WO2017138471A1 - Anticorps monoclonal anti-cd70 du chien - Google Patents

Anticorps monoclonal anti-cd70 du chien Download PDF

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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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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.

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Abstract

La présente invention concerne une méthode permettant de diagnostiquer un lymphome chez un chien. Ladite méthode permettant de diagnostiquer un lymphome chez un chien consiste à : mesurer les CD70 exprimés dans les lymphocytes dans le sang périphérique collecté du chien à l'aide d'un anticorps monoclonal anti-CD70 du chien qui peut se lier aux CD70 canins ou d'un fragment fonctionnel dudit anticorps monoclonal qui peut se lier aux CD70 canins ; et déterminer que le chien souffre d'un lymphome lorsque des CD70 sont exprimés dans les lymphocytes.
PCT/JP2017/004147 2016-02-08 2017-02-06 Anticorps monoclonal anti-cd70 du chien WO2017138471A1 (fr)

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JP2016022105A JP2017141172A (ja) 2016-02-08 2016-02-08 抗イヌｃｄ７０モノクローナル抗体
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WO2022002019A1 (fr) 2020-06-30 2022-01-06 江苏恒瑞医药股份有限公司 Anticorps anti-cd70 et son application
CN113754769A (zh) * 2021-10-13 2021-12-07 宜明昂科生物医药技术（上海）有限公司 靶向cd70的抗体及其制备和用途
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