression was detected in any of the cells. (FIG. 6).
  • Example 9 Analysis of CD70 expression in various organs of healthy dogs using anti-CD70 antibodies Immunostaining was performed on tumor tissues collected from 29 cases from canine lymphoma cases. Expression of canine CD70 was detected in 12 cases It was done.
  • FIG. 7 shows a typical immunostained image.
  • the anti-canine CD70 antibody of the present invention can be used for diagnosis and treatment of canine lymphoma.

Abstract

Provided is a method for diagnosing lymphoma in a dog. A method for diagnosing lymphoma in a dog, comprising: measuring CD70 expressed in lymphocytes in peripheral blood collected from the dog using an anti-dog CD70 monoclonal antibody which can bind to canine CD70 or a functional fragment of the monoclonal antibody which can bind to canine CD70; and determining that the dog is suffering from lymphoma when CD70 is expressed in the lymphocytes.

Description

抗イヌＣＤ７０モノクローナル抗体Anti-canine CD70 monoclonal antibody
　本発明は、イヌリンパ腫の治療及び診断のために用いる抗イヌCD70抗体及びその利用法に関する。 The present invention relates to an anti-canine CD70 antibody used for treatment and diagnosis of canine lymphoma and a method for using the same.
　イヌの寿命の延長に伴い、ヒトと同様、がんの発生が増加している。従来から用いられている3大療法（外科手術、放射線療法、化学療法）では限界があり、それに代わる又は同時に用いることができる新たな治療法が必要である。 伴 い With the extension of the lifespan of dogs, the incidence of cancer is increasing as in humans. The three major therapies conventionally used (surgery, radiation therapy, chemotherapy) are limited and new therapies that can be used instead or simultaneously are needed.
　ヒトにおいては、抗体医薬として、腫瘍細胞表面に発現する分子に対する抗体を担ガン患者へ投与することによる腫瘍の治療が近年行われており、新規治療法として注目されている。 In humans, treatment of tumors by administering to a cancer-bearing patient an antibody against a molecule expressed on the surface of a tumor cell as an antibody drug has been performed in recent years, and has attracted attention as a novel therapeutic method.
　例えば腫瘍壊死因子（TNF)スーパーファミリーの一員であるCD70に対する抗体をヒトの癌等の治療に用いることが報告されている（特許文献１を参照）。 For example, it has been reported that an antibody against CD70, which is a member of the tumor necrosis factor (TNF) superfamily, is used for the treatment of human cancer or the like (see Patent Document 1).
　しかしイヌに比較的多い血液系腫瘍であるリンパ腫について抗体医薬の候補となる分子の同定はほとんどなされていない。 However, there has been little identification of molecules that are candidates for antibody drugs for lymphoma, which is a blood tumor that is relatively common in dogs.
特表2009-509510号公報Special table 2009-509510
　本発明は、イヌのリンパ腫に対する新しい診断及び病勢モニターマーカーとしてCD70分子を同定し、それを用いた治療法及び診断法の提供を目的とする。 The present invention aims to identify a CD70 molecule as a new diagnostic and disease monitoring marker for lymphoma in dogs, and to provide a therapeutic method and a diagnostic method using the same.
　イヌに比較的多い血液系腫瘍であるリンパ腫について抗体医薬の候補となる分子の同定はほとんどなされていなかった。本発明者らは、イヌのリンパ腫に特異的に発現する分子の同定を行い、腫瘍壊死因子（TNF)スーパーファミリーの一員であるCD70が特異的に発現していることを見出した。 There has been little identification of molecules that are candidates for antibody drugs for lymphoma, which is a relatively large blood system tumor in dogs. The present inventors have identified molecules that are specifically expressed in canine lymphomas and found that CD70, a member of the tumor necrosis factor (TNF) superfamily, is specifically expressed.
　本発明者らは、CD70に対するモノクローナル抗体を作製し、該モノクローナル抗体を用いることにより、イヌのリンパ球におけるCD70の発現を測定することにより、イヌがリンパ腫に罹患しているか否かを診断できることを見出した。 The present inventors have prepared a monoclonal antibody against CD70, and by using the monoclonal antibody, by measuring the expression of CD70 in canine lymphocytes, it is possible to diagnose whether or not a dog suffers from lymphoma. I found it.
　さらに、抗CD70抗体をイヌのリンパ腫の治療に用い得ることを見出し本発明を完成させるに至った。 Furthermore, the present inventors have found that an anti-CD70 antibody can be used for the treatment of canine lymphoma and have completed the present invention.
　すなわち、本発明は以下のとおりである。
[１]　配列番号２で表されるアミノ酸配列からなる重鎖可変領域及び配列番号４で表されるアミノ酸配列からなる軽鎖可変領域を含む、イヌCD70に結合する抗イヌCD70モノクローナル抗体又はイヌCD70に結合するその機能的断片。
[２]　配列番号２で表されるアミノ酸配列と90％以上の配列同一性を有するアミノ酸配列からなり、イヌCD70への結合活性を有する重鎖可変領域、及び配列番号４で表されるアミノ酸配列と90％以上の配列同一性を有するアミノ酸配列からなり、イヌCD70への結合活性を有する軽鎖可変領域を含む、イヌCD70に結合する抗イヌCD70モノクローナル抗体又はイヌCD70に結合するその機能的断片。
[３]　配列番号１で表される塩基配列からなるDNAがコードする重鎖可変領域及び配列番号３で表される塩基配列からなるDNAがコードする軽鎖可変領域を含む、イヌCD70に結合する抗イヌCD70モノクローナル抗体又はイヌCD70に結合するその機能的断片。
[４]　配列番号１で表される塩基配列と90％以上の配列同一性を有する塩基配列がコードするイヌCD70への結合活性を有する重鎖可変領域、及び配列番号３で表される塩基配列と90％以上の配列同一性を有する塩基配列がコードするイヌCD70への結合活性を有する軽鎖可変領域を含むイヌCD70に結合する抗イヌCD70モノクローナル抗体又はイヌCD70に結合するその機能的断片。
[５]　重鎖定常領域及び軽鎖定常領域がイヌIgG抗体の定常領域である、[１]～[４]のいずれかのイヌCD70に結合する抗イヌCD70モノクローナル抗体又はイヌCD70に結合するその機能的断片。
[６]　機能的断片がFab、Fab’、F(ab’)2、ジスルフィド結合Fv(dsFv) 、二量体化V領域（diabody）、一本鎖Fv（scFv）及びCDRからなる群から選択されるペプチド断片である[１]～[５]のいずれかのイヌCD70に結合する抗イヌCD70モノクローナル抗体又はイヌCD70に結合するその機能的断片。
[７]　配列番号２で表されるアミノ酸配列からなる、抗イヌCD70モノクローナル抗体の重鎖可変領域であるポリペプチド。
[８]　配列番号４で表されるアミノ酸配列からなる、抗イヌCD70モノクローナル抗体の軽鎖可変領域であるポリペプチド。
[９]　配列番号１で表されるDNA配列からなる、抗イヌCD70モノクローナル抗体の重鎖可変領域をコードするポリヌクレオチド。
[１０]　配列番号３で表されるDNA配列からなる、抗イヌCD70モノクローナル抗体の軽鎖可変領域をコードするポリヌクレオチド。
[１１]　[９]のポリヌクレオチド、[１０]のポリヌクレオチド、又は[９]のポリヌクレオチドと[１０]のポリヌクレオチドを含むベクター。
[１２]　[１１]に記載のベクターを含む細胞。
[１３]　配列番号１で表されるDNA配列からなる、抗イヌCD70モノクローナル抗体の重鎖可変領域をコードするDNAを抗体の重鎖定常領域をコードするDNAと連結し、配列番号３で表されるDNA配列からなる、抗イヌCD70モノクローナル抗体の軽鎖可変領域をコードするDNAを抗体の軽鎖定常領域をコードするDNAと連結し、得られたDNAコンストラクトを発現ベクターに挿入し、該ベクターで宿主細胞又は宿主動物を形質転換し、該宿主細胞又は宿主動物により抗体を産生させることを含む、抗イヌCD70モノクローナル抗体の製造方法。
[１４]　[１]～[６]のいずれかのイヌCD70に結合する抗イヌCD70モノクローナル抗体又はイヌCD70に結合するその機能的断片を用いて、イヌ末梢血のリンパ球に発現するCD70を測定し、リンパ球にCD70が発現している場合に、リンパ腫に罹患していると判断するイヌリンパ腫の診断方法。
[１５]　フローサイトメーターを用いて行う、[１４]の診断方法。
[１６]　[１]～[６]のいずれかのイヌCD70に結合する抗イヌCD70モノクローナル抗体又はイヌCD70に結合するその機能的断片を含む、イヌリンパ腫の検出試薬。 That is, the present invention is as follows.
[1] An anti-canine CD70 monoclonal antibody or canine CD70 that binds to canine CD70, comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 4 Its functional fragment that binds to.
[2] A heavy chain variable region comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 and having binding activity to canine CD70, and an amino acid sequence represented by SEQ ID NO: 4 An anti-canine CD70 monoclonal antibody that binds to canine CD70 or a functional fragment thereof that binds to canine CD70, comprising a light chain variable region having an amino acid sequence having 90% or more sequence identity and binding activity to canine CD70 .
[3] Binds to canine CD70, which includes a heavy chain variable region encoded by DNA consisting of the base sequence represented by SEQ ID NO: 1 and a light chain variable region encoded by DNA consisting of the base sequence represented by SEQ ID NO: 3. An anti-canine CD70 monoclonal antibody or a functional fragment thereof that binds to canine CD70.
[4] A heavy chain variable region having binding activity to canine CD70 encoded by a base sequence having 90% or more sequence identity with the base sequence represented by SEQ ID NO: 1, and a base sequence represented by SEQ ID NO: 3 A canine CD70 monoclonal antibody that binds to canine CD70 or a functional fragment thereof that binds to canine CD70, comprising a light chain variable region having binding activity to canine CD70 encoded by a nucleotide sequence having 90% or more sequence identity.
[5] The anti-canine CD70 monoclonal antibody that binds to the canine CD70 of any one of [1] to [4], wherein the heavy chain constant region and the light chain constant region are constant regions of a canine IgG antibody, or that that binds to a canine CD70 Functional fragment.
[6] The functional fragment is selected from the group consisting of Fab, Fab ′, F (ab ′) 2 , disulfide bond Fv (dsFv), dimerization V region (diabody), single chain Fv (scFv) and CDR An anti-canine CD70 monoclonal antibody that binds to canine CD70 according to any one of [1] to [5], or a functional fragment that binds to canine CD70.
[7] A polypeptide which is a heavy chain variable region of an anti-canine CD70 monoclonal antibody consisting of the amino acid sequence represented by SEQ ID NO: 2.
[8] A polypeptide that is a light chain variable region of an anti-canine CD70 monoclonal antibody, comprising the amino acid sequence represented by SEQ ID NO: 4.
[9] A polynucleotide encoding the heavy chain variable region of an anti-canine CD70 monoclonal antibody, comprising the DNA sequence represented by SEQ ID NO: 1.
[10] A polynucleotide encoding the light chain variable region of an anti-canine CD70 monoclonal antibody, comprising the DNA sequence represented by SEQ ID NO: 3.
[11] A vector comprising the polynucleotide of [9], the polynucleotide of [10], or the polynucleotide of [9] and the polynucleotide of [10].
[12] A cell comprising the vector according to [11].
[13] A DNA encoding the heavy chain variable region of the anti-canine CD70 monoclonal antibody consisting of the DNA sequence represented by SEQ ID NO: 1 is ligated with the DNA encoding the heavy chain constant region of the antibody, and represented by SEQ ID NO: 3. DNA encoding the light chain variable region of an anti-canine CD70 monoclonal antibody consisting of a DNA sequence comprising: a DNA sequence encoding the light chain constant region of the antibody; and inserting the resulting DNA construct into an expression vector; A method for producing an anti-canine CD70 monoclonal antibody, comprising transforming a host cell or host animal and producing an antibody by the host cell or host animal.
[14] Using the anti-canine CD70 monoclonal antibody that binds to canine CD70 according to any one of [1] to [6] or a functional fragment thereof that binds to canine CD70, CD70 expressed in canine peripheral blood lymphocytes is measured. A method for diagnosing canine lymphoma, wherein CD70 is expressed in lymphocytes and it is judged that the patient is affected by lymphoma.
[15] The diagnostic method according to [14], which is performed using a flow cytometer.
[16] A detection reagent for canine lymphoma, comprising the anti-canine CD70 monoclonal antibody that binds to canine CD70 according to any one of [1] to [6] or a functional fragment thereof that binds to canine CD70.
　本明細書は本願の優先権の基礎となる日本国特許出願番号2016-022105号の開示内容を包含する。 This specification includes the disclosure of Japanese Patent Application No. 2016-022105, which is the basis of the priority of the present application.
　本発明の抗イヌCD70抗体により、イヌのリンパ腫を診断することができる。また、本発明の抗イヌCD70抗体は、イヌのリンパ腫の治療のためにも有効である。 The canine lymphoma can be diagnosed by the anti-canine CD70 antibody of the present invention. The anti-canine CD70 antibody of the present invention is also effective for the treatment of canine lymphoma.
イヌリンパ腫におけるCD70の発現解析の結果を示す図である。It is a figure which shows the result of the expression analysis of CD70 in canine lymphoma. イヌCD70及びCD27をコードする遺伝子の塩基配列を示す図である。It is a figure which shows the base sequence of the gene which codes canine CD70 and CD27. 抗イヌCD70モノクローナル抗体とNRK/cCD70との結合性を示す図である。It is a figure which shows the binding property of anti-canine CD70 monoclonal antibody and NRK / cCD70. cCD27-hIgとNRK/cCD70との結合性を示す図である。It is a figure which shows the binding property of cCD27-hIg and NRK / cCD70. 抗イヌCD70モノクローナル抗体がイヌCD70とイヌCD27の結合阻害効果を有しないことを示す図である。It is a figure which shows that an anti-canine CD70 monoclonal antibody does not have the binding inhibitory effect of canine CD70 and canine CD27. イヌリンパ腫腫瘍細胞株9例におけるCD70の発現解析の結果を示す図である。It is a figure which shows the result of the expression analysis of CD70 in 9 canine lymphoma tumor cell lines. イヌリンパ腫症例2例(B細胞型)、慢性リンパ性白血病症例1例より採取した腫瘍細胞におけるCD70の発現解析の結果を示す図である。It is a figure which shows the result of the expression analysis of CD70 in the tumor cell extract | collected from 2 canine lymphoma cases (B cell type) and a chronic lymphocytic leukemia case. イヌの末梢血リンパ球におけるCD70の発現解析の結果を示す図である。It is a figure which shows the result of the expression analysis of CD70 in the peripheral blood lymphocyte of a dog. イヌの各種臓器におけるCD70の発現解析の結果を示す図である。It is a figure which shows the result of the expression analysis of CD70 in various organs of a dog.
　以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
　本発明はイヌCD70に特異的に結合するモノクローナル抗体4F2-12の重鎖可変領域及び軽鎖（κ鎖）可変領域を含むイヌCD70に特異的に結合する抗イヌCD70モノクローナル抗体である。 The present invention is an anti-canine CD70 monoclonal antibody that specifically binds to canine CD70 including the heavy chain variable region and light chain (κ chain) variable region of monoclonal antibody 4F2-12 that specifically binds to canine CD70.
　さらに、本発明は前記4F2-12抗体の重鎖可変領域若しくは軽鎖（κ鎖）可変領域であるポリペプチド並びにそれらの可変領域をコードするポリヌクレオチドも包含する。 Furthermore, the present invention also includes polypeptides that are the heavy chain variable region or light chain (κ chain) variable region of the 4F2-12 antibody, and polynucleotides encoding these variable regions.
　本発明の抗体は、抗体の機能的断片又はその修飾物も包含する。例えば、抗体の機能的断片は、抗体の断片であって抗原に特異的に結合し得る断片である。機能的断片としては、Fab、F（ab'）2、Fv、1個のFabと完全なFcを有するFab/c、Ｈ鎖若しくはＬ鎖のFvを適当なリンカーで連結させたシングルチェインFv（scFv）又はCDR等が挙げられる。ポリヌクレオチドは、DNAもRNAも包含する。 The antibody of the present invention includes a functional fragment of an antibody or a modified product thereof. For example, a functional fragment of an antibody is a fragment of an antibody that can specifically bind to an antigen. Functional fragments include Fab, F (ab ′) 2, Fv, a single chain Fv in which Fab / c having one Fc and a complete Fc, H chain or L chain Fv are linked by an appropriate linker ( scFv) or CDR. Polynucleotide includes both DNA and RNA.
　4F2-12抗体の重鎖可変領域をコードするDNAの塩基配列は配列番号１で表される塩基配列からなり、重鎖可変領域のアミノ酸配列は配列番号２で表されるアミノ酸配列からなる。また、4F2-12抗体の軽鎖可変領域をコードするDNAの塩基配列は配列番号３で表される塩基配列からなり、軽鎖可変領域のアミノ酸配列は配列番号４で表されるアミノ酸配列からなる。 The base sequence of the DNA encoding the heavy chain variable region of the 4F2-12 antibody consists of the base sequence represented by SEQ ID NO: 1, and the amino acid sequence of the heavy chain variable region consists of the amino acid sequence represented by SEQ ID NO: 2. Further, the base sequence of the DNA encoding the light chain variable region of the 4F2-12 antibody is composed of the base sequence represented by SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is composed of the amino acid sequence represented by SEQ ID NO: 4. .
　重鎖可変領域は配列番号２で表わされるアミノ酸配列からなる重鎖可変領域のみならず、該アミノ酸配列において、１若しくは数個、例えば、１～１０個、好ましくは１～５個、さらに好ましくは１若しくは２個、さらに好ましくは１個のアミノ酸が欠失、置換、付加されたアミノ酸配列からなり、抗体の重鎖可変領域の活性、すなわちイヌCD70への結合活性を有するタンパク質からなる重鎖可変領域も含む。軽鎖可変領域は配列番号４で表わされるアミノ酸配列からなる軽鎖可変領域のみならず、該アミノ酸配列において１若しくは数個、例えば、１～１０個、好ましくは１～５個、さらに好ましくは１若しくは２個、さらに好ましくは１個のアミノ酸が欠失、置換、付加されたアミノ酸配列からなり、抗体の軽鎖可変領域の活性、すなわちイヌCD70への結合活性を有するタンパク質からなる軽鎖可変流域も含む。 The heavy chain variable region includes not only the heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 2, but also one or several, for example, 1 to 10, preferably 1 to 5, more preferably in the amino acid sequence. A heavy chain variable consisting of an amino acid sequence in which one or two, more preferably one amino acid has been deleted, substituted, or added, and consisting of an antibody heavy chain variable region activity, that is, a protein having a binding activity to canine CD70. Including area. The light chain variable region includes not only the light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 4, but also 1 or several, for example, 1 to 10, preferably 1 to 5, more preferably 1 in the amino acid sequence. Or a light chain variable basin comprising a protein having an amino acid sequence in which two, more preferably one amino acid has been deleted, substituted, or added, and having the activity of the light chain variable region of an antibody, ie, the binding activity to canine CD70. Including.
　このような配列番号２又は４のアミノ酸配列において１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列として、配列番号２又は４のアミノ酸配列と、BLAST（Basic Local Alignment Search Tool at the National Center for Biological Information（米国国立生物学情報センターの基本ローカルアラインメント検索ツール））等（例えば、デフォルトすなわち初期設定のパラメータ）を用いて計算したときに、少なくとも85％以上、好ましくは90％以上、さらに好ましくは95％以上、特に好ましくは97％以上の配列同一性を有しているものが挙げられる。 As an amino acid sequence in which one or several amino acids are deleted, substituted or added in such an amino acid sequence of SEQ ID NO: 2 or 4, an amino acid sequence of SEQ ID NO: 2 or 4 and BLAST (Basic Local Alignment Search Tool at the (E.g., National Center for Biological Information (Basic National Alignment Search Tool of the National Center for Biological Information))) (for example, default or default parameters), at least 85%, preferably 90% or more, More preferred are those having a sequence identity of 95% or more, particularly preferably 97% or more.
　このような配列番号２又は４のアミノ酸配列において１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有するタンパク質は配列番号２又は４のアミノ酸配列を有するタンパク質と実質的に同一である。 A protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2 or 4 is substantially the same as a protein having the amino acid sequence of SEQ ID NO: 2 or 4. is there.
　また、上記の配列番号１又は３で表される配列からなる塩基配列とBLAST（Basic Local Alignment Search Tool at the National Center for Biological Information（米国国立生物学情報センターの基本ローカルアラインメント検索ツール））等（例えば、デフォルトすなわち初期設定のパラメータ）を用いて計算したときに、少なくとも85％以上、好ましくは90％以上、さらに好ましくは95％以上、特に好ましくは97％以上の配列同一性を有している塩基配列からなるDNAであって、抗体の重鎖可変領域又は軽鎖可変領域の活性、すなわちイヌCD70への結合活性を有するタンパク質をコードするDNAも本発明の抗体の重鎖可変領域又は軽鎖可変領域をコードするDNAに含まれる。 In addition, the base sequence consisting of the sequence represented by SEQ ID NO: 1 or 3 above and BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information), etc.) ( For example, the sequence identity is at least 85% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 97% or more when calculated using default or default parameters. A DNA comprising a base sequence, which also encodes a protein having the activity of the heavy chain variable region or light chain variable region of the antibody, that is, the binding activity to canine CD70, is also the heavy chain variable region or light chain of the antibody of the present invention Included in the DNA encoding the variable region.
　また、上記の配列番号１又は３で表される配列からなるDNAと相補的な配列からなるDNAとストリンジェントな条件下でハイブリダイズすることができるDNAであって抗体の重鎖可変領域又は軽鎖可変領域の活性、すなわちイヌCD70への結合活性を有するタンパク質をコードするDNAも本発明の重鎖可変領域又は軽鎖可変領域をコードするDNAに含まれる。すなわち、DNAを固定したフィルターを用いて、0.7～1.0MのNaCl存在下、68℃でハイブリダイゼーションを行った後、0.1～2倍濃度のSSC溶液（1倍濃度のSSCとは150mM NaCl、15mM クエン酸ナトリウムからなる）を用い、68℃で洗浄することにより同定することができる条件をいう。あるいは、サザンブロッティング法によりニトロセルロース膜上にDNAを転写、固定後、ハイブリダイゼーション緩衝液〔50％ フォルムアミド、４×SSC、50mM HEPES(pH7.0)、10×デンハルツ（Denhardt, s）溶液、100μg/mlサケ精子DNA〕中で42℃で一晩反応させることによりハイブリッドを形成することができるDNAである。 Further, it is a DNA that can hybridize under stringent conditions with a DNA consisting of a sequence complementary to the DNA consisting of the sequence represented by SEQ ID NO: 1 or 3, wherein the heavy chain variable region or light chain of the antibody. DNA encoding a protein having the activity of the chain variable region, that is, the binding activity to canine CD70, is also included in the DNA encoding the heavy chain variable region or the light chain variable region of the present invention. That is, after hybridization at 68 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA is immobilized, a 0.1 to 2 fold concentration SSC solution (1 fold concentration SSC is 150 mM NaCl, 15 mM) This is a condition that can be identified by washing at 68 ° C. using sodium citrate. Alternatively, after transferring and immobilizing DNA on a nitrocellulose membrane by the Southern blotting method, hybridization buffer [50% formamide, 4 × SSC, 50 mM HEPES (pH 7.0), 10 × Denhardt , s) solution, 100 μg / ml salmon sperm DNA], which can form a hybrid by reacting overnight at 42 ° C.
　本発明のイヌCD70に特異的に結合する抗イヌCD70モノクローナル抗体の重鎖可変領域又は軽鎖可変領域は、イヌCD70を免疫原として用いて、マウスやラットの細胞を用いて、公知の方法により抗イヌCD70抗体産生ハイブリドーマを取得し、該ハイブリドーマから重鎖可変領域をコードするDNA又は軽鎖可変領域をコードするDNAを単離し、該DNAを発現させることにより得ることができる。 The heavy chain variable region or the light chain variable region of the anti-canine CD70 monoclonal antibody that specifically binds to the canine CD70 of the present invention can be obtained by a known method using canine CD70 as an immunogen, using mouse or rat cells. It can be obtained by obtaining an anti-canine CD70 antibody-producing hybridoma, isolating a DNA encoding a heavy chain variable region or a DNA encoding a light chain variable region from the hybridoma, and expressing the DNA.
　また、前記の重鎖可変領域及び軽鎖可変領域を含むイヌCD70に特異的に結合する抗イヌCD70モノクローナル抗体は、上記の重鎖可変領域及び重鎖定常領域並びに上記の軽鎖可変領域及び軽鎖定常領域とから構成される。重鎖定常領域は、３個のドメインCH1、CH2及びCH3から構成されている。重鎖定常領域は、IgG1、IgG2、IgG3、IgG4、IgA、IgE、IgM又はIgD定常領域であってもよいが、最も好適には、IgG1又はIgG4定常領域である。軽鎖定常領域は、１個のドメインCLで構成されている。軽鎖定常領域は、κ又はλ定常領域である。 The anti-canine CD70 monoclonal antibody that specifically binds to canine CD70 containing the heavy chain variable region and the light chain variable region includes the heavy chain variable region and the heavy chain constant region, and the light chain variable region and the light chain. It consists of a chain constant region. The heavy chain constant region is composed of three domains C H 1, C H 2 and C H 3. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is most preferably an IgG1 or IgG4 constant region. The light chain constant region is comprised of one domain C L. The light chain constant region is a kappa or lambda constant region.
　抗体をコードするDNAは、重鎖可変領域をコードするDNAと重鎖定常領域をコードするDNAを連結し、さらに軽鎖可変領域をコードするDNAと軽鎖定常領域をコードするDNAを連結することにより重鎖をコードするDNA及び軽鎖をコードするDNAとして得られる。可変領域の由来生物種と定常領域の由来生物種は異なっていてもよく、本発明の抗イヌCD70抗体は、可変領域の由来生物種と定常領域の由来生物種が異なるキメラ抗体も含む。例えば、配列番号１で表されるDNAにコードされる重鎖可変領域及び配列番号３で表されるDNAにコードされる軽鎖可変領域はラット由来であるが、これをイヌ由来の抗体の定常領域をコードするDNAと連結させてラット由来の可変領域とイヌ由来の定常領域を含むキメラ抗体を作製することができる。 The antibody-encoding DNA is obtained by linking the DNA encoding the heavy chain variable region and the DNA encoding the heavy chain constant region, and further linking the DNA encoding the light chain variable region and the DNA encoding the light chain constant region. To obtain DNA encoding a heavy chain and DNA encoding a light chain. The species derived from the variable region may be different from the species derived from the constant region, and the anti-canine CD70 antibody of the present invention includes a chimeric antibody in which the species derived from the variable region and the species derived from the constant region are different. For example, the heavy chain variable region encoded by the DNA represented by SEQ ID NO: 1 and the light chain variable region encoded by the DNA represented by SEQ ID NO: 3 are derived from rat. A chimeric antibody comprising a rat-derived variable region and a dog-derived constant region can be prepared by ligation with a region-encoding DNA.
　本発明の重鎖可変領域又は軽鎖可変領域は、重鎖可変領域をコードするDNA又は軽鎖可変領域をコードするDNAを発現ベクターに挿入し、発現用の宿主細胞を該ベクターを用いて形質転換し、宿主細胞を培養することにより細胞に産生させることができる。 In the heavy chain variable region or light chain variable region of the present invention, DNA encoding a heavy chain variable region or DNA encoding a light chain variable region is inserted into an expression vector, and a host cell for expression is transformed using the vector. It can be transformed and cultivated by culturing host cells.
　本発明の抗イヌCD70モノクローナル抗体は、上記の重鎖をコードするDNA及び軽鎖をコードするDNAを発現ベクターに挿入し、該ベクターを用いて宿主細胞を形質転換し、該宿主細胞を培養して産生させることができる。この際、上記の重鎖をコードするDNA及び軽鎖をコードするDNAを同じ発現ベクターに挿入し、該ベクターを用いて宿主細胞を形質転換してもよいし、重鎖をコードするDNAと軽鎖をコードするDNAを別々のベクターに挿入し、２つのベクターを用いて宿主細胞を形質転換してもよい。この際、特定のアイソタイプの重鎖定常領域及び軽鎖定常領域をコードするDNAを予め挿入したベクターに重鎖可変領域及び軽鎖可変領域をコードするDNAを挿入してもよい。また、該ベクターは宿主細胞からの抗体の分泌を促進するシグナルペプチドをコードするDNAを含んでいてもよい。この場合、シグナルペプチドをコードするDNAと抗体をコードするDNAをインフレームで連結するようにする。抗体が産生された後にシグナルペプチドを除去し、抗体を成熟タンパク質として得ることができる。 The anti-canine CD70 monoclonal antibody of the present invention inserts the DNA encoding the heavy chain and the DNA encoding the light chain into an expression vector, transforms a host cell using the vector, and cultures the host cell. Can be produced. At this time, the DNA encoding the heavy chain and the DNA encoding the light chain may be inserted into the same expression vector, and the host cell may be transformed using the vector, or the DNA encoding the heavy chain and the light chain may be lightly mixed. The DNA encoding the strands may be inserted into separate vectors and the two cells may be used to transform host cells. At this time, DNA encoding the heavy chain variable region and the light chain variable region may be inserted into a vector into which DNA encoding the heavy chain constant region and the light chain constant region of a specific isotype has been inserted in advance. The vector may also contain DNA encoding a signal peptide that promotes antibody secretion from the host cell. In this case, the DNA encoding the signal peptide and the DNA encoding the antibody are linked in frame. After the antibody is produced, the signal peptide can be removed to obtain the antibody as a mature protein.
　この際、重鎖可変領域をコードするDNA、軽鎖可変領域をコードするDNA、重鎖可変領域をコードするDNAと重鎖定常領域をコードするDNAを連結したDNA、軽鎖可変領域をコードするDNAと軽鎖定常領域をコードするDNAを連結したDNAをプロモータ、エンハンサー、ポリアデニル化シグナル等のエレメントと機能的に連結してもよい。ここで機能的に連結とは、エレメントがその機能を果たすように連結することをいう。 In this case, DNA encoding the heavy chain variable region, DNA encoding the light chain variable region, DNA encoding the heavy chain variable region and DNA encoding the heavy chain constant region, encoding the light chain variable region A DNA obtained by ligating DNA and DNA encoding a light chain constant region may be operably linked to elements such as a promoter, an enhancer, and a polyadenylation signal. Here, functionally connected means that elements are connected so as to perform their functions.
　プロモータ及びエンハンサーとしては、サイトメガロウイルス（CMV)、シミアンウイルス40(SV40）、アデノウイルス由来のプロモータ及びエンハンサーを用いることができる。 As the promoter and enhancer, a cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus-derived promoter and enhancer can be used.
　本発明の遺伝子を挿入するためのベクターは、動物細胞、細菌、酵母等の宿主中で複製可能なものであれば特に限定されず、例えば、プラスミド、ファージ等が挙げられる。発現ベクターの構築に用いられるベクターとしては、公知のものを用いることができる。例えば、Flexi（登録商標）ベクター（プロメガ社）、pUC19、pTV118 N (宝酒造社製)、pUEX2（アマシャム社製）、pGEX-4T、pKK233-2(ファルマシア社製)、pMAM-neo（クロンテック社製）等が挙げられる。 The vector for inserting the gene of the present invention is not particularly limited as long as it can be replicated in a host such as an animal cell, a bacterium, or a yeast, and examples thereof include a plasmid and a phage. A well-known thing can be used as a vector used for construction of an expression vector. For example, Flexi (registered trademark) vector (Promega), pUC19, pTV118 N (Takara Shuzo), pUEX2 (Amersham), pGEX-4T, pKK233-2 (Pharmacia), pMAM-neo (Clontech) ) And the like.
　発現ベクターは公知の方法で宿主細胞に導入し、宿主細胞を形質転換することができる。例えば、エレクトロポレーション法、リン酸カルシウム沈殿法、DEAE－デキストラントランスフェクション法等がある。 The expression vector can be introduced into a host cell by a known method to transform the host cell. For example, electroporation method, calcium phosphate precipitation method, DEAE-dextran transfection method and the like.
　宿主細胞としては、大腸菌、枯草菌等の原核細胞も酵母、動物細胞等の真核細胞も用いることができるが、真核細胞を用いることが好ましい。動物細胞としては、ヒト胎児腎細胞株であるHEK293細胞、チャイニーズ・ハムスター・卵巣（CHO）細胞、カイコ等の鱗翅目昆虫細胞であるSf 21細胞、Sf 9細胞やTN5細胞、サルCOS細胞、マウス線維芽細胞等が挙げられ、酵母としてはサッカロマイセス・セレビィシエ等が挙げられる。また、本発明の可変領域又は抗体は、カイコ虫体等の動物体を用いて産生することもできる。カイコ虫体を用いての産生は公知の方法により行うことができる。 As a host cell, prokaryotic cells such as Escherichia coli and Bacillus subtilis and eukaryotic cells such as yeast and animal cells can be used, but eukaryotic cells are preferably used. Animal cells include human fetal kidney cell line HEK293 cells, Chinese hamster ovary (CHO) cells, lepidopteran insect cells such as silkworms, Sf 21 cells, Sf 9 cells and TN5 cells, monkey COS cells, mice Examples include fibroblasts, and examples of yeast include Saccharomyces cerevisiae. The variable region or antibody of the present invention can also be produced using an animal body such as a silkworm worm. Production using a silkworm body can be performed by a known method.
　発現、産生された抗体の精製は、通常のタンパク質で使用されている分離、精製方法を使用すればよい。例えば、アフィニティークロマトグラフィー、その他のクロマトグラフィー、フィルター、限外濾過、塩析、透析等を適宜選択、組み合わせることにより、抗体を分離、精製することができる（Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988）。また、抗イヌCD70抗体をアフィニティータグ付きの状態で製造した後、アフィニティータグを利用したアフィニティークロマトグラフィーにより精製することもできる。アフィニティータグ配列としては、例えば、２～１２個、好ましくは４個以上、さらに好ましくは４～７個、さらに好ましくは５個若しくは６個のヒスチジンからなるポリヒスチジン配列が挙げられる。この場合、ニッケルをリガンドとしたニッケルキレートカラムクロマトグラフィーを利用することにより合成タンパク質を精製することができる。また、ポリヒスチジンに対する抗体をリガンドとして固定化したカラムを用いたアフィニティークロマトグラフィーによっても精製することができる。その他、ヒスチジンを含む配列からなるHATタグ、HNタグ等も用いることができる。さらに、他のアフィニティータグとして、V5タグ、Xpressタグ、AU1タグ、T7タグ、VSV-Gタグ、DDDDKタグ、Sタグ、CruzTag09、CruzTag22、CruzTag41、Glu-Gluタグ、Ha.11タグ、KT3タグ等がある。本発明は、これらのタグが結合した抗イヌCD70抗体も包含する。 The purification of the expressed and produced antibody may be carried out using the separation and purification methods used for ordinary proteins. For example, antibodies can be separated and purified by appropriately selecting and combining affinity chromatography, other chromatography, filters, ultrafiltration, salting out, dialysis, etc. (Antibodies A Laboratory Manual. Ed Harlow, David Lane , Cold Spring Harbor Laboratory, 1988). In addition, after producing an anti-canine CD70 antibody with an affinity tag, it can be purified by affinity chromatography using an affinity tag. Examples of the affinity tag sequence include a polyhistidine sequence comprising 2 to 12, preferably 4 or more, more preferably 4 to 7, more preferably 5 or 6 histidines. In this case, the synthetic protein can be purified by using nickel chelate column chromatography using nickel as a ligand. It can also be purified by affinity chromatography using a column in which an antibody against polyhistidine is immobilized as a ligand. In addition, HAT tags and HN tags composed of sequences containing histidine can also be used. In addition, other affinity tags include V5 tag, Xpress tag, AU1 tag, T7 tag, VSV-G tag, DDDDK tag, S tag, CruzTag09, CruzTag22, CruzTag41, Glu-Glu tag, Ha.11 tag, KT3 tag, etc. There is. The present invention also includes anti-canine CD70 antibodies to which these tags are bound.
　本発明のイヌCD70モノクローナル抗体は、イヌのリンパ腫の検出、診断及びイヌのリンパ腫の治療の両方に用いることができるが、治療剤として用いる場合は、イヌの体内に投与した場合に免疫反応を惹起しないものが望ましい。このような抗体として、抗体の定常領域としてイヌの定常領域を含むキメラ抗体、定常領域と超可変領域を除く全ての可変領域をイヌの配列に置き換えたイヌ化抗体等が挙げられる。 The canine CD70 monoclonal antibody of the present invention can be used for both detection and diagnosis of canine lymphoma and treatment of canine lymphoma. When used as a therapeutic agent, the canine CD70 monoclonal antibody elicits an immune response when administered into the body of a dog. What you do not want is desirable. Examples of such an antibody include a chimeric antibody containing a canine constant region as an antibody constant region, and a canine antibody in which all variable regions except the constant region and the hypervariable region are replaced with a canine sequence.
　本発明の抗イヌCD70モノクローナル抗体は、イヌのリンパ腫の診断に用いることができる。すなわち、本発明の抗イヌCD70モノクローナル抗体を用いてイヌのリンパ球表面に発現しているCD70を免疫学的に検出することによりイヌがリンパ腫に罹患しているか否かを診断することができる。イヌ被験体のリンパ球にCD70が発現している場合に、該イヌ被験体はリンパ腫に罹患していると判断することができる。リンパ球におけるイヌCD70の検出は、イヌ被験体の末梢血中のリンパ球表面のCD70抗原の発現の有無を検出すればよい。 The anti-canine CD70 monoclonal antibody of the present invention can be used for diagnosis of canine lymphoma. That is, it is possible to diagnose whether or not a dog suffers from lymphoma by immunologically detecting CD70 expressed on the lymphocyte surface of a dog using the anti-canine CD70 monoclonal antibody of the present invention. If CD70 is expressed in the lymphocytes of a canine subject, the canine subject can be determined to be suffering from a lymphoma. Detection of canine CD70 in lymphocytes may be performed by detecting the presence or absence of expression of CD70 antigen on the surface of lymphocytes in the peripheral blood of a canine subject.
　リンパ球表面のCD70抗原の発現は、例えば、抗CD70抗体であって、発色酵素、蛍光化合物等で標識した抗体を用いて細胞が染色されたか否かを顕微鏡観察等により決定することができる。例えば、これらの抗体を用いて細胞を免疫染色して、表面抗原の有無を決定することができ、また該抗体を結合させた磁性ビーズを用いても決定することができる。また、FACS又はフローサイトメーターを用いても表面抗原があるかどうか決定することができる。フローサイトメーターとしては例えばFACSAria(ベクトン・ディッキンソン社製)、FACS vantage(ベクトン・ディッキンソン社製)、FACS Calibur(ベクトン・ディッキンソン社製)等を用いることができる。 The expression of the CD70 antigen on the surface of lymphocytes can be determined by, for example, microscopic observation as to whether or not the cells are stained with an anti-CD70 antibody labeled with a chromogenic enzyme, a fluorescent compound, or the like. For example, cells can be immunostained using these antibodies to determine the presence or absence of surface antigens, or can be determined using magnetic beads to which the antibodies are bound. It can also be determined whether there is a surface antigen using a FACS or flow cytometer. As the flow cytometer, for example, FACSAria (manufactured by Becton Dickinson), FACS vantage (manufactured by Becton Dickinson), FACS Calibur (manufactured by Becton Dickinson) and the like can be used.
　これらの表面抗原が陰性とは、上記のようにFACSを用いて分析した場合に、陽性細胞としてソーティングされないこと、あるいはRT-PCRにより発現を調べた場合に、発現が認められないことをいい、これらの手法により検出できない程度発現していたとしても、本発明においては陰性とする。また、上記マーカーが陽性であることが公知の細胞と同時に測定を行い、これらの陽性細胞と比較して、ほとんど検出されないか、あるいは有意に発現量が低い場合に陰性としてもよい。 When these surface antigens are negative, it means that they are not sorted as positive cells when analyzed using FACS as described above, or that expression is not observed when expression is examined by RT-PCR, Even if it is expressed to such an extent that it cannot be detected by these techniques, it is considered negative in the present invention. Alternatively, the measurement may be performed simultaneously with cells known to be positive for the marker, and may be negative if the expression level is hardly detected or significantly lower than those positive cells.
　この際、あらかじめ健常イヌ個体から採取したリンパ球のCD70の発現レベルを測定しておき、測定値からカットオフ値を定め、イヌ被験体のリンパ球のCD70の発現レベルをカットオフ値と比較して診断することもできる。 At this time, the CD70 expression level of lymphocytes collected from healthy dogs in advance was measured, a cut-off value was determined from the measured value, and the CD70 expression level of lymphocytes in the dog subject was compared with the cut-off value. Can also be diagnosed.
　本発明は、イヌ血液中のリンパ球のイヌCD70の検出を可能にするキットの提供をも目的とするが、該キットは少なくとも抗イヌCD70モノクローナル抗体を含む。 The present invention is also intended to provide a kit that enables detection of canine CD70 in lymphocytes in canine blood, and the kit contains at least an anti-canine CD70 monoclonal antibody.
　本発明の検出方法が対象とする、イヌリンパ腫には、ホジキンリンパ腫、非ホジキンリンパ腫、バーキットリンパ腫、大細胞型Ｂ細胞リンパ腫、マントル細胞リンパ腫、ＭＡＬＴリンパ腫、ろ胞性リンパ腫等が含まれる。 The canine lymphomas targeted by the detection method of the present invention include Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, large B-cell lymphoma, mantle cell lymphoma, MALT lymphoma, follicular lymphoma, and the like.
　本発明の抗CD70モノクローナル抗体をイヌリンパ腫の治療に用いることも可能である。 The anti-CD70 monoclonal antibody of the present invention can also be used for the treatment of canine lymphoma.
　本発明の抗体を含む製剤の投与形態は限定されず、経口、非経口、経粘膜（例えば舌下又は口腔投与）、局所、経皮、直腸、吸入（例えば鼻又は肺奥吸入）等により投与することができる。非経口投与として、静脈内、皮下、筋肉内注射等が挙げられる。局所又は経皮製剤は粉末、エマルジョン、懸濁液、スプレー、クリーム、軟膏、ローション及びペースト等の形態で用いられ、又は薬用膏薬、パッチ又は膜の形で用いられる。治療に用いるに必要な本発明の抗体の量は、治療する病状の性質、被験体の年齢と状態で変わり、最終的には担当獣医が決めることができる。例えば、抗体を１日あたり0.05～２mgの抗体を投与すればよい。所定の投与量は１回の投与で与えてもよいし、１日当たり２回、３回、４回又はそれ以上の分割投与の適当な間隔で与えてもよい。 The dosage form of the preparation containing the antibody of the present invention is not limited, and it is administered orally, parenterally, transmucosally (for example, sublingual or buccal administration), topical, transdermal, rectal, inhalation (for example, nasal or deep lung inhalation), etc. can do. Parenteral administration includes intravenous, subcutaneous, intramuscular injection and the like. Topical or transdermal formulations are used in the form of powders, emulsions, suspensions, sprays, creams, ointments, lotions and pastes, or in the form of medicated salves, patches or films. The amount of the antibody of the present invention required for treatment will vary depending on the nature of the condition being treated, the age and condition of the subject, and can ultimately be determined by the attending veterinarian. For example, the antibody may be administered at 0.05 to 2 mg of antibody per day. The predetermined dose may be given as a single dose, or may be given at appropriate intervals of 2, 3, 4 or more divided doses per day.
　本発明は、抗イヌCD70モノクローナル抗体を有効成分として含む、イヌのリンパ腫治療剤も包含し、さらに、抗イヌCD70モノクローナル抗体をイヌに投与することを含むイヌのリンパ腫の治療法も包含する。さらにイヌCD70を分子標的とした治療剤も本発明の範囲に含まれる。 The present invention also includes a therapeutic agent for canine lymphoma comprising an anti-canine CD70 monoclonal antibody as an active ingredient, and further includes a method for treating canine lymphoma comprising administering an anti-canine CD70 monoclonal antibody to a dog. Furthermore, therapeutic agents targeting canine CD70 as a molecular target are also included in the scope of the present invention.
　イヌのリンパ腫の治療において、CD70発現腫瘍細胞の成長が阻害されるように腫瘍細胞と抗CD70抗体を接触させればよい。 In the treatment of canine lymphoma, the tumor cells may be brought into contact with the anti-CD70 antibody so that the growth of CD70-expressing tumor cells is inhibited.
　本発明を以下の実施例によって具体的に説明するが、本発明はこれらの実施例によって限定されるものではない。
実施例１　イヌリンパ腫におけるCD70の発現解析
　山口大学動物医療センターに来院したイヌのT細胞型リンパ腫3症例より腫瘍細胞を採取し、用いるまで-80℃で保存した。一方コントロールとして、健常ビーグル3頭よりそれぞれ末梢血液を採取し、末梢血単核球を分離後、抗イヌCD3抗体（AbD Serotec社）を用いた磁気ビーズ法によりT細胞を分離し、正常Tリンパ球とした。それぞれのサンプルよりTRI Reagent(Molecular Research Center社)を用いてトータルRNAを抽出し、cDNAマイクロアレイをMiltenyi社に依頼し、腫瘍細胞と正常細胞において発現量に差のある遺伝子群を同定した。その中で同定された遺伝子イヌCD70についてreal time PCR（Superscript IIIおよびQunatiTect SYBR Green PCR Kit）を用いてさらに遺伝子発現を検討した。 The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.
Example 1 CD70 Expression Analysis in Canine Lymphoma Tumor cells were collected from 3 cases of canine T-cell lymphomas who visited the Yamaguchi University Animal Medical Center and stored at −80 ° C. until use. On the other hand, as a control, peripheral blood was collected from each of three healthy beagles, and after isolating peripheral blood mononuclear cells, T cells were separated by magnetic bead method using anti-canine CD3 antibody (AbD Serotec), and normal T lymphocytes were isolated. A sphere. Total RNA was extracted from each sample using TRI Reagent (Molecular Research Center), and a cDNA microarray was commissioned to Miltenyi to identify gene groups with different expression levels in tumor cells and normal cells. The gene expression of the canine CD70 identified therein was further examined using real time PCR (Superscript III and QunatiTect SYBR Green PCR Kit).
　cDNAマイクロアレイの結果、発現上昇が認められた遺伝子中の中から選択したCD70のイヌのリンパ腫における発現量をRealtime PCRによって検討した。10種類のイヌのリンパ腫腫瘍細胞株（GL-1, CL-1, Ema, UL-1, Nody-1, CLBL-1. CLGL90, 17-71, CLL1390, 3132）及び38例のイヌのリンパ腫由来症例においては、健常イヌ由来無刺激リンパ球、刺激Tリンパ球及び刺激Bリンパ球と比較してCD70の発現上昇が認められるサンプルが存在した。一方で、健常イヌ由来無刺激リンパ球、刺激Tリンパ球及び刺激Bリンパ球とリンパ腫腫瘍細胞株及びリンパ腫由来症例のCD70遺伝子発現量の比較を行ったところ、有意差を認めなかった（図１）。
実施例２　イヌCD70のCD70分子の遺伝子クローニング及び塩基配列の決定
　健常イヌより採取した肺及びConA活性化リンパ球より抽出したRNAを用いてsuperscript III cDNA合成キットを用いて作製したcDNAを鋳型に、YTM946（ggagggctgacgtttcatc（配列番号５））及びYTM947（aagagaaaaacacattctcccaat（配列番号６））、YTM1277（agctcccctgagtgagaacc（配列番号７））及びYTM1278（tgggatagaggctgaggttt（配列番号８））をプライマーとして用いて、KOD Plus neo によってイヌCD70分子及びイヌCD27分子の遺伝子クローニングを行ない、塩基配列を決定した（図２）。
実施例３　イヌCD70分子をラット腎臓細胞株NRKに過剰発現させた細胞株（NRK/cCD70）の作製及び抗イヌCD70モノクローナル抗体の作製
　イヌのCD70分子をpMx-IPベクター（東京大学医科学研究所北村俊雄博士より分与）に組み込み、PLAT-gp細胞（東京大学医科学研究所北村俊雄博士より分与）にpCVSVGとともに遺伝子導入したあと産生されたレトロウイルスを用いて、ラット腎臓細胞株NRKに感染させることにより、イヌCD70過剰発現細胞(NRK/cCD70)を得た。NRK/cCD70をSDラットに免疫することにより、イヌCD70に対するラットモノクローナル抗体(4F2-12)を作製した。これら抗体をprotein Aカラムで精製後、ラットIgG2a, kappa鎖であることを確認した。またNRK/cCD70細胞と各種濃度に希釈した4F12-E6抗体をインキュベートし、さらに二次抗体を反応させ、その後フローサイトメトリー（Becton Dickinson社、BD Accuri）により蛍光強度を測定することにより、濃度依存的にNRK/cCD70に結合することを確認した（図３）。
実施例４　イヌCD70リガンド(CD27)とヒトIg Fc領域との融合蛋白 cCD27-hIgの作製
　CD70のリガンドであるイヌCD27の細胞外領域をPCRによって増幅し、pFUSE-hIgF2-Fc (Invivogen社)ベクターに組み込み、それをHEK293T細胞にトランスフェクとし、培養上清を回収した。さらに、それら培養上清より、Protein Aビーズを用いて、ヒトIgG2のFc領域の融合蛋白cCD27-hIgを作製した。これら融合蛋白を用いて、実施例3と同様にして、NRK/cCD70との結合を確認した。その結果、cCD27-hIgは濃度依存的にNRK/cCD70に結合することが確認できた（図４Ａ）。
実施例５　抗イヌCD70モノクローナル抗体(4F2-12)の性質の検定
　さらに実施例３で得られた抗イヌCD70抗体 (4F2-12)がcCD27-hIgとNRK/cCD70の結合を阻害できるかを、実施例3と同様にして検討した。すなわちNRK/cCD70に4F2-12を1-10μg/mlで反応させ、そのあとcCD27-hIgを100μg/mlで反応させたところ、4F2-12の濃度にかかわらず、cCD27-hIgはNRK/cCD70に結合可能であることが明らかとなった。この結果より、4F2-12抗体は、イヌCD70とイヌCD27の結合阻害効果は認められなかった（図４Ｂ）。
実施例６　抗体の重鎖及び軽鎖の可変領域の塩基配列の決定
　実施例３において得られた各抗体の重鎖及び軽鎖の可変領域の塩基配列を決定した。塩基配列は以下の方法で決定した。 As a result of cDNA microarray, the expression level of CD70 selected from among genes whose expression was increased was examined in canine lymphoma by Realtime PCR. 10 canine lymphoma tumor cell lines (GL-1, CL-1, Ema, UL-1, Nody-1, CLBL-1. CLGL90, 17-71, CLL1390, 3132) and 38 canine lymphomas In the case, there was a sample in which increased expression of CD70 was observed as compared with unstimulated lymphocytes, stimulated T lymphocytes and stimulated B lymphocytes derived from healthy dogs. On the other hand, when the expression levels of CD70 gene in healthy dog-derived unstimulated lymphocytes, stimulated T lymphocytes and stimulated B lymphocytes, and lymphoma tumor cell lines and lymphoma-derived cases were compared, no significant difference was observed (FIG. 1). ).
Example 2 Cloning of the gene of the CD70 molecule of canine CD70 and determination of the nucleotide sequence cDNA prepared using the superscript III cDNA synthesis kit using RNA extracted from lungs and ConA-activated lymphocytes collected from healthy dogs as a template, Using YTM946 (ggagggctgacgtttcatc (SEQ ID NO: 5)) and YTM947 (aagagaaaaacacattctcccaat (SEQ ID NO: 6)), YTM1277 (agctcccctgagtgagaacc (SEQ ID NO: 7)) and YTM1278 (tgggatagaggctgaggttt (SEQ ID NO: 8)) as primers, KOD Plus neo Gene cloning of CD70 molecule and canine CD27 molecule was performed, and the nucleotide sequence was determined (FIG. 2).
Example 3 Production of a cell line (NRK / cCD70) in which a canine CD70 molecule was overexpressed in a rat kidney cell line NRK and production of an anti-canine CD70 monoclonal antibody A canine CD70 molecule was transformed into a pMx-IP vector (Institute of Medical Science, University of Tokyo) Incorporated into Drat Toshio Kitamura) and retrovirus produced after gene transfer with pCVSVG into PLAT-gp cells (distributed by Dr. Toshio Kitamura, Institute of Medical Science, The University of Tokyo) By infecting, canine CD70 overexpressing cells (NRK / cCD70) were obtained. A rat monoclonal antibody (4F2-12) against canine CD70 was produced by immunizing SD rats with NRK / cCD70. These antibodies were purified with a protein A column and confirmed to be rat IgG2a and kappa chains. In addition, NRK / cCD70 cells and 4F12-E6 antibody diluted to various concentrations are incubated, further reacted with a secondary antibody, and then the fluorescence intensity is measured by flow cytometry (Becton Dickinson, BD Accuri). Was confirmed to bind to NRK / cCD70 (FIG. 3).
Example 4 Production of Fusion Protein cCD27-hIg of Canine CD70 Ligand (CD27) and Human Ig Fc Region The extracellular region of canine CD27, which is a ligand for CD70, was amplified by PCR, and pFUSE-hIgF2-Fc (Invivogen) vector And transferred to HEK293T cells, and the culture supernatant was collected. Furthermore, a fusion protein cCD27-hIg of the Fc region of human IgG2 was prepared from these culture supernatants using Protein A beads. Using these fusion proteins, binding to NRK / cCD70 was confirmed in the same manner as in Example 3. As a result, it was confirmed that cCD27-hIg bound to NRK / cCD70 in a concentration-dependent manner (FIG. 4A).
Example 5 Assay for Properties of Anti-Canine CD70 Monoclonal Antibody (4F2-12) Further, whether the anti-canine CD70 antibody (4F2-12) obtained in Example 3 can inhibit the binding between cCD27-hIg and NRK / cCD70, The examination was performed in the same manner as in Example 3. That is, when 4F2-12 was reacted with NRK / cCD70 at 1-10 μg / ml and then cCD27-hIg was reacted at 100 μg / ml, cCD27-hIg was reacted with NRK / cCD70 regardless of the concentration of 4F2-12. It became clear that they could be combined. From these results, the 4F2-12 antibody did not show the binding inhibitory effect between canine CD70 and canine CD27 (FIG. 4B).
Example 6 Determination of base sequence of variable region of antibody heavy chain and light chain The base sequence of the variable region of heavy chain and light chain of each antibody obtained in Example 3 was determined. The base sequence was determined by the following method.
　抗イヌCD70モノクローナル抗体を産生するハイブリドーマからSV total RNA isolation (promega)を用いてtotal RNAを抽出し、これを鋳型としてSMARTerlM RACE cDNA Ampllittion Kit(TaKaRa)とラット抗体(IgG)定常領域に特異的なプライマーを用いて逆転写反応を実施した。 Total RNA was extracted from hybridomas producing anti-canine CD70 monoclonal antibody using SV total RNA isolation (promega), and this was used as a template for SMARTerlM RACE DNA cDNA Ampllittion Kit (TaKaRa) and rat antibody (IgG) specific regions. Reverse transcription reaction was performed using primers.
　得られたcDNAを鋳型としてPrimeSTAR GXL DNA polymerase(TaKaRa)とラットIgG定常領域に特異的なプライマーを使用してRACE PCRを実施した。得られたPCR産物をpGEM easy T-vector(Promega)にクローニングした後、ランダムにピックアップしたクローンについて遺伝子配列の解析を実施した。 Using the obtained cDNA as a template, RACE PCR was performed using PrimeSTAR® GXL DNA polymerase (TaKaRa) and primers specific to the rat IgG constant region. The obtained PCR product was cloned into pGEM easy T-vector (Promega), and then the gene sequence of the randomly picked clone was analyzed.
　逆転写には、以下に記すH-RT1およびL-RT1をプライマーとして使用した。 For reverse transcription, H-RT1 and L-RT1 described below were used as primers.
　PCRにはH-PCR-N1、H-PCR-N2およびL-PCRをプライマーとして使用した。 In PCR, H-PCR-N1, H-PCR-N2 and L-PCR were used as primers.
　表１にプライマーの配列を示す。 Table 1 shows the primer sequences.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　抗体の重鎖可変領域及び軽鎖可変領域の塩基配列をそれぞれ、配列番号１及び３に示し、それぞれの推定アミノ酸配列を配列番号２及び４に示す。
実施例７　抗CD70抗体を用いてフローサイトメトリーによりイヌのリンパ腫症例より採取した腫瘍細胞におけるCD70の発現解析
　イヌリンパ腫腫瘍細胞株9例（T細胞型 Ema, Nody-1, CLC, UL-1, CLGL90, CLK; B細胞型 CLBL-1, GL-1, 17-71）、イヌリンパ腫症例2例(B細胞型)、慢性リンパ性白血病（ＣＬＬ）症例1例より採取した腫瘍細胞を用いてフローサイトメトリー（Becton Dickinson社、BD Accuri）によりイヌCD70の発現を検討したところ、腫瘍細胞株では5例において（図５Ａ）、症例では3例全てにおいて（図５Ｂ）イヌCD70の発現が検出された。
実施例８　抗CD70抗体を用いてフローサイトメトリーによりイヌの末梢血リンパ球におけるCD70の発現解析
　健常ビーグル4頭の末梢血より分離したリンパ球、さらにそれをConA刺激（5μg/ml）又はLPS (5μg/ml)及びCD40リガンドで2日間刺激した細胞を用いてフローサイトメトリー（Becton Dickinson社、BD Accuri）によりイヌCD70の発現を検討したところ、いずれの細胞においてもイヌCD70の発現は検出されなかった（図６）。
実施例９　抗CD70抗体を用いて、健常イヌの各種臓器におけるCD70の発現解析
　イヌリンパ腫症例より29例より採取した腫瘍組織を用いて免疫染色を行ったところ、12例においてイヌCD70の発現が検出された。図７に典型的な免疫染色像を示す。 The nucleotide sequences of the heavy chain variable region and the light chain variable region of the antibody are shown in SEQ ID NOs: 1 and 3, respectively, and the deduced amino acid sequences are shown in SEQ ID NOs: 2 and 4, respectively.
Example 7 Expression analysis of CD70 in tumor cells collected from canine lymphoma cases by flow cytometry using anti-CD70 antibody Nine canine lymphoma tumor cell lines (T cell types Ema, Nody-1, CLC, UL-1, CLGL90, CLK; B cell type CLBL-1, GL-1, 17-71), 2 canine lymphoma cases (B cell type), chronic lymphocytic leukemia (CLL) 1 tumor flow When the expression of canine CD70 was examined by cytometry (Becton Dickinson, BD Accuri), expression of canine CD70 was detected in 5 cases in the tumor cell line (FIG. 5A) and in all 3 cases (FIG. 5B). .
Example 8 Analysis of CD70 Expression in Canine Peripheral Blood Lymphocytes by Flow Cytometry Using Anti-CD70 Antibody Lymphocytes isolated from the peripheral blood of 4 healthy beagle heads, and further treated with ConA stimulation (5 μg / ml) or LPS ( 5 μg / ml) and cells stimulated with CD40 ligand for 2 days were examined for canine CD70 expression by flow cytometry (Becton Dickinson, BD Accuri). No canine CD70 expression was detected in any of the cells. (FIG. 6).
Example 9 Analysis of CD70 expression in various organs of healthy dogs using anti-CD70 antibodies Immunostaining was performed on tumor tissues collected from 29 cases from canine lymphoma cases. Expression of canine CD70 was detected in 12 cases It was done. FIG. 7 shows a typical immunostained image.
　本発明の抗イヌCD70抗体をイヌリンパ腫の診断及び治療に用いることができる。 The anti-canine CD70 antibody of the present invention can be used for diagnosis and treatment of canine lymphoma.
配列番号５～１３　プライマー
　本明細書で引用した全ての刊行物、特許及び特許出願はそのまま引用により本明細書に組み入れられるものとする。 SEQ ID NOs: 5-13 Primer All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims (16)

  1. 　配列番号２で表されるアミノ酸配列からなる重鎖可変領域及び配列番号４で表されるアミノ酸配列からなる軽鎖可変領域を含む、イヌCD70に結合する抗イヌCD70モノクローナル抗体又はイヌCD70に結合するその機能的断片。 An anti-canine CD70 monoclonal antibody that binds to canine CD70 or a canine CD70, comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 4 Its functional fragment.
  2. 　配列番号２で表されるアミノ酸配列と90％以上の配列同一性を有するアミノ酸配列からなり、イヌCD70への結合活性を有する重鎖可変領域、及び配列番号４で表されるアミノ酸配列と90％以上の配列同一性を有するアミノ酸配列からなり、イヌCD70への結合活性を有する軽鎖可変領域を含む、イヌCD70に結合する抗イヌCD70モノクローナル抗体又はイヌCD70に結合するその機能的断片。 A heavy chain variable region comprising an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 2 and having binding activity to canine CD70, and 90% with the amino acid sequence represented by SEQ ID NO: 4 An anti-canine CD70 monoclonal antibody that binds to canine CD70 or a functional fragment thereof that binds to canine CD70, comprising a light chain variable region comprising the amino acid sequence having the above sequence identity and having binding activity to canine CD70.
  3. 　配列番号１で表される塩基配列からなるDNAがコードする重鎖可変領域及び配列番号３で表される塩基配列からなるDNAがコードする軽鎖可変領域を含む、イヌCD70に結合する抗イヌCD70モノクローナル抗体又はイヌCD70に結合するその機能的断片。 An anti-canine CD70 that binds to canine CD70, comprising a heavy chain variable region encoded by DNA consisting of the base sequence represented by SEQ ID NO: 1 and a light chain variable region encoded by DNA consisting of the base sequence represented by SEQ ID NO: 3. A monoclonal antibody or a functional fragment thereof that binds to canine CD70.
  4. 　配列番号１で表される塩基配列と90％以上の配列同一性を有する塩基配列がコードするイヌCD70への結合活性を有する重鎖可変領域、及び配列番号３で表される塩基配列と90％以上の配列同一性を有する塩基配列がコードするイヌCD70への結合活性を有する軽鎖可変領域を含むイヌCD70に結合する抗イヌCD70モノクローナル抗体又はイヌCD70に結合するその機能的断片。 A heavy chain variable region having binding activity to canine CD70 encoded by a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence represented by SEQ ID NO: 1, and 90% with the nucleotide sequence represented by SEQ ID NO: 3 An anti-canine CD70 monoclonal antibody that binds to canine CD70 or a functional fragment that binds to canine CD70, comprising a light chain variable region having a binding activity to canine CD70 encoded by the base sequence having the above sequence identity.
  5. 　重鎖定常領域及び軽鎖定常領域がイヌIgG抗体の定常領域である、請求項１～４のいずれか１項に記載のイヌCD70に結合する抗イヌCD70モノクローナル抗体又はイヌCD70に結合するその機能的断片。 The anti-canine CD70 monoclonal antibody that binds to canine CD70 or the function thereof that binds to canine CD70 according to any one of claims 1 to 4, wherein the heavy chain constant region and the light chain constant region are constant regions of a canine IgG antibody. Fragment.
  6. 　機能的断片がFab、Fab’、F(ab’)2、ジスルフィド結合Fv(dsFv) 、二量体化V領域（diabody）、一本鎖Fv（scFv）及びCDRからなる群から選択されるペプチド断片である請求項１～５のいずれか１項に記載のイヌCD70に結合する抗イヌCD70モノクローナル抗体又はイヌCD70に結合するその機能的断片。 Peptides wherein the functional fragment is selected from the group consisting of Fab, Fab ′, F (ab ′) 2 , disulfide bond Fv (dsFv), dimerization V region (diabody), single chain Fv (scFv) and CDR The anti-canine CD70 monoclonal antibody that binds to canine CD70 or a functional fragment thereof that binds to canine CD70 according to any one of claims 1 to 5, which is a fragment.
  7. 　配列番号２で表されるアミノ酸配列からなる、抗イヌCD70モノクローナル抗体の重鎖可変領域であるポリペプチド。 A polypeptide which is a heavy chain variable region of an anti-canine CD70 monoclonal antibody consisting of the amino acid sequence represented by SEQ ID NO: 2.
  8. 　配列番号４で表されるアミノ酸配列からなる、抗イヌCD70モノクローナル抗体の軽鎖可変領域であるポリペプチド。 A polypeptide which is a light chain variable region of an anti-canine CD70 monoclonal antibody consisting of the amino acid sequence represented by SEQ ID NO: 4.
  9. 　配列番号１で表されるDNA配列からなる、抗イヌCD70モノクローナル抗体の重鎖可変領域をコードするポリヌクレオチド。 A polynucleotide encoding the heavy chain variable region of an anti-canine CD70 monoclonal antibody comprising the DNA sequence represented by SEQ ID NO: 1.
  10. 　配列番号３で表されるDNA配列からなる、抗イヌCD70モノクローナル抗体の軽鎖可変領域をコードするポリヌクレオチド。 A polynucleotide encoding the light chain variable region of the anti-canine CD70 monoclonal antibody consisting of the DNA sequence represented by SEQ ID NO: 3.
  11. 　請求項９に記載のポリヌクレオチド、請求項１０に記載のポリヌクレオチド、又は請求項９に記載のポリヌクレオチドと請求項１０に記載のポリヌクレオチドを含むベクター。 A polynucleotide comprising the polynucleotide according to claim 9, the polynucleotide according to claim 10, or the polynucleotide according to claim 9 and the polynucleotide according to claim 10.
  12. 　請求項１１に記載のベクターを含む細胞。 A cell comprising the vector according to claim 11.
  13. 　配列番号１で表されるDNA配列からなる、抗イヌCD70モノクローナル抗体の重鎖可変領域をコードするDNAを抗体の重鎖定常領域をコードするDNAと連結し、配列番号３で表されるDNA配列からなる、抗イヌCD70モノクローナル抗体の軽鎖可変領域をコードするDNAを抗体の軽鎖定常領域をコードするDNAと連結し、得られたDNAコンストラクトを発現ベクターに挿入し、該ベクターで宿主細胞又は宿主動物を形質転換し、該宿主細胞又は宿主動物により抗体を産生させることを含む、抗イヌCD70モノクローナル抗体の製造方法。 A DNA sequence comprising the DNA sequence represented by SEQ ID NO: 1 and ligating the DNA encoding the heavy chain variable region of the anti-canine CD70 monoclonal antibody with the DNA encoding the heavy chain constant region of the antibody, and the DNA sequence represented by SEQ ID NO: 3 The DNA encoding the light chain variable region of the anti-canine CD70 monoclonal antibody is ligated with the DNA encoding the light chain constant region of the antibody, and the resulting DNA construct is inserted into an expression vector, A method for producing an anti-canine CD70 monoclonal antibody, comprising transforming a host animal and producing the antibody by the host cell or host animal.
  14. 　請求項１～６のいずれか１項に記載のイヌCD70に結合する抗イヌCD70モノクローナル抗体又はイヌCD70に結合するその機能的断片を用いて、イヌ末梢血のリンパ球に発現するCD70を測定し、リンパ球にCD70が発現している場合に、リンパ腫に罹患していると判断するイヌリンパ腫の診断方法。 Using the anti-canine CD70 monoclonal antibody that binds to canine CD70 or a functional fragment thereof that binds to canine CD70 according to any one of claims 1 to 6, CD70 expressed in lymphocytes of canine peripheral blood is measured. A method for diagnosing canine lymphoma, which is judged to be affected by lymphoma when CD70 is expressed in lymphocytes.
  15. 　フローサイトメーターを用いて行う、請求項１４記載の診断方法。 The diagnostic method according to claim 14, which is performed using a flow cytometer.
  16. 　請求項１～６のいずれか１項に記載のイヌCD70に結合する抗イヌCD70モノクローナル抗体又はイヌCD70に結合するその機能的断片を含む、イヌリンパ腫の検出試薬。 A canine lymphoma detection reagent comprising the anti-canine CD70 monoclonal antibody that binds to canine CD70 or a functional fragment thereof that binds to canine CD70 according to any one of claims 1 to 6.
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