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WO2010047117A1 - Nouvel anticorps monoclonal et son utilisation - Google Patents

Nouvel anticorps monoclonal et son utilisation Download PDF

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Publication number
WO2010047117A1
WO2010047117A1 PCT/JP2009/005547 JP2009005547W WO2010047117A1 WO 2010047117 A1 WO2010047117 A1 WO 2010047117A1 JP 2009005547 W JP2009005547 W JP 2009005547W WO 2010047117 A1 WO2010047117 A1 WO 2010047117A1
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Prior art keywords
monoclonal antibody
btn3
lymphocytes
antibody
transmembrane molecule
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Japanese (ja)
Inventor
山城宏道
只木敏雅
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Medinet Co Ltd
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Medinet Co Ltd
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Priority to JP2010534713A priority Critical patent/JP5686600B2/ja
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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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation

Definitions

  • the present invention relates to a novel monoclonal antibody that specifically recognizes butyrophilin (BTN) 3 antigen. Furthermore, it is related with the use of the said monoclonal antibody.
  • BTN butyrophilin
  • a cell is created by creating a narrow channel through which cells pass one by one, detecting scattered light and fluorescence generated when a laser beam strikes the cells flowing through the cell, and analyzing the obtained information. Can know the situation.
  • a method of performing flow cytometry analysis by staining cells with an antibody labeled with a fluorescent substance is generally known.
  • cell surface antigen molecules monoclonal antibodies against hematopoietic cells are classified into clusters based on reaction specificity under the supervision of WHO (World Health Organization) at an international workshop on human leukocyte differentiation antigens. of Differentiation) number.
  • the CD number of each antibody corresponds to a specific cell surface antigen molecule.
  • BTN antigen is known as one of cell surface antigens.
  • BTN antigens belong to the immunoglobulin superfamily and are transmembrane molecules classified into BTN1, BTN2, and BTN3 by subfamily. More specifically, there is a member classification. For example, BTN3 is classified as BTN3A1, BTN3A2, and BTN3A3.
  • BTN3A1 (butyrophilin, subfamily 3, member A1) has been identified as CD277 with a molecular weight of 56 kD, and this antigen is a general lymphocyte such as T cells, B cells, NK (natural killer) cells, monocytes, and dendritic cells. Expression has been confirmed. A monoclonal antibody against this has already been acquired by a group in France and has been commercialized as a reagent by eBioscience (Affinity purified anti-human CD277 (B7 Family, Butyrophilin), trade name: eBioBT3.1). However, the feature only recognizes BTN3 specifically, and the function and characteristics of BTN3 have not been clarified (Non-patent Documents 1 and 2).
  • BTN3A3 antibody As a mouse polyclonal antibody against BTN3A3, for example, (BTN3A3 antibody; abcam) (Non-patent Document 3) obtained by an Italian group has been commercialized as a reagent, similar to BT3.1.
  • BTN3 It is important to understand the functions and properties of BTN3 that have not been clarified so far. By using the properties of BTN3, it is possible to further improve the medical field such as treatment of autoimmune diseases, immune cell therapy, and organ transplantation. Development can be expected.
  • Examples of methods for suppressing cell proliferation include orthoclone OKT3 injection (Muromonab-CD3 injection solution; Janssen Pharma) consisting of anti-CD3 antibody as an immunosuppressive agent, but this method uses only T cells that express CD3. It is a thing to suppress. There are no reports of molecules involved in the suppression of proliferation of lymphocytes in general.
  • Examples of a method for distinguishing activated cells from inactive cells include molecules expressed by lymphocyte activation such as CD25 and CD69, which are known as activation markers. However, there are few known examples of molecules that are expressed in inactive lymphocytes and decreased in activated lymphocytes.
  • the present invention has been made in view of the above circumstances, and an object of the present invention is to provide a novel monoclonal antibody that specifically recognizes BTN3 present on the cell surface and a method of using the same. Furthermore, it is an object to provide a reagent, a medicine and the like prepared using the antibody.
  • the inventors of the present invention have intensively studied to solve the above problems, and have completed the present invention by obtaining a monoclonal antibody that specifically recognizes BTN3 (hereinafter referred to as the present antibody).
  • BTN3 which has not been clarified so far by further research using the antibody of the present invention, is involved in the suppression of cell proliferation, and the activation of lymphocytes and the expression of BTN3 are inversely correlated.
  • the present invention has been completed. That is, the present invention provides the following means.
  • (6) a method for measuring the proportion of activated or inactive lymphocytes in a population of lymphocytes, the step of binding a monoclonal antibody that specifically recognizes the transmembrane molecule BTN3 to the population of lymphocytes; A step of detecting a monoclonal antibody bound to lymphocytes, and a step of calculating a ratio of activated lymphocytes or inactive lymphocytes in a population of lymphocytes, wherein the lymphocytes bound to the monoclonal antibodies are inactive lymphocytes, A measuring method consisting of (7) the measurement method according to (6), wherein the transmembrane molecule BTN3 is BTN3A1, BTN3A2 or BTN3A3; (8) the monoclonal antibody is 35 from the N-terminus of the transmembrane molecule BTN3; The method according to (6) or (7), characterized by recognizing -40 amino acids as an antigen; (9) the monoclonal antibody produced from a hybridoma having the microorganism deposit
  • (23) A method of suppressing lymphocyte proliferation using a monoclonal antibody that specifically recognizes the transmembrane molecule BTN3; (24) the transmembrane molecule BTN3 is BTN3A1, BTN3A2, or BTN3A3 ( (25) The method according to (23) or (24), wherein the monoclonal antibody recognizes 35-40 amino acids from the N-terminus of the transmembrane molecule BTN3 as an antigen.
  • the monoclonal antibody acts to suppress proliferation of lymphocytes;
  • the monoclonal antibody comprises: The same antigen recognition as the antibody produced from the hybridoma of microorganism deposit number FERM BP-11177 or FERM BP-11178 The method suppression of any one of (26) from the constitution (23) to have a site.
  • a lymphocyte proliferation inhibitor comprising a monoclonal antibody that specifically recognizes the transmembrane molecule BTN3;
  • the transmembrane molecule BTN3 is BTN3A1, BTN3A2 or BTN3A3 (28)
  • the monoclonal antibody recognizes 35-40 amino acids from the N-terminus of the transmembrane molecule BTN3 as an antigen, according to (28) or (29), (31)
  • (32) Is the monoclonal antibody a hybridoma having the microorganism deposit number FERM BP-11177 or FERM BP-11178? Growth inhibitor of lymphocytes according to any one of (31) from and having an antibody of the same antigen recognition site produced (28).
  • An inversely correlated marker of activated lymphocytes comprising a transmembrane molecule BTN3; (34) the marker according to (33), wherein the transmembrane molecule BTN3 is BTN3A1, BTN3A2 or BTN3A3; A method for detecting inactive lymphocytes, comprising binding the marker according to (33) or (34) to a monoclonal antibody that specifically binds to the marker, and examining the expression level of the marker; 36) The inactive lymphocyte according to (35), wherein the monoclonal antibody has the same antigen recognition site as an antibody produced from a hybridoma having the microorganism accession number FERM BP-11177 or FERM BP-11178 Detection method.
  • the antibody of the present invention has an affinity that is 8 to 9 times that of the currently available anti-CD277 antibody (eBioBT3.1, hereinafter referred to as BT3.1), so that the expression of BTN3 can be detected more clearly. It is.
  • the antibody of the present invention has an inhibitory effect on lymphocytes. Therefore, for example, when developed as a monoclonal antibody drug, it may be used as an immunosuppressant after autoimmune disease or organ transplantation.
  • the present inventors have found that the expression of BTN3 is decreased in lymphocytes that have enjoyed an activation signal, as a result of research using the antibody of the present invention.
  • This phenomenon can be observed to some extent using the above-mentioned BT3.1, but can be more clearly identified by using the antibody of the present invention having a higher affinity.
  • how many cells stimulate the activation in vitro. It is possible to immediately determine whether or not it has been received. For example, when performing immune cell therapy in which lymphocytes collected from patient blood are proliferated and activated in large quantities in vitro and administered to the patient's body, they are used as a measurement method / device to know the proportion of activated lymphocytes It is possible.
  • BTN3 molecule expression decreases in activated lymphocytes, for example, by applying it to an adsorption negative selection column using the antibody of the present invention, a cell population effective for activated anti-tumor immunity can be selectively selected. Can be recovered.
  • FIG. 7 is a diagram showing the results of experiments similar to FIG. 1 performed using IgG2a, 232-5 antibody, and 34-7 antibody.
  • the fluorescence intensity of the antibody of the present invention decreased depending on the concentration of BT3.1. It is the figure which showed that both BT3.1 and this invention antibody recognize the epitope of BTN3 antigen vicinity.
  • FIG. 5 is a diagram showing the results of an experiment similar to FIG. 4 performed using BT3.1, 232-5 antibody and 34-7 antibody.
  • a plasmid for estimating an antigen recognition site in the Vset region of the BTN3 antigen was prepared and subjected to Western blotting.
  • FIG. 7 is a diagram showing the results of experiments similar to FIG. 6 performed using BT3.1, 232-5 antibody and 34-7 antibody. Antigen affinity for BTN3 antigen was compared.
  • FIG. 5 is a view showing that the antibody of the present invention has an affinity of 8 to 9 times at most as compared with BT3.1. It is the figure which verified the expression of BTN3 antigen in various lymphocyte populations with the flow cytometer. The antibody of the present invention recognized most (> 95%) of the cell surface of each lymphocyte population.
  • FIG. 11 is a diagram showing the results of experiments similar to FIG. 10 performed using IgG2a, BT3.1, 232-5 antibody, and 34-7 antibody.
  • A It is the figure which activated only a T cell selectively and confirmed that the expression reduction of BTN3 occurred only in the activated T cell.
  • B It is the figure which confirmed that the expression reduction of BTN3 occurred only in the activated B cell by selectively activating only B cell.
  • FIG. 2 is a diagram in which PBMCs were stimulated with OKT3 / rIL-2 for 72 hours, mRNA was prepared, and the expression of BTN3A1, BTN3A2, and BTN3A3 was verified using cDNA obtained therefrom.
  • the expression of the three types of BTN3 molecules was maintained in PBMCs that were not treated or cultured only in the medium, but the expression decreased by culturing with OKT3 / rIL-2. It is a figure showing that CD8 positive T cells are more markedly upregulated in activation marker CD25 and decreased in expression of BTN3 than CD4 positive T cells.
  • the antibody of the present invention against BTN3 molecule is a monoclonal antibody that specifically recognizes BTN3, which is a transmembrane molecule.
  • the transmembrane molecule BTN3 means butyrophilin, subfamily 3, which belongs to the immunoglobulin superfamily and is classified into 1 to 3 by subfamily. Further classified into members is BTN3A1 (member A1), which has been identified as CD277 having a molecular weight of 56 kD. Expression of this antigen has been confirmed in all lymphocytes such as T cells, B cells, NK cells, monocytes, and dendritic cells.
  • SEQ ID Nos. 1 and 2 in the sequence listing show the gene sequence and amino acid sequence of BTN3A1, SEQ ID Nos. 3 and 4 show the gene sequence and amino acid sequence of BTN3A2, and SEQ ID Nos. 5 and 6 show the gene sequence and amino acid sequence of BTN3A3, respectively. ing.
  • the extracellular domain is the same for all three molecules, and is 1 to 248 of the amino acid sequence shown in the Sequence Listing (SEQ ID NO: 2, 4 or 6 respectively).
  • BTN3 recognized by the antibody of the present invention is often expressed in an inactive state in lymphocytes, but it has been revealed that the expression decreases upon receiving an activation signal.
  • the antibody of the present invention has the property of specifically binding to inactive lymphocytes.
  • the antibody of the present invention may be any cloned immunoglobulin antibody, and the animal species from which the antibody is derived, the type and subtype of the immunoglobulin, and the antibody production method are not limited. Also included are those obtained by fragmenting an antibody to leave an immune reaction site, a repaired product of these fragments, a modified product of the antibody itself, a chimeric antibody in which two types of antibodies are bound, and the like.
  • the antibody of the present invention can be prepared by appropriately using a conventionally known method. Specifically, for example, plasmid DNA (deoxyribonucleic acid) incorporating a gene sequence encoding a target antigen is prepared, and animals are immunized. B cells obtained from the immunized animal and established myeloma cells are fused, and a hybridoma that reacts with the target antigen is purely cloned and established. By extracting and purifying the antibody produced from the hybridoma, the necessary amount can be obtained when necessary.
  • the animal species of the immunized animal is not particularly limited, but generally mice, rats, hamsters, rabbits and the like are used.
  • immunogens for immunizing animals such as mice and rats
  • a BTN3 protein or peptide fragment thereof, a vector into which a gene encoding BTN3 or a fragment thereof has been introduced, a transfectant expressing BTN3, and the like can be mentioned.
  • any of the above-described immunogens can be used.
  • the extramembrane region is used as an immunogen, or in particular, the Vset region (SEQ ID NO: 2, 4 or 6). Or a region of 26 to 139 of the amino acid sequence described in 1).
  • the antibody of the present invention is an antibody characterized by recognizing 35-40 amino acids from the N-terminus of BTN3 as an antigen
  • a peptide containing 35-40 amino acids from the N-terminus of BTN3 can also be used as an immunogen.
  • the 35-40 amino acids from the N-terminus of BTN3 refer to the amino acid sequence from the 35th to the 40th amino acid sequence described in SEQ ID NO: 2, 4 or 6. Since the 35th to 40th amino acid sequences are common to BTN3A1, BTN3A2, and BTN3A3, using a peptide containing this part as an immunogen results in an antibody that recognizes three BTN3 molecules.
  • Immunization of immunized animals eg, mice
  • a mixture of a vector in which a gene sequence encoding BTN3 is inserted and an immunostimulant such as colloidal gold is introduced with a gene gun at 8-10 weeks of age in Balb / c mice and bred for about 2 to 3 months.
  • the increase in the antibody titer against BTN3 can be confirmed by the following procedure. 1) The gene insertion vector is introduced into an appropriate cell (for example, CHO cell) to produce a cell in which BTN3 is transiently expressed. 2) Mix with sera collected from immunized animals (containing polyclonal antibody against BTN3). 3) Perform flow cytometer analysis using a fluorescently labeled anti-mouse polyclonal antibody (secondary antibody). As described above, it is possible to confirm whether or not an antibody that recognizes the three-dimensional structure outside the cell membrane of BTN3 is obtained by confirming the antibody titer using a cell in which BTN3 is transiently expressed. it can.
  • the immunized animal injects cells (for example, CHO cells) in which BTN3 is transiently expressed into the abdominal cavity to perform final immunization.
  • cells for example, CHO cells
  • BTN3 is transiently expressed into the abdominal cavity to perform final immunization.
  • spleen cells of the immunized animal are taken out and fused with myeloma cells (for example, SP2 / 0 mouse myeloma cells) using polyethylene glycol (eg, PEG 1500) to produce a hybridoma.
  • myeloma cells for example, SP2 / 0 mouse myeloma cells
  • polyethylene glycol eg, PEG 1500
  • a hybridoma producing an antibody against the target BTN3 from the prepared hybridoma cell line group is prepared by mixing cells transiently expressing BTN3 with the hybridoma culture supernatant and then fluorescently labeled anti-polyclonal mouse antibody ( It can be selected by performing FCM analysis using a secondary antibody.
  • the other strain was named Anti-human BTN3 Mouse hybridoma 34-7 (hereinafter referred to as 34-7) and was deposited internationally on September 17, 2009 (reception number FERM BP-11178). By using these hybridomas, the antibody of the present invention can be easily obtained.
  • Examples of the method for purifying the obtained antibody include salting-out using ammonium sulfate, sodium sulfate, polyethylene glycol, etc., ion exchange chromatography, gel filtration, affinity column, etc., but any method may be used.
  • Lymphocyte proliferation can be inhibited by using the antibody of the present invention.
  • proliferation of lymphocytes can be suppressed by adding the antibody of the present invention to a solution in which lymphocytes are suspended and culturing.
  • the effect of suppressing the proliferation appears by co-culturing at an added concentration of 1 to 10 ⁇ g / ml for 3 days or more.
  • the antibody of the present invention can be used as a medicine by utilizing the function of inhibiting lymphocyte proliferation. For example, by administering to a patient with an autoimmune disease and suppressing the activation of lymphocytes, the symptoms of the autoimmune disease can be suppressed. Moreover, it becomes possible to suppress rejection after organ transplantation by administering to a patient who has undergone organ transplantation and suppressing the proliferation of lymphocytes in the patient's body.
  • Inactive lymphocytes are lymphocytes that have not been stimulated by active factors immediately after recovery from PBMC (Peripheral Blood Mononuclear Cell). Point to a sphere. The shape is small and spherical. In contrast, activated lymphocytes refer to those stimulated by some activator. The shape is large, has cytokine production ability, and does not necessarily have a spherical shape but includes many distorted shapes.
  • the antibody of the present invention can be used as an apparatus / method for separating activated lymphocytes and inactive lymphocytes.
  • the separation device / method of the present invention is a method / device utilizing the fact that the expression level of BTN3 decreases when lymphocytes receive an activation signal.
  • the apparatus / method for separating activated lymphocytes and non-activated lymphocytes of the present invention comprises, for example, binding the antibody of the present invention and a polymer compound such as beads and filling it in a column or the like, thereby Activated lymphocytes or inactive lymphocytes can be selectively separated efficiently from a population of lymphocytes.
  • the polymer compound here is selected from a low toxicity polymer that does not dissolve even when it comes into contact with body fluids, and does not harm when it comes into contact with blood cell components, such as polyurethane, polystyrene, polysulfone, polyvinyl chloride, Examples thereof include acrylic resins, polyamide resins, phenoxy resins, urethane resins, fluorine resins, silicon resins, cellulose resins, chitin, chitosan, agarose, dextran, and the like. In addition, these polymer materials may be used alone, or may be composed of a copolymer, a composite, or a mixture.
  • the above-mentioned polymer compound to which the antibody of the present invention is bound is packed in a column, and after culturing, the inactive lymphocytes are adsorbed and removed to pass through the cells.
  • an antibody having a low affinity with an antigen cannot be adsorbed sufficiently, so that it is not suitable for the separation and an antibody having a high affinity is required. Since the antibody of the present invention has an affinity 8 to 9 times that of a commercially available product, it is possible to efficiently separate inactive lymphocytes from activated lymphocytes.
  • the bond between the antibody of the present invention and these polymer compounds is fixed by a chemical bond.
  • the chemical bond there are a covalent bond, an ionic bond, a hydrophobic bond, and the like. preferable.
  • these polymer compounds preferably have an appropriate functional group, such as amino group, hydroxyl group, carboxyl group, thiol group, glycidyl group, isocyanate group, halogen group, etc. It is preferable to have. Among them, for example, a tosyl group or an epoxy group is preferably used.
  • the shape of the packing material when the column of the antibody of the present invention and the polymer compound is packed is not particularly limited, and examples include beads, films, fibers, hollow fibers, gels and the like.
  • the amount of the antibody of the present invention immobilized on the polymer compound if the antibody of the present invention is too small, the removal effect may be insufficient, and if it is too large, the antibodies may overlap and cause steric hindrance. Therefore, it is preferably immobilized in the range of 1 pmol to 10 pmol per 1 g of the polymer compound.
  • the antibody of the present invention is added to a suspension containing a population of lymphocytes, and the lymphocyte is bound to the antibody of the present invention.
  • magnetic beads on which anti-mouse IgG or protein G is immobilized are added. Collect the magnetic beads using a magnet. Thereby, inactive lymphocytes are collected in the magnetic beads, and the lymphocytes remaining in the solution become activated lymphocytes.
  • the magnetic beads on which the antibody of the present invention and anti-mouse IgG or protein G are immobilized may be added to a suspension containing a lymphocyte population in a state in which they are bound in advance.
  • the non-activated lymphocyte separation apparatus / method of the present invention configured as described above efficiently uses lymphocytes that have not been adsorbed to the column as activated lymphocytes and adsorbed cells as inactive lymphocytes. Can be separated.
  • the antibody of the present invention can be used as an apparatus / method for measuring the ratio of activated lymphocytes to inactive lymphocytes.
  • a fluorescently labeled antibody of the present invention is added to a suspension of a lymphocyte population (PBMC or the like), and the BTN3 molecule expressed in an inactive lymphocyte is bound to the antibody of the present invention. After removing unbound antibody (centrifugation, etc.), the proportion of inactive lymphocytes in the lymphocyte population is measured by measuring fluorescently labeled cells with a flow cytometer or the like. be able to.
  • the apparatus / method for measuring the ratio of activated lymphocytes and inactive lymphocytes using the antibody of the present invention can be used for, for example, immune cell therapy.
  • Immune cell therapy is a treatment method in which lymphocytes are nonspecifically activated by culturing patient lymphocytes together with cytokines and administered to patients. At that time, a part of cultured lymphocytes can be mixed with the antibody of the present invention before administration, and the ratio of activated lymphocytes can be determined by measuring with a flow cytometer or the like, and the quality of immune cells can be measured Is.
  • the inverse correlation marker of activated lymphocytes of the present invention consists of a transmembrane molecule BTN3.
  • the transmembrane molecule BTN3 is expressed on the cell surface of non-activated lymphocytes and monocytes, and when the lymphocytes receive an activation signal, the expression level of BTN3 decreases and is inversely correlated with lymphocyte activation. Yes. Therefore, it can be used as an inverse correlation marker for activated lymphocytes that distinguish activated lymphocytes from inactivated lymphocytes.
  • transmembrane molecule BTN3 The above-mentioned inversely correlated marker of activated lymphocytes (transmembrane molecule BTN3) binds a monoclonal antibody that specifically binds to the marker, and transmembrane molecule BTN3.
  • the expression level of the (marker) it can be used as a method for detecting inactive lymphocytes. For example, for patients undergoing immune cell therapy, a part of the blood obtained by blood collection before and after treatment is mixed with an anti-correlation marker-specific monoclonal antibody of activated lymphocytes, and the activated lymphocytes are mixed with a flow cytometer or the like.
  • the increase or decrease in activated lymphocytes can be confirmed as a numerical value in the patient's body before and after treatment, which can be a new indicator of the effect of immune cell therapy It is.
  • a part of the lymphocyte group is mixed with an anti-correlation marker-specific monoclonal antibody of activated lymphocytes to activate activated lymphocytes.
  • plasmid DNA was prepared in order to produce a hybridoma cell according to the production of the monoclonal antibody of the present invention.
  • the human full-length BTN3A3 molecule was cloned from a cDNA (complementary DNA) library derived from human peripheral blood mononuclear cells (hereinafter PBMCs) using the following two primers.
  • PBMCs peripheral blood mononuclear cells
  • PCR polymerase chain reaction
  • the BTN3A3 full-length cDNA obtained by the above PCR (SEQ ID NO: 5, amino acid sequence is described in SEQ ID NO: 6) is inserted into the HindIII / XbaI region of the cytomegalovirus-derived plasmid vector (pRC-CMV) (INVITROGEN).
  • pRC-CMV cytomegalovirus-derived plasmid vector
  • plasmid DNA was completed.
  • BALB / 3T3 cells expressing BTN3A3 were prepared by forcibly expressing BTN3A3 using pRC-CMV-BTN3A3. Lipofectamine 2000 (Invitrogen) was used for gene introduction at that time.
  • a mouse is immunized with the prepared plasmid DNA to obtain a polyclonal antibody. Specifically, 30 ⁇ g of the above plasmid DNA was transferred subcutaneously into 8-10 week old female Balb / c mice. This operation was performed 4 times every 2 weeks.
  • pTracer-BTN3A3-GFP a vector expressing both BTN3A3 molecule and GFP (green fluorescent protein) molecule; INVITROGEN
  • pTracer-BTN3A3-GFP a vector expressing both BTN3A3 molecule and GFP (green fluorescent protein) molecule; INVITROGEN
  • pTracer-BTN3A3-GFP a vector expressing both BTN3A3 molecule and GFP (green fluorescent protein) molecule; INVITROGEN
  • 75 ml of Lipofectamine 2000 was added to 30 mg of the plasmid vector and reacted at room temperature for 20 minutes.
  • 4 ⁇ 10 6 BALB / 3T3 cells that had been seeded in a 75 cm 2 flask (Sumilon) were added to the cells and collected after 48 hours were used as transfectants.
  • the collected serum was added to the suspension in which the 1 ⁇ 10 6 transfectants were suspended at a ratio of 1/100 or 1/1000, and reacted at 4 ° C. for 30 minutes.
  • PBS 0.5 ml of PE-labeled anti-mouse IgG + IgM polyclonal antibody was added and reacted at 4 ° C. for 30 minutes.
  • analysis was performed with a flow cytometer. It was confirmed by a flow cytometer analysis that even a 1/1000 reaction occurred, and it was confirmed that a polyclonal antibody against BTN3A3 molecule was sufficiently produced.
  • the target mouse 1 ⁇ 10 8 spleen cells and 2 ⁇ 10 7 SP2 / 0 myeloma cells were fused using polyethylene glycol (PEG 1500; SIGMA).
  • the fused cells were seeded in a DMEM medium supplemented with 15% FCS (fetal bovine serum), HAT (hypoxanthine 100 ⁇ M, aminopterin 0.4 ⁇ M, thymidine 16 ⁇ M: Sigma-Aldrich), and 10% methylcellulose (Stem Cell Technologies).
  • FCS fetal bovine serum
  • HAT hyperxanthine 100 ⁇ M, aminopterin 0.4 ⁇ M, thymidine 16 ⁇ M: Sigma-Aldrich
  • 10% methylcellulose Stem Cell Technologies
  • the formed hybridoma colonies were picked up and screened by a flow cytometer in the same manner as the polyclonal antibody measurement.
  • the selection of the monoclonal antibody-producing hybridoma was performed by developing the detection result of the flow cytometer with the binding amount of GFP and the anti-mouse IgG + IgM polyclonal antibody, and selecting the one having a direct relationship between the expression level of GFP and the binding amount of the antibody.
  • Hybridomas 232-5 and 34-7 were obtained during the screening.
  • 232-5 or 34-7 cell supernatant was purified with PROTEIN G column (GE Healthcare Science), and 232-5 or 34-7 derived monoclonal antibody (hereinafter, each antibody is referred to as 232-5 antibody). , 34-7 antibody).
  • the obtained subclass of the antibody of the present invention was confirmed to be immunoglobulin G2a (IgG2a) by Isostrip kit (Roche).
  • pTracer-BTN3A3-GFP or pTracer mock gene was transiently expressed in CHO-K1 and reacted with the antibody of the present invention (the 232-5 antibody) or control IgG2a.
  • FIG. 1 shows the results of measuring cells with a flow cytometer after washing.
  • CHO-K1 cells expressing mock gene (pseudogene) neither the control IgG2a nor the antibody of the present invention (the 232-5 antibody) reacted.
  • the antibody of the present invention only the antibody of the present invention (antibody 232-5) reacted in CHO-K1 cells expressing BTN3A3. From these results, the antibody of the present invention (the 232-5 antibody) was shown to be a monoclonal antibody specific for BTN3A3.
  • FIG. 2 shows the results of a similar experiment performed using IgG2a, 232-5 antibody, and 34-7 antibody.
  • the 232-5 antibody is a BTN3A3-specific monoclonal antibody
  • the 34-7 antibody is also a BTN3A3-specific monoclonal antibody.
  • PBMCs were reacted with 0, 0.1, 1, 10, 100, 1000 ng / ml of BT3.1 antibody, washed and reacted with FITC-labeled 232-5 antibody at a concentration of 1000 ng / ml, followed by a flow cytometer. was used to measure the fluorescence intensity of FITC.
  • FIG. 3 is a diagram showing the results of flow cytometer analysis.
  • the fluorescence intensity of the antibody of the present invention (antibody 232-5) decreased depending on the concentration of BT3.1. This indicates that the BT3.1 and 232-5 antibodies recognize epitopes very close to the BTN3 antigen.
  • the BTN3 antigen has a Vset region and a Cset region belonging to the immunoglobulin superfamily in the extracellular region.
  • a vector expressing only Cset of BTN3A3 or both Vset and Cset (pDisplay-Cset or pDisplay-ALL) was made.
  • HA Tag is inserted.
  • COS-7 cells were transiently transfected with pDisplay-Cset or pDisplay-ALL plasmid, and reacted with BT3.1 or an antibody of the present invention (antibody 232-5). Furthermore, in order to confirm the expression of the pDisplay vector, staining was also performed with an anti-HA (hepatitis A virus) antibody.
  • Fig. 4 shows the results of the flow cytometer.
  • BT3.1 and 232-5 no fluorescence was detectable with pDisplay-Cset.
  • fluorescence was detected with both antibodies in the pDisplay-ALL transfectant. From these results, it became clear that the antigen recognition site by BT3.1 and the antibody of the present invention (the 232-5 antibody) exists in the Vset region on the N-terminal side of the BTN3 antigen.
  • FIG. 5 shows the results of a similar experiment performed using BT3.1, 232-5 antibody and 34-7 antibody. It became clear that the 34-7 antibody also recognizes the Vset region on the N-terminal side of the BTN3 antigen, like the other two antibodies.
  • the primers used in preparing the plasmid are: V-all: 5′-GTT GGG GCA ACG CCG CCC TCT TTT GGA GGG TT-3 ′ (SEQ ID NO: 9) V-35: 5′-TCA ATG GCC CAG CCG GCC GGA CCC TCT GGG CCC AT-3 ′ (SEQ ID NO: 10), V-40: 5′-TCA ATG GCC CAG CCG GCC ATC CTG GCC ATG GTG GG-3 ′ (SEQ ID NO: 11), V-50: 5′-TCA ATG GCC CAG CCG GCC CTG CCC TGT CAC CTG TT-3 ′ (SEQ ID NO: 12) V-60: 5′-TCA ATG GCC CAG CCG GCC GCA GAG ACC ATG GAG CT-3 ′ (SEQ ID NO: 13) And the common reverse primer is 5′-TCA ATG TCG ACA AGA TCA GAA CCC AAT GCT G-3 ′ (SEQ ID
  • PCR products DNA fragments of the Vset region
  • PBMCs-derived cDNA library DNA as a template.
  • PCR was performed for 25 cycles at 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 30 seconds.
  • the plasmid was inserted into the SFi / Sal site of the pDisplay vector and transiently forced to express in COS-7. After 24 hours, cells were collected and expression was confirmed by Western blotting. Specifically, it was dissolved in SDS-PAGE (sodium dodecyl sulfate / polyacrylamide gel electrophoresis) sample buffer, heat-treated (100 ° C., 5 minutes), and electrophoresed in 12.5% SDS-PAGE. After electrophoresis, the gel was transferred to a PVDF (polyvinylidene fluoride) membrane (BIO-RAD), and the membrane was blocked with 3% BSA (bovine serum albumin).
  • SDS-PAGE sodium dodecyl sulfate / polyacrylamide gel electrophoresis
  • BT3.1 or the antibody of the present invention was added to the membrane at 1 ⁇ g / ml and reacted at room temperature for 1 hour. After washing with PBS-tween (phosphate buffered saline-surfactant), sheep anti-mouse IgG-HRP was diluted 2000 times and reacted at room temperature for 30 minutes. After washing with PBS-tween, ECL (GE Healthcare Science), which is a substrate for HRP, was added and exposed to an X-ray film.
  • PBS-tween phosphate buffered saline-surfactant
  • sheep anti-mouse IgG-HRP was diluted 2000 times and reacted at room temperature for 30 minutes.
  • ECL GE Healthcare Science
  • FIG. 6 shows the result of the Western blot.
  • BT3.1 bands were detected in V-all, V-35, and V-40, but not in V-50 and V60. Therefore, it was suggested that BT3.1 recognizes the amino acid sequence between 40-50 of BTN3 antigen.
  • FIG. 7 shows the results of a similar experiment performed using BT3.1, 232-5 antibody and 34-7 antibody.
  • the 34-7 antibody was estimated to recognize the amino acid sequence between 35-40 of the BTN3 antigen.
  • the amino acid sequence between 35 and 40 was identical in BTN3A1, BTN3A2, and BTN3A3, and it was estimated that the antibody of the present invention is a monoclonal antibody that recognizes any molecule of BTN3.
  • PBMCs were stained with BT3.1 or 232-5 antibody (10 ⁇ g / ml), reacted with FITC-goat anti-mouse IgG (BD Bioscience), and analyzed with a flow cytometer.
  • the final concentrations of BT3.1, 232-5 antibody were adjusted to 0.01, 0.1, 1, 10, 100, 100 ng / ml. These solutions were reacted with PBMCs, and the fluorescence intensity was measured with a flow cytometer after the reaction.
  • Example 5 ⁇ Expression of BTN3 antigen on the surface of various lymphocyte subset cells>
  • the antibody of the invention was shown to stain about 95% of PBMCs, but the expression of BTN3 antigen in each subset of lymphocytes was verified.
  • FITC-labeled 232-5 antibody and various lymphocyte markers PE-CD3, CD4, CD8, CD19, CD56, ⁇ TCR antibody (above, manufactured by Beckman Coulter) were used. Flow cytometer analysis was performed. In addition, FITC-IgG2a (manufactured by Beckman Coulter) was used as a negative control for FITC-232-5.
  • CFSE carboxyfluorescein diacetate succinimidyl ester
  • the medium is RPMI1640 containing 10% FCS and rhIL-2 (recombinant human interleukin-2) added at 50 U / ml. After culturing in a 37 ° C., 5% CO 2 incubator for 3 days, analysis was performed with a flow cytometer. At this time, phycoerythrin (PE) -anti-human CD4 (13B.2; Beckman Coulter) or PE-anti-human CD8a (B9.11; Beckman Coulter) was used.
  • PE phycoerythrin
  • FIG. CFSE has the property that the fluorescence intensity decreases with the rate of cell division.
  • FIG. 11 shows the results of a similar experiment performed using the BT3.1, 232-5 antibody and the 34-7 antibody. Similar to the 232-5 antibody, suppression of cell division was observed with the 34-7 antibody. From these results, it was revealed that the antibodies of the present invention (antibodies 232-5 and 34-7) have an effect of suppressing cell proliferation.
  • PBMCs ⁇ Reduction of BTN3 expression upon stimulation of PBMCs>
  • OKT3 5 ⁇ g / ml
  • FCS 10% FCS
  • 50 U / ml rhIL-2 Cultivation was performed in RPMI 1640 medium at 37 ° C. in a 5% CO 2 incubator.
  • lipopolysaccharide lipopolysaccharide: LPS
  • lipopolysaccharide: LPS lipopolysaccharide
  • LPS lipopolysaccharide
  • FITC-232-5 / PE-human CD3 UCHT1; Beckman Coulter
  • FITC-232-5 / PE-human CD4 FITC-232-5 / PE-human CD8a
  • FITC-232- 5 / PE-human CD19 J4.119; Beckman Coulter
  • FITC-232-5 / PE-human CD56 N901; Beckman Coulter
  • FITC-232-5 / PE- human ⁇ TCR IMMU510; Beckman Coulter
  • Example 8 ⁇ Decrease in BTN3 expression upon stimulation of PBMCs (analysis of mRNA)>
  • BTN3A1, BTN3A2, and BTN3A3 constituting BTN3 molecule is affected. It cannot be verified by analysis using antibodies. Therefore, mRNA was extracted when lymphocytes were activated, and PCR was performed after cDNA synthesis using specific primers for BTN3A1, BTN3A2, and BTN3A3.
  • BTN3A1 5′-GCA TCT CGG GGA GAG AGA CA-3 ′ (SEQ ID NO: 15), 5′-GAA TAT GCG ATC CAT CCA CA-3 ′ (SEQ ID NO: 16), BTN3A2: 5′-GAT-GGA-GTG-GGC-CTA-TAT-GA-3 ′ (SEQ ID NO: 17), 5′-TCA-GGC-TGA CTT ATT G-3 ′ (SEQ ID NO: 18), BTN3A3: 5′-ATG GCT CGT GGA GAG AAG TC-3 ′ (SEQ ID NO: 19), 5′-AGA TAT GAG ATC CAT CTG TG-3 ′ (SEQ ID NO: 20), G3PDH: 5′-ACC ACA GTC CAT CTC ATC AC-3 ′ (SEQ ID NO: 21), 5′-TCC ACC ACC CTG TTG CTG TA-3 ′ (SEQ ID NO: 22) PCR conditions were as follows: 35
  • PBMCs immediately after blood collection expressed BTN3A1, BTN3A2, and BTN3A3 mRNA. Their expression was maintained even when PBMCs were cultured in the medium alone. On the other hand, in PBMCs stimulated with OKT3 / rIL-2, the expression of all mRNAs of BTN3A1, BTN3A2, and BTN3A3 was decreased. From these facts, it was found that when lymphocytes were activated, mRNAs of all three molecules of BTN3A1, BTN3A2, and BTN3A3 constituting the BTN3 molecule decreased.
  • Example 8 ⁇ Inverse correlation 1 between decrease in BTN3 expression and activation marker>
  • lymphocyte activation reduced the expression of BTN3 molecules. Therefore, the correlation with the IL-2 receptor ⁇ chain (CD25) that is strongly expressed when lymphocytes are activated was examined.
  • PBMCs 1 ⁇ 10 6 PBMCs were seeded on OKT3 (5 ⁇ g / ml) pre-coated 24 well plates.
  • Example 8 ⁇ Inverse correlation 2 between decreased expression of BTN3 and activation marker>
  • BTN3 is expressed in most PBMCs
  • the expression of BTN3 is partially reduced in the memory T cell fraction expressing CD45RO.
  • Example 8 it was described that a decrease in BTN3 occurs in activated lymphocytes. However, 100% of BTN3 was expressed in order to counteract the possibility that the fraction in which BTN3 decreased was proliferated before stimulation.
  • the naive T cells (CD4 + CD25 ⁇ CD45RO ⁇ ) were isolated and stimulated.
  • CD4 + CD25 ⁇ T cells were first obtained from PBMCs by negative selection using CD4 + CD25 + regulatory Tcell isolation kit (Miltenyi Biotec).
  • CD45RO microbead (Miltenyi Biotec) is added to 1 ⁇ 10 7 CD4 + CD25 ⁇ T cells, washed with MACS buffer (1% BSA, 2 mM EDTA in PBS), and then passed through a magnetic field column. CD4 + CD25 ⁇ CD45RO ⁇ naive T cells were obtained.
  • naive T cells thus obtained were seeded on OKT3 (5 ⁇ g / ml) pre-coated 24 well plate, and cultured in a medium containing 10% FCS, 50 U / ml rhIL-2, 5 ⁇ g / ml anti-human CD28 antibody. Cultured for 6 days. After 6 days, cells were harvested and stained with FITC-232-5 / PE-human CD25 (2A3; BD Bioscience), FITC-232-5 / PE-human CD45RO (UCHL1; Beckman Coulter). BT3.1 was also subjected to FITC conversion (DOJINDO), the same staining was performed, and a flow cytometer analysis was performed.
  • DOJINDO FITC conversion
  • FIG. 15 shows the results of this test.
  • CD25 is known as an activation marker and CD45RO is known as a memory marker.
  • CD45RO is known as a memory marker.
  • BTN3 molecules were shown to decrease with lymphocyte activation, but some lymphocytes did not express BTN3 molecules, so we used naive CD4 positive T cells that expressed almost 100% BTN3 molecules. Went.
  • CD4 + CD25-CD45RO-na ⁇ ve T cells were obtained from healthy human PBMCs using human CD4 isolation kit (Miltenyi biotec), human CD25 beads (Miltenyi biotec), and CD45RO beads (Miltenyi biotec). These cells were stimulated with immobilized OKT3 (5 ⁇ g / ml), rIL-2 (50 U / ml), ⁇ CD28 antibody (5 ⁇ g / ml, BD Bioscience) for 7 days, and then subjected to flow cytometer analysis.
  • OKT3 5 ⁇ g / ml
  • rIL-2 50 U / ml
  • ⁇ CD28 antibody 5 ⁇ g / ml
  • FITC-232-5, FITC-34-7, FITC-BT3.1, FITC-IgG2a (Beckman Coulter), PE-CD25 (Miltenyi biotec), PE-CD45RO (Beckman Coulter), PE-CD70 (BD Bioscience) ), PE-CD103 (BD Bioscience), PE-ICOS (BD Bioscience), and PE-PD-1 (BD Bioscience) were used.
  • the antibody of the present invention has a characteristic of specifically recognizing BTN3.
  • lymphocytes when lymphocytes are activated, the expression of BTN3 decreases.
  • activated lymphocytes and inactive lymphocytes can be efficiently distinguished. For example, by applying to an antibody adsorption column or the like, activated lymphocytes and inactive lymphocytes can be efficiently separated from the activated lymphocyte population.
  • the antibody of the present invention since the antibody of the present invention has the property of inhibiting the proliferation of lymphocytes, it is considered to function as an immunosuppressant when developed as an antibody drug. There is a possibility that it can be applied to.

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Abstract

La présente invention concerne un nouvel anticorps monoclonal capable d'identifier de manière spécifique le BTN3 situé à la surface d'une cellule (par exemple, un lymphocyte). L'invention concerne également un procédé d'utilisation de l'anticorps monoclonal. L'anticorps peut être produit par la préparation d'un hybridome qui peut produire un anticorps monoclonal capable d'identifier de manière spécifique le BTN3. Il devient alors possible de distinguer ou de séparer un lymphocyte activé d'un lymphocyte non activé grâce à l'utilisation de l'anticorps. Il devient également possible d'inhiber la prolifération d'un lymphocyte par l'ajout à un milieu de culture pour le lymphocyte.
PCT/JP2009/005547 2008-10-22 2009-10-22 Nouvel anticorps monoclonal et son utilisation Ceased WO2010047117A1 (fr)

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CN109750002A (zh) * 2017-11-02 2019-05-14 北京蛋白质组研究中心 杂交瘤细胞株及其分泌的具有抑制肿瘤进程活性的单克隆抗体与应用

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WO2016058148A1 (fr) * 2014-10-13 2016-04-21 李小彦 Anticorps anti-butyrophiline-3 humanisé et ses utilisations
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CN109750002A (zh) * 2017-11-02 2019-05-14 北京蛋白质组研究中心 杂交瘤细胞株及其分泌的具有抑制肿瘤进程活性的单克隆抗体与应用
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