WO2020171171A1 - Anticorps anti-hla-dr et son utilisation dans le traitement du cancer - Google Patents

Anticorps anti-hla-dr et son utilisation dans le traitement du cancer Download PDF

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WO2020171171A1
WO2020171171A1 PCT/JP2020/006822 JP2020006822W WO2020171171A1 WO 2020171171 A1 WO2020171171 A1 WO 2020171171A1 JP 2020006822 W JP2020006822 W JP 2020006822W WO 2020171171 A1 WO2020171171 A1 WO 2020171171A1
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antibody
cells
seq
cancer
cancer cells
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Japanese (ja)
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徳弘 中村
由貴江 笹倉
りさ 野澤
雄二 三嶋
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ブライトパス・バイオ株式会社
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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 antibody having a cytotoxic activity specifically against a cancer expressing HLA-DR, and a composition and a method for cancer treatment and cancer test containing the antibody.
  • Cancer is the leading cause of death in the Japanese people, with one in two suffering from cancer once in their lives and one in three dying due to cancer (2013 Ministry of Health, Labor and Welfare data. ). Therefore, although new cancer therapeutic agents are being developed both in Japan and overseas, satisfactory therapeutic results have not yet been obtained.
  • the HLA-DR antigen has been reported to be highly expressed in many cancers (leukemia, malignant lymphoma, glioblastoma, melanoma, breast cancer, colon cancer, lung cancer, etc.) and is considered to be a target for cancer treatment. Therefore, preparation of an antibody against this antigen has been attempted, a plurality of antibodies have been commercially available, and preparation of a new antibody has also been attempted (Patent Documents 1 and 2).
  • Non-Patent Document 1 drugs that target HLA-DR, such as IMMU-114 (humanized L243 antibody) and Hu1D10, have been developed (Patent Document 1), their efficacy is part of Phase 1 trials. Only partial response was observed in the cases, and no results that meet the needs of the medical field have been obtained (Non-Patent Document 1).
  • Non-Patent Document 2 a typical hemolytic uremic syndrome
  • HLA-DP HLA-DP
  • HLA-DQ HLA-DR
  • HLA-DR cytotoxicity of these antibodies in 3 types of Hodgkin lymphoma cells.
  • This antibody has a characteristic effect of making a huge hole in a cancer cell in a very short time, and has a damaging activity independent of effector cells or complement (Patent Document 2).
  • Patent Document 2 no information is known as to whether this research group is developing with this antibody.
  • the present invention provides a binding substance for an HLA-DR antigen capable of exerting cytotoxicity against a cancer cell expressing the HLA-DR antigen, and a binding substance for such an HLA-DR antigen. It is an object to provide a therapeutic drug for a tumor containing
  • the present inventors have developed a binding substance for HLA-DR antigens, which has heavy chain complementarity determining regions (CDRs) 1 to 3 and light chain CDRs 1 to 3 of a specific amino acid sequence, particularly an antibody or human form against HLA-DR antigens. It has been clarified that the above problems can be solved by providing an antibody derivative, and the present invention has been completed. The present inventors have also revealed that cancer can be treated by utilizing such a binding substance for the HLA-DR antigen.
  • CDRs heavy chain complementarity determining regions
  • [1] Complementarity determining region of heavy chain, CDR1 (SEQ ID No.: 1), CDR2 (SEQ ID No.: 2), CDR3 (SEQ ID No.: 3), Light chain complementarity determining regions, CDR1 (SEQ ID No.: 4), CDR2 (SEQ ID No.: 5), CDR3 (SEQ ID No.: 6),
  • An antibody or a humanized antibody derivative having a binding property to an HLA-DR antigen and inducing toxicity to a cancer cell comprising: [2]: The human antibody derivative is selected from humanized antibody variants selected from humanized antibodies, chimeric antibodies, multivalent antibodies, and multispecific antibodies or functional fragments thereof, [1] Antibody or humanized antibody derivative; [3]: The antibody or human antibody derivative according to [1] or [2], wherein the functional fragment is F(ab′)2; [4]: The amino acid sequence of the heavy chain variable region VH domain
  • a pharmaceutical composition for treating cancer comprising the antibody or humanized antibody derivative according to any one of [1] to [8]; [10]: The pharmaceutical composition according to [9], wherein the cancer is selected from the group consisting of Hodgkin lymphoma, lung cancer, and melanoma; [11]: a step of contacting the cancer cells collected from the subject with the antibody or humanized antibody derivative according to any one of [1] to [8] in vitro, Under culture conditions, a step of measuring whether the cell viability of cancer cells is reduced, or a step of measuring whether the secretion of immunostimulatory substances is enhanced, A method of measuring cytotoxicity against cancer cells, comprising: [12]: In the presence of peripheral blood lymphocytes of the same subject, it is measured whether the cell viability of cancer cells is reduced or whether immune cells derived from peripheral blood lymphocytes are activated, [ [11] The method for measuring cytotoxicity against cancer cells according to [11]; [13]: The antibody or humanized antibody derivative according to any one of [
  • Cytotoxicity of the antibody or human antibody derivative according to any one of [1] to [8] to cancer cells collected from a subject, secretion of an immunostimulator, or immunity A measurement kit containing the above-mentioned antibody for measuring cell activation in vitro.
  • the binding substance for the HLA-DR antigen of the present invention reduces or eliminates a tumor for the treatment of a cancer expressing the HLA-DR antigen and for inhibiting the growth of cancer cells.
  • This binding substance, particularly an antibody or a human antibody derivative can bind to the HLA-DR antigen, immunization of the HLA-DR antigen in vivo as a test application of cancer cells expressing the HLA-DR antigen. It can be used for biological detection (ELISA, Western blotting, flow cytometry, etc.).
  • the cancer cells are treated in the subject. It can be examined whether it has cytotoxicity against.
  • the binding substance of the present invention particularly an antibody or a humanized antibody derivative
  • FIG. 1 is a diagram showing the cell survival rate after treating two Hodgkin lymphoma cell lines (L428 cells and KM-H2 cells) with antibody LN-3.
  • FIG. 2 is a diagram showing cell death induction (relative value based on dead cell staining) after treating a lung cancer cell line (Calu-1) with antibody LN-3.
  • Figure 3 shows the cell survival rate of HLA-DR expressing cells ((A) L428 cells, (B) KM-H2 cells) and non-expressing cells ((C) Jurkat cells) treated with antibody LN-3.
  • FIG. FIG. 4 shows the amino acid sequences ((A) heavy chain, (B) light chain) of the variable region of antibody LN-3.
  • FIG. 5-1 is a diagram showing (A) antibody binding strength to each peptide by epitope mapping using a peptide array designed based on the human HLA-DR ⁇ 1 derived sequence.
  • FIG. 5-2 is a diagram showing a region in the HLA-DR ⁇ 1 three-dimensional structure of the (B) antibody-binding peptide by epitope mapping using a peptide array designed based on the human HLA-DR ⁇ 1-derived sequence.
  • FIG. 6 is a diagram showing that the BP1206 chimeric antibody has higher cytotoxicity than the antibody LN-3.
  • FIG. 7-1 is a diagram showing the effect of BP1206 chimeric antibody on tumor growth in a L428 cell subcutaneous transplant model ((A) tumor volume, (B) body weight).
  • FIG. 7-2 is a diagram showing the effect of BP1206 chimeric antibody on tumor growth in a L428 cell subcutaneous transplant model ((C) tumor weight at autopsy after 42 days).
  • FIG. 8-1 is a graph showing the dose response of the BP1206 chimeric antibody to tumor growth in the L428 cell subcutaneous transplant model ((A) tumor volume, (B) body weight).
  • FIG. 8-2 is a graph showing the dose responsiveness of the BP1206 chimeric antibody to tumor growth in the L428 cell subcutaneous transplant model ((C) tumor weight at necropsy after 42 days).
  • FIG. 8-1 is a graph showing the dose response of the BP1206 chimeric antibody to tumor growth in the L428 cell subcutaneous transplant model ((A) tumor volume, (B) body weight).
  • FIG. 8-2 is a graph showing the dose responsiveness of the BP1206 chimeric antibody to tumor growth in the L428 cell subcutaneous transplant model ((C) tumor weight at necropsy after 42 days).
  • FIG. 9 is a diagram showing the effect of a surrogate antibody (anti-mouse MHC class II antibody) against the BP1206 chimeric antibody on tumor growth in a mouse T cell lymphoma cell line E.G7 cell subcutaneous transplant model ((A) tumor Volume, (B) body weight).
  • FIG. 10-1 is a diagram showing that immunity induction to the model antigen (Ova) occurs in the mouse individual body of the anti-mouse MHC class II antibody administration group.
  • FIG. 10-2 is a diagram showing that administration of anti-mouse MHC class II antibody did not alter the composition of dendritic cells (DC) in the spleen of mouse individuals.
  • DC dendritic cells
  • FIG. 11 is a diagram showing a cell presence ratio when PBMCs of healthy subjects were treated with the BP1206 chimeric antibody, as compared with those before treatment.
  • FIG. 12 is a diagram showing that the antibody of the present invention has cytotoxic activity against two types of human melanoma-derived cell lines and two types of human lung cancer-derived cell lines.
  • FIG. 13 is a diagram showing that the antibody of the present invention has cytotoxic activity against three types of human melanoma-derived cell lines, and the combined effect of the antibody of the present invention and vemurafenib.
  • Figure 14 is a diagram showing cell death induction after treatment of a melanoma cell line with a BP1206 chimeric antibody (relative value based on viable cell mass measurement) ((A) HT144 cells, BP1206 chimeric antibody in combination with cisplatin, ( (B) HT144 cells, BP1206 antibody in combination with Vemurafenib, (C) A375 cells, BP1206 antibody in combination with Vemurafenib).
  • FIG. 15 is a diagram showing changes in cell shape when a Hodgkin lymphoma cell line (L428 cell) was treated with a BP1206 chimeric antibody.
  • FIG. 1 Hodgkin lymphoma cell line
  • FIG. 16 shows the effect of BP1206 chimeric antibody on HMGB1 secretion in the L428 cell subcutaneous transplant model (left) and infiltration of the antibody into tumor tissue (right).
  • FIG. 17-1 is a diagram showing a variable region amino acid sequence ((A) heavy chain) of a humanized antibody.
  • FIG. 17-2 is a diagram showing a variable region amino acid sequence ((B) light chain) of a humanized antibody.
  • FIG. 18 is a diagram showing cell death induction (relative value based on dead cell staining) when a Hodgkin lymphoma cell line (L428 cell) was treated with a BP1206 chimeric antibody or a BP1206 humanized antibody.
  • FIG. 17-1 is a diagram showing a variable region amino acid sequence ((A) heavy chain) of a humanized antibody.
  • FIG. 17-2 is a diagram showing a variable region amino acid sequence ((B) light chain) of a humanized antibody.
  • FIG. 18 is
  • FIG. 19 is a diagram showing that expression of the HLA-DR antigen was observed in nearly half of the tumor tissues examined, although there were variations in the amount of expression and the proportion of expressing cells. ..
  • FIG. 20 shows that in various cancer types, a correlation was observed between the expression level of the target antigen of the antibody of the present invention in cell lines and the induction of cell death by the addition of the antibody of the present invention.
  • FIG. 6 shows that the higher the target antigen expression level in E. coli, the higher the cell death inducing activity.
  • HLA-DR one of the major molecules that make up HLA (human leukocyte antigen) Class II, which is an antigen-presenting molecule
  • B Human antibody derivative: a human antibody variant selected from a humanized antibody or a chimeric antibody, or a functional fragment thereof
  • C humanized antibody: an antibody produced in a non-human animal body by recombination of a portion other than the heavy chain and light chain complementarity determining regions (CDR) into a human antibody gene
  • CDR chimeric antibody: an antibody produced recombinantly so that the variable region of the antibody produced in the non-human animal body is bound to the constant region of the human antibody gene
  • E Functional fragment of human antibody variant: means a part (partial fragment) of a human antibody variant (humanized antibody or chimeric antibody), which retains the action of the antibody on the antigen ( For example, specifically includes F(ab')2, Fab', Fab, single chain Fv (F(ab')2, Fab', Fab, single chain Fv
  • the present invention provides, in one aspect, a binding substance for an HLA-DR antigen, particularly an antibody or a humanized antibody derivative, which has a binding property for an HLA-DR antigen and induces toxicity to cancer cells.
  • the binding substance of the present invention particularly an antibody or a humanized antibody derivative, is used for treatment of cancer cells expressing HLA-DR antigen, for inhibiting the growth of cancer cells, and for reducing or eliminating tumors. be able to.
  • the present invention provides antibodies or human antibody derivatives having binding properties to HLA-DR antigens.
  • the antibody or human-type antibody derivative of the present invention comprises a total of 6 positions of complementarity determining regions (CDRs) 1 to 3 of heavy chain and CDRs 1 to 3 of light chain which are the same as those of the antibody having the binding property to HLA-DR antigen. It is characterized by having the amino acid sequence of. Examples of such amino acid sequences of CDRs at 6 positions of an antibody having binding properties to HLA-DR antigens include CDR1 (SEQ ID No.: 1) and CDR2 (SEQ ID No.: 2) of heavy chain.
  • CDR3 (SEQ ID No.: 3), and light chain CDR1 (SEQ ID No.: 4), CDR2 (SEQ ID No.: 5), CDR3 (SEQ ID No.: 6) Include an antibody or human-type antibody derivative specified by a combination of CDRs other than the above-mentioned combination of CDRs, as long as it has the characteristic of binding to the HLA-DR antigen.
  • the antibody or humanized antibody derivative of the present invention is also characterized in that it has the ability to induce toxicity to cancer cells expressing the HLA-DR antigen.
  • the toxicity to the cancer cells expressing the HLA-DR antigen may occur in vitro or in vivo. Whether or not it has the ability to induce cytotoxicity to cancer cells depends on the ability to actually induce cytotoxicity to cancer cells from among antibodies having binding properties to HLA-DR antigen. It can be obtained by screening.
  • the screening of the ability of the antibody or human antibody derivative to induce cytotoxicity to cancer cells can be performed in vivo or in vitro.
  • In-vivo screening is performed by transplanting target cancer cells into animals such as immunodeficient mice such as nude mice and SCID mice, and tumors in the body when the antibody or humanized antibody derivative of the present invention is administered. This can be done by measuring the change in mass size.
  • Screening performed in vitro can be performed by contacting a target cancer cell with the antibody or human antibody derivative of the present invention under culture conditions and examining whether the cancer cell causes cell death.
  • the cytotoxicity of the antibody of the present invention or the human antibody derivative against cancer cells is that the antibody or the human antibody derivative of the present invention bound to the cancer cell is a natural killer cell (NK cell), macrophage, neutrophil, Antibody-dependent cellular cytotoxicity induced by stimulating effector cells selected from the group consisting of eosinophils, complement-dependent cytotoxicity induced by stimulating the complement system, or these external factors It may be due to any aspect of physical destruction of cells due to independent antibody binding.
  • NK cell natural killer cell
  • macrophage macrophage
  • neutrophil neutrophil
  • Antibody-dependent cellular cytotoxicity induced by stimulating effector cells selected from the group consisting of eosinophils, complement-dependent cytotoxicity induced by stimulating the complement system, or these external factors It may be due to any aspect of physical destruction of cells due to independent antibody binding.
  • the antibody may be derived from any animal species of mammals, and the species from which the antibody is derived are not limited to humans, and include mouse, rat, guinea pig, hamster, It may be a rabbit or the like.
  • a human antibody derivative of the above-mentioned antibody is also a human antibody derivative of the above-mentioned antibody, as long as it has a binding property to HLA-DR antigen and has a functional characteristic of inducing toxicity to cancer cells.
  • a human antibody derivative in the present invention in one aspect, it has the amino acid sequence of the CDR at 6 positions of the above antibody, and the amino acid sequence of the constant region derived from a human antibody, and the other amino acid sequences are derived from the original antibody. It is possible to provide a derivative of an antibody, which is characterized by being a combination of the amino acid sequence of and the amino acid sequence of human antibody.
  • antibody derivatives include humanized antibodies in which amino acid sequences derived from human antibodies have been substituted for the regions other than the complementarity determining region (CDR) of the above-mentioned "antibody", or the variable regions of the above antibodies are the constant regions of human antibodies.
  • Chimeric antibodies such as those linked to regions, multivalent antibodies in which one type of antibody has multiple antigen binding sites, and multispecific antibodies in which one type of antibody has multiple specificities (bispecific antibodies) are included However, other than these are also included.
  • examples of the amino acid sequence of the heavy chain variable region VH domain of the antibody or humanized antibody derivative of the present invention include: SEQ ID No.:8, SEQ ID No.:12, SEQ ID No.:16, SEQ Amino acid sequence described in either ID No.: 20, 20, SEQ ID No.: 24, SEQ ID No.: 28, SEQ ID No.: 32, SEQ ID No.: 36, or SEQ ID No.: 40 Can be raised.
  • examples of the amino acid sequence of the light chain variable region VL domain of the antibody or humanized antibody derivative of the present invention include SEQ ID No.: 10, SEQ ID No.: 14, SEQ ID No.: 18, SEQ Amino acid sequence described in any of ID No.:22, SEQ ID No.:26, SEQ ID No.:30, SEQ ID No.:34, SEQ ID No.:38, or SEQ ID No.:42. Can be raised.
  • the human antibody derivative of the present invention also includes the functional fragment of the above-mentioned antibody or human antibody derivative.
  • Functional fragments of the antibody or human antibody derivative of the present invention include F(ab')2, Fab', Fab, single-chain Fv (scFv) and the like.
  • the functional fragment of the present invention may be any of these as long as it is characterized by being capable of inducing toxicity to cancer cells, and for example, F(ab')2 fragment or the like can be used. , Can be used as such a functional fragment.
  • the antibody or human antibody derivative of the present invention can be obtained by culturing antibody-producing cells collected from the animal body of the above-mentioned species of origin, but the antibody or human antibody derivative can also be obtained. It is also possible to design a vector for protein expression containing a DNA sequence capable of defining the amino acid sequence of, to introduce the vector into cells for protein production, and obtain it recombinantly.
  • the DNA sequence capable of defining the amino acid sequence of the antibody or human antibody derivative of the present invention is a method for obtaining it from cells producing the desired antibody or human antibody derivative, and the animal species used in the expression system based on the amino acid sequence. It can be prepared by a method of designing based on the optimized codon or a method of using these methods in combination.
  • Obtained by incorporating the prepared DNA sequence into an expression vector suitable for the cell type for protein expression (such as CHO cells) for which the antibody or human antibody derivative is to be expressed, and introducing it into the cell type for protein expression can be obtained using a method well known to those skilled in the art.
  • DNA sequences defining the amino acid sequences of the heavy chain variable region VH domain of the antibody or human antibody derivative of the present invention exemplified above are, for example, as follows:
  • DNA sequence defining the amino acid sequence of the light chain variable region VL domain of the antibody or human antibody derivative of the present invention exemplified above is, for example, as follows:
  • the antibody or human antibody derivative of the present invention is characterized in that it has the ability to induce cytotoxicity against cancer cells described above, and in one embodiment, for treating or preventing cancer.
  • a pharmaceutical composition comprising the antibody or humanized antibody derivative of the present invention, which induces toxicity to cancer cells in a subject in need thereof, can be provided.
  • Cancer cells that can be targeted in the present invention include, for example, leukemia (including chronic lymphocytic leukemia and acute lymphocytic leukemia), lymphoma (non-Hodgkin's lymphoma, Hodgkin's lymphoma, T-cell lymphoma, B-cell lymphoma, Burkitt lymphoma, malignant lymphoma, diffuse lymphoma, follicular lymphoma), myeloma (including multiple myeloma), melanoma, lung cancer, breast cancer, colon cancer, kidney cancer, gastric cancer, ovarian cancer, pancreas Cancer, cervical cancer, endometrial cancer, endometrial cancer, esophageal cancer, liver cancer, head and neck cancer, head and neck squamous cell carcinoma, skin cancer, urinary tract cancer, prostate Cancer, choriocarcinoma, pharyngeal cancer, laryngeal cancer, capsular tumor, male embryoma, endometri
  • the cancer cell is preferably a cancer cell that expresses an HLA-DR antigen, for example Hodgkin lymphoma, lung cancer, melanoma, cervical cancer, endometrial cancer, ovarian cancer, head. Cells derived from cervical cancer are preferred.
  • the antibody or humanized antibody derivative of the present invention is characterized in that it induces cytotoxicity to target cancer cells as described above, but does not induce cytotoxicity to normal cells. That is, when administered to a living body, cytotoxicity is induced only in cancer cells that express the target HLA-DR antigen, and in the case of normal cells that express the HLA-DR antigen, clinically, It is required not to induce potentially problematic cytotoxicity.
  • the antibody or humanized antibody derivative of the present invention can be provided as a composition in combination with another antibody or another drug such as an anticancer agent.
  • the antibody or humanized antibody derivative of the present invention can be bound to a drug to form an antibody drug complex (ADC).
  • ADC antibody drug complex
  • a formulation containing the antibody or humanized antibody derivative of the present invention together with a physiologically acceptable diluent or carrier can also be provided.
  • suitable carriers include buffers (phosphate buffer, citrate buffer, acetate buffer, etc.), salts (sodium chloride, etc.), sugars (glucose, trehalose, mannitol, sorbitol, etc.), additives (arginine, etc.). Amino acids, surfactants such as polysorbates, etc.), but are not limited to these.
  • the antibody or humanized antibody derivative of the present invention can be reconstituted and used by freeze-drying (freeze-drying) and adding a buffer aqueous solution as described above when necessary.
  • the preparation containing the antibody or human antibody derivative of the present invention can be administered in various dosage forms, for example, a parenteral preparation such as an injection or a drip.
  • the dose of the antibody or humanized antibody derivative of the present invention varies depending on the symptoms, age, body weight, etc., but usually for parenteral administration, 0.01 mg to 1000 mg per dose, preferably 1 mg to 10 mg per body weight per day. Depending on the type of cancer, it can be administered via an appropriate administration route such as intraperitoneal injection, subcutaneous injection, intramuscular injection, intratumoral injection, or intravenous injection.
  • the present invention is based on the above-mentioned characteristic of having an ability to induce cytotoxicity to cancer cells, and in another aspect, a subject in need of treatment or prevention of cancer is treated with the antibody or human of the present invention.
  • Methods of treating or preventing cancer in a subject can also be provided that include administering an effective amount of a type antibody derivative. Treatment or prevention of cancer with the antibody or human antibody derivative of the present invention occurs when the antibody or human antibody derivative causes cytotoxicity to cancer cells in the body.
  • an adjuvant for example, Clin. Microbiol
  • an adjuvant for example, Clin. Microbiol
  • a liposomal preparations particulate preparations bound to beads with a diameter of several ⁇ m, preparations bound with lipids, etc. It can also be administered in the form of dosage forms.
  • the antibody or human-type antibody derivative of the present invention is for detecting the HLA-DR antigen in a sample based on the characteristic that it has a binding property to the HLA-DR antigen.
  • the antibody or humanized antibody derivative of the present invention is a purification method using an antibody such as an immunoprecipitation method, an agglutination reaction, or a magnetic bead method for a sample containing cancer cells collected from a subject. It can be used in carrying out various detection methods that can be performed using an antibody, such as an ELISA method, a Western blot, an immunoassay such as immunohistochemistry, and immunocytochemistry such as flow cytometry.
  • the antibody or humanized antibody derivative of the present invention can be detected using a detection label generally known to those skilled in the art (eg, fluorescence, DAB, enzyme, etc.).
  • the antibody or human antibody derivative of the present invention also has a binding property to the HLA-DR antigen and induces toxicity to cancer cells, based on the characteristics of the antibody or human antibody derivative of the present invention. , Can be used to measure cytotoxicity against cancer cells in a subject.
  • the method for measuring the cytotoxicity against cancer cells in such a subject includes the following steps: A step of contacting a cancer cell collected from a subject with an antibody of the present invention or a humanized antibody derivative under culture conditions (that is, in vitro), Under culture conditions, a step of measuring whether the cell viability of cancer cells is reduced, or a step of measuring whether the secretion of immunostimulatory substances is enhanced, Examples of the method for measuring cytotoxicity against cancer cells using the antibody or humanized antibody derivative of the present invention containing
  • the secretion of the immunostimulatory substance is enhanced as a result of activation of peripheral blood lymphocyte-derived immune cells by the antibody of the present invention or the humanized antibody derivative under culture conditions is determined by the same test. It can be carried out by measuring whether or not immune cells derived from peripheral blood lymphocytes contacted with the antibody or humanized antibody derivative of the present invention are activated in the presence of peripheral blood lymphocytes in the body. By this method, by measuring the increase in the secretion of the immunostimulatory substance, when the antibody or humanized antibody derivative of the present invention is administered to the subject, cytotoxicity in the subject (in vivo) It is possible to measure activation of immune cells such as lymphocytes possessed and the resulting increase in cytotoxicity to cancer cells.
  • the present invention also comprises in vitro measurement of cytotoxicity against cancer cells collected from a subject, secretion of an immunostimulatory substance, or activation of immune cells, including the antibody or humanized antibody derivative of the present invention.
  • a measurement kit for doing so can also be provided.
  • Example 1 Identification of antibody clone having cytotoxicity
  • an antibody having cytotoxicity against human cancer cell-derived cells was identified from among existing anti-HLA-DR antibodies.
  • L428 cells DSMZ, ACC197
  • KM-H2 cells DSMZ, ACC8
  • 2x10e6 cells/ml of cells in RPMI1640 medium Wako Pure Chemical 189-02025) containing 10% FBS (Equitech-bio.Inc SFBM30-0500) and 1% penicilin-streptmycin (Nacalai Tesque 09367-34).
  • RPMI1640 medium Wako Pure Chemical 189-02025
  • FBS Equitech-bio.Inc SFBM30-0500
  • penicilin-streptmycin Nacalai Tesque 09367-34
  • Antibody LN-3 showed the action of reducing the survival rate of L428 cells to 11.3% and the survival rate of KM-H2 cells to 23.2% (Fig. 1).
  • the survival rate was calculated with the survival rate of each cell treated with the isotype control antibody as 100%.
  • L428 cells and KM-H2 cells as a result of examination by flow cytometry, since they show the same level of HLA-DR expression, the results of this Example, due to the difference in affinity between the antibody and cells, It was expected that the cytotoxic activity of the antibodies would be different.
  • Example 2 Evaluation of cytotoxicity against solid cancer cells
  • the cytotoxicity of existing anti-HLA-DR antibodies against solid cancer cells was examined.
  • the lung cancer cell line Calu-1 (ATCC HTB-46), in which high expression of HLA-DR was confirmed in the presence of IFN ⁇ , was adopted.
  • Calu-1 cells were prepared to 1 ⁇ 10 5 cells/ml using MyCoy's 5a Medium (SH30200.01, Hyclone) containing 10% FBS, and IFN- ⁇ was added to a final concentration of 2 ng/ml. Then, 100 ⁇ l of each was seeded on a 96-well plate. IncuCyte (registered trademark) Annexin V Red Reagent (Essen Bio, 4641) and antibody LN-3 (final concentration 1 ⁇ g/ml) were added and subjected to analysis by an Incucyte real-time analyzer (Essen Bio). Since apoptotic cells were stained with Annexin V Red Reagent, cell death induction was evaluated based on the change in fluorescence intensity after 15 hours.
  • IncuCyte registered trademark
  • Annexin V Red Reagent Essen Bio, 4641
  • antibody LN-3 final concentration 1 ⁇ g/ml
  • Example 3 Evaluation of target specificity
  • the antibody LN-3 which was found to be cytotoxic in Example 2, was evaluated for target specificity of cytotoxicity.
  • cytotoxicity test was performed on L428 cells, KM-H2 cells, which are human Hodgkin lymphoma cell lines that express HLA-DR, and Jurkat cells (ATCC TIB-152), which are HLA-DR non-expressing cells. Cytotoxicity was measured by the same method as in Example 1 except for the antibody concentration.
  • the antibody LN-3 shows dose-dependent cytotoxicity against HLA-DR expressing cells (Fig. 3(A) and (B) for L428 cells and KM-H2 cells, respectively) and to Jurkat cells. On the other hand, it showed no cytotoxicity (FIG. 3(C)), indicating that the induced cytotoxicity was HLA-DR specific (FIG. 3).
  • Example 4 Determination of antibody amino acid sequence
  • the amino acid sequence of antibody LN-3 was analyzed by mass spectrometry. Samples for mass spectrometric analysis were prepared by two methods, in gel digestion and in solution digestion.
  • MS/MS analysis was performed on the fragmented peptide using Thermo Fisher Q-Exactive, and the sequence was determined by de novo.
  • the leucine and the isoleucine having the same mass were distinguished from each other by using w-ion having different masses generated by HCD cleavage of a peptide fragment having the amino acid at the N-terminus.
  • FIG. 4(A) shows the heavy chain variable region amino acid sequence of antibody LN-3
  • FIG. 4(B) shows the light chain variable region amino acid sequence of antibody LN-3.
  • Example 5 Determination of epitope
  • the epitope in the amino acid sequence of HLA-DR to which antibody LN-3 binds was analyzed by peptide array.
  • a secondary antibody (anti-mouse antibody, Antimouse IgG (H+L), Thermo_84545) labeled with fluorescence was reacted and detected with a fluorescence scanner.
  • Example 6 Production of chimeric antibody
  • a human Fc chimeric recombinant antibody hereinafter referred to as BP1206 chimeric antibody
  • BP1206 chimeric antibody a human Fc chimeric recombinant antibody designed based on the antibody LN-3 was expressed and purified.
  • pCI-neo has a light chain expression element (EF-1 ⁇ promoter, secretion signal, light chain variable region, light chain constant region linked in tandem), heavy chain expression element (EF-1 ⁇ promoter, secretion)
  • EF-1 ⁇ promoter secretion signal
  • light chain variable region variable region
  • light chain constant region linked in tandem
  • heavy chain expression element EF-1 ⁇ promoter, secretion
  • the DNA sequences of the heavy chain variable region and the light chain variable region are based on the amino acid sequences of the antibody variable regions (Fig. 4, SEQ ID No.: 8 for heavy chains, SEQ ID No.: 10 for light chains). In addition, it was designed based on the codon optimized for the hamster expression system (SEQ ID No.:7 and SEQ ID No.:9).
  • the amino acid sequences of the heavy chain constant region portion and the light chain constant region portion the known amino acid sequences of the heavy chain constant region portion and light chain constant region portion of a human antibody, or a DNA sequence defining a variant thereof can be used.
  • ExpiCHO cells (invitrogen A2910002) were transfected with the plasmid using the Gibco TM ExpiCHO TM Expression System (GIbco A29129), cultured for 14 days, and the culture supernatant was collected.
  • the antibody was obtained by purifying the culture supernatant using a protein A column (MonoSpin ProA, GL Science 7510-11314).
  • Example 7 Drug effect on cancer-bearing mice (1)
  • the antitumor effect of the BP1206 chimeric antibody obtained in Example 6 on tumor in vivo was examined.
  • Hodgkin lymphoma cell line L428 cells (5 ⁇ 10e6 cells) were transplanted subcutaneously into the flank of a mouse NOD/Shi-scid, IL-2R ⁇ KOJic (generic name: NOG mouse, in vivo science).
  • the tumor mass reached 100 mm3
  • Body weight and tumor diameter were measured twice a week for 42 days from the first administration day of the BP1206 chimeric antibody. Further, after the observation period of 42 days was over, the tumor was excised and its weight was measured.
  • the isotype control antibody group continued to increase until Day 42, while the BP1206 chimeric antibody group continued to decrease, and compared to the isotype control antibody group after Day 5. It showed a statistically significant low value (Fig. 7(A)). Further, in one example of the BP1206 chimeric antibody group, tumor cells in the body disappeared.
  • Example 8 Drug effect on cancer-bearing mice (2) (dose-response test)
  • dose-responsiveness of the anti-tumor effect of the BP1206 chimeric antibody obtained in Example 6 on tumor in vivo was examined.
  • Hodgkin lymphoma cell line L428 cells (5x10e6 cells) were transplanted subcutaneously into the flank of mouse NOD/Shi-scid, IL-2R ⁇ KOJic.
  • the tumor mass reaches 100 mm3
  • Body weight and tumor diameter were measured twice a week for 42 days from the first administration day of the BP1206 chimeric antibody. Further, after the observation period of 42 days was over, the tumor was excised and its weight was measured.
  • the isotype control antibody group continued to increase until Day 42, while it decreased in the BP1206 chimeric antibody 10 mg/kg administration group and 5 mg/kg administration group, and the isotype It showed a statistically significant lower value than the control antibody group.
  • the BP1206 chimeric antibody 1 mg/kg administration group showed a statistically significant low value as compared with the isotype control antibody group on the 35th day during the observation period (FIG. 8(A)).
  • Example 9 Drug effect on cancer-bearing mice (3) (confirmation using surrogate antibody)
  • a surrogate antibody against the BP1206 chimeric antibody obtained in Example 6 antibody that crosses animal antigen, anti-mouse MHC class II antibody
  • the effect on cancer cells of the BP1206 chimeric antibody but was affected by species differences between the host and the transplanted cancer cells.
  • Mouse C57BL/6 (CLEA Japan, 7 weeks old, female) was subcutaneously implanted with the mouse T cell lymphoma cell line E.G7 cell mutant (5 ⁇ 10e6 cells) in the flank.
  • the cells used in this example are mutants prepared by forcibly expressing CIITA, which is a transcription factor that induces the expression of MHC Class II, in EG7 cells (ATCC (registered trademark) CRL-2113 TM ).
  • mice Three days after transplantation of E.G7 cells, one group will receive anti-mouse MHC class II antibody (M5/114 clone, Bioxcell) that is a surrogate antibody, and another group will receive 10 mg/kg of isotype control antibody.
  • Phosphate buffer solution (PBS) was administered to the remaining group as a negative control on the same schedule.
  • Body weight and tumor diameter were measured twice a week for 21 days from the first administration of anti-mouse MHC class II antibody.
  • the isotype control antibody-administered group and the PBS-administered group continued to increase significantly until Day 21, while the anti-mouse MHC class II antibody-administered group showed an increase in volume. It was relatively suppressed, and showed a statistically significant low value at Day 10 as compared with the isotype control antibody administration group and the PBS administration group (FIG. 9(A)). In addition, tumor cells in the body disappeared in one of the anti-mouse MHC class II antibody-administered groups.
  • DC dendritic cells
  • cDC1 particularly contributes to the process of recognizing a foreign antigen and activating TIL.
  • the effect of antibody administration on DC in spleen was determined based on the ratio of DC to total cells in the spleen, the ratio of cDC1 cells in DC, and the ratio of cDC2 cells in mice treated with antibody. was evaluated.
  • the DC ratio was calculated based on the expression of DR and CD11a-positive cells, and the ratio of cDC1 and cDC2 in DC was calculated based on the expression ratio of XCR1 and CD172a.
  • the percentages of cDC1 cells and cDC2 cells were 2.17 ⁇ 0.1% and 2.0 ⁇ 0.1%, 70.64 ⁇ 2.6% and 73.4 ⁇ 1.3%, 11.04 ⁇ in the isotype control antibody-administered group and the surrogate antibody-administered group, respectively. It was 1.3% and 11.4 ⁇ 1.3%.
  • Example 10 Effect on normal cells
  • the effect of the BP1206 chimeric antibody on normal cells expressing HLA-DR was evaluated.
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • B cells CD19 positive cells
  • T cells CD3 positive cells
  • the BP1206 chimeric antibody reduced the abundance of B cells (CD19 positive cells) by about 60%, while not affecting the abundance of T cells (CD3 positive cells).
  • rituximab a monoclonal antibody consisting of anti-human CD20 human/mouse chimeric antibody
  • rituximab reduces the abundance ratio of B cells by 90% (Blood. 2010 Jun 24; 115(25 ): 5180-5190), suggesting that the B cell suppression of the BP1206 chimeric antibody was less than that of rituximab.
  • Example 11 Evaluation of cytotoxicity against solid cancer cells (1)
  • the cytotoxic effect of the BP1206 chimeric antibody on solid cancer cells of various origins was examined.
  • solid cancer cells in this example, two human melanoma-derived cell lines (HT144 (ATCC, HTB-63) and A375 (ATCC, CRL-1619)) and two human lung cancer-derived cell lines (COR- L105 (ECACC, 92031918) and LU65 (JCRB, 0079)) were used to assess the cytotoxic effect of the BP1206 chimeric antibody on these cells using a three-dimensional culture method suitable for culturing these cells. ..
  • ⁇ For culture of HT144 cells MyCoy's 5A medium (GE, SH30200.01)
  • DMEM Dulbecco's Modified Eagle's Medium
  • RPMI1640 Wako, 189-02025 for culturing COR-L105
  • RPMI1640 Wako, 189-02025) was used with 10% FBS (gibco, 10270-106) added.
  • Colony culture was performed using a 24-well plate. Add 0.3 mL of 0.5% agar-containing medium to the well to form a Bed layer, and add 0.3 mL of 0.3% agar-containing medium containing cells and test substance on it, and incubate at 37°C in 5% CO 2 incubator for 5 days. I went.
  • the ratio of viable cells was measured using MTT reagent (Cell Proliferation Kit I, Sigma, 11465007001) or Calcein-AM (Dojindo, C396).
  • MTT reagent Cell Proliferation Kit I, Sigma, 11465007001
  • Calcein-AM Calcein-AM
  • Figure 12 shows the results when the colony culture method was adopted.
  • the survival rate of tumor cells was obtained by adding the BP1206 chimeric antibody. It has become clear that Among these cells, especially for human melanoma-derived cells, HT144 cells, a remarkable effect of reducing the ratio of viable cells to about 40% was shown.
  • Example 12 Evaluation of cytotoxicity against solid cancer cells (2)
  • the cytotoxic effect of BP1206 chimeric antibody on solid cancer cells of various origins and the combined effect with anticancer agents were examined.
  • Hs852.T ATCC CRL-7585
  • C32 ATCC CRL-1585
  • SK-MEL-24 ATCC HTB-71
  • Hs 852.T cells L-Glutamine-containing Dulbecco's Modified Eagle's Medium (DMEM) and High Glucose (Wako, 044-29765)
  • DMEM Dulbecco's Modified Eagle's Medium
  • Wako 044-29765
  • EMEM Eagle's Minimum Essential Medium
  • SK-MEL-24 culture Eagle's Minimum Essential Medium (EMEM) (Wako, 055-08975) was used with 10% FBS (gibco, 10270-106) added.
  • Vemurafenib SIGMA, S1267
  • SIGMA Vemurafenib
  • the LDH test is a test method that quantitatively evaluates cell death based on the activity of LDH (lactate dehydrogenase) that leaks out of cells due to cell membrane damage accompanying cell death.
  • LDH lactate dehydrogenase
  • lactic acid which is a substrate of LDH
  • NAD+ which is a cofactor
  • WST-8 tetrazolium salt which is a chromogenic substrate are added to the culture supernatant, and WST-8 is produced by NADH produced in the catalytic process of the enzyme.
  • the amount of dead cells was indirectly quantified from the absorbance (450 nm) after coloring using the principle that tetrazolium salt was reduced and colored.
  • Example 13 Evaluation of dose response of cytotoxicity to solid cancer cells (three-dimensional culture method) In this example, the dose response of the cytotoxic effect of BP1206 chimeric antibody on solid cancer cells was examined.
  • HT144 ATCC, HTB-63
  • A375 ATCC, CRL-1619
  • MyCoy's 5A medium GE Healthcare, SH30200.01
  • Agar Difco, 526-00054
  • FBS gibco, 10270-106
  • DMEM Wired Chemical Industries, 044-29765
  • BP1206 chimeric antibody were prepared at 6 ⁇ 10 4 cells/ml using the same medium as the bed layer, and added at 0.3 ml/well on the bed layer.
  • the specified dose is cisplatin (SIGMA, PHR1624, 2 ⁇ M) or vemurafenib (SIGMA, S1267, HT144 cells 300 nM, A375 cells 600 nM) together with the BP1206 chimeric antibody.
  • SIGMA cisplatin
  • PHR1624, 2 ⁇ M cisplatin
  • SIGMA vemurafenib
  • the mixture was mixed in the same manner, and similarly added at 0.3 ml/well on the bed layer. After cooling at 4°C to solidify, the temperature was returned to room temperature, and the cells were cultured at 37°C in a 5% CO 2 incubator for 5 days.
  • Figure 14 shows the results of cell death induction (relative value based on viable cell mass measurement) after treating cells with the BP1206 chimeric antibody.
  • A HT144 cells, BP1206 chimeric antibody and cisplatin combination
  • B HT144 cells, BP1206 chimeric antibody and Vemurafenib combination
  • C A375 cells, BP1206 chimeric antibody and Vemurafenib combination
  • the cell death rate induced by is shown.
  • the BP1206 chimeric antibody showed a dose-dependent survival inhibitory effect on both cell lines (Fig. 14). The survival inhibitory effect was enhanced more than additively by using cisplatin or vemurafenib in combination with the BP1206 chimeric antibody.
  • Example 14 Observation of cell morphological change associated with cell death induction
  • the BP1206 chimeric antibody induces cell death of cancer cells
  • cell morphological changes caused by cancer cells were shown. ..
  • L428 cells were suspended in the medium and seeded in a PCR tube (Nippon Genetics, FG-008FC) at a concentration of 50,000 cells/50 ⁇ l, and then the BP1206 chimeric antibody solution (50 ⁇ l) was added to a final concentration of 10 ⁇ g/mL. And incubated at 37°C for 1 hour. The cells were collected by centrifugation, suspended in a 1% glutaraldehyde solution (Nacalai, 1700392) and fixed overnight, and then the cell shape was analyzed with a tabletop electron microscope Miniscope TM3030Plu (Hitachi) (Fig. 15).
  • Example 15 Immunological cell death (Immunogenic cell death) induction evaluation
  • immunological cell death in tumor immunological cell death in tumor (Immunogenic cell death, hereinafter also referred to as ICD) Based on the secretion of the marker (described), the ICD-inducing ability of the antibody was evaluated.
  • Mouse CB17.Cg-PrkdcscidLystbg-J/CrlCrlj (generic name: SCID-Beige) was subcutaneously transplanted with Hodgkin lymphoma cell line L428 cells (5 ⁇ 10e6 cells) into the flank, and the tumor mass exceeded 100 mm 3 .
  • BP1206 chimeric antibody (10 mg/kg), isotype control antibody (10 mg/kg) or PBS was administered once to an individual (administration route: intraperitoneal, dosage form: solution). The number of experimental cases was 3 for the BP1206 chimeric antibody administration group and 3 for the isotype control antibody administration group, and 1 for the PBS administration group.
  • HMGB1 is a protein that originally exists in the nucleus, but is known to be released into the cytoplasm with cell death, and since it serves as an ICD marker, it can be labeled with an anti-HMGB1 antibody to produce a BP1206 chimeric antibody.
  • the effect on HMGB1 secretion can be investigated.
  • immunostaining using an anti-human IgG antibody was performed for the purpose of confirming whether the BP1206 chimeric antibody actually infiltrated into the tissue in the animal body.
  • Dyeing was performed according to the following procedure. After preparing thin slices, submerge in BOND Dewax Solution (Reica, AR9222) and 100% ethanol to deparaffinize them, wash with 0.01M PBS, and use BOND Epitope Retrieval Solution1 (Reica, AR9961). Then, the antigen activation treatment was performed at 98°C for 30 minutes. After washing with 0.01M PBS, react with anti-HMGB1 antibody (GeneTex, GTX628834, 750-fold diluted) or rabbit anti-human IgG (H+L) antibody (Bethyl, A80-118A, 10,000-fold diluted) at room temperature for 30 minutes I went.
  • BOND Dewax Solution Reica, AR9222
  • BOND Epitope Retrieval Solution1 Reica, AR9961
  • the antigen activation treatment was performed at 98°C for 30 minutes. After washing with 0.01M PBS, react with anti-HMGB1 antibody (GeneTex, GTX628834,
  • 16(A) and (B) are BP1206 chimeric antibody-administered mice
  • (C) and (D) are isotype control antibody-administered mice
  • (E) and (F) are PBS-administered mice, respectively.
  • (C) and (E) are whole tumor views
  • (B), (D) and (F) are enlarged views of strongly stained sites.
  • the effect of BP1206 chimeric antibody on HMGB1 secretion in the L428 cell subcutaneous transplant model (left) and tumor infiltration (right) are shown.
  • Example 16 Humanization of antibody
  • a humanized recombinant antibody hereinafter referred to as BP1206 humanized antibody
  • BP1206 humanized antibody was designed based on the structure of the BP1206 chimeric antibody prepared in Example 6, and its expression/purification I went.
  • Example 17 Functional evaluation of humanized antibody
  • the cytotoxic activity of the eight types of BP1206 humanized antibodies prepared in Example 13 against cancer cells was clarified.
  • the BP1206 humanized antibody prepared in Example 13 was evaluated for in vitro cytotoxicity against Hodgkin lymphoma cell line L428 cells.
  • L428 cells were prepared in RPMI medium containing 10% FBS at 1 ⁇ 10 6 cells/ml, and Propidium iodide (PI; DOJINDO, FE159) was added to a final concentration of 2 ⁇ M, then 50,000 cells/50 ⁇ l The cells were seeded in a PCR tube (Fast gene, FG-1700) at the concentration of.
  • Eight types of BP1206 humanized antibody solutions (50 ⁇ l) obtained in Example 13 were added to each tube (final antibody concentration 10 ⁇ g/mL) and incubated at 37° C. for 1 hour.
  • Example 18 Search for indications for solid cancer
  • indications for solid cancer of the BP1206 humanized antibody obtained in the present invention were searched.
  • the above-mentioned sample was spotted on a slide glass and carried out using a tumor tissue microarray (manufactured by Pantomics). After activating the antigens on the microarray by the pressure cooker method, they are sequentially reacted with the LN3 antibody, peroxidase-labeled polymer, and chromogenic substrate (diaminobenzidine (DAB)), and the expression of HLA-DR antigen is evaluated based on the color development of DAB. did.
  • DAB diaminobenzidine
  • Example 19 Correlation between target expression and cytotoxicity
  • various cancer cell lines are used to express target HLA-DR antigen and cytotoxicity that can be induced against the cells. The correlation between the two was investigated.
  • 16 cell lines ⁇ Kasumi-1: Derived from human acute myelogenous leukemia (AML) (ATCC CRL-2724); ⁇ OCI-LY-19 cells: derived from human diffuse large B-cell lymphoma (DLBCL) (DSMZ, ACC 528); ⁇ Nalm-6 cells: derived from human acute lymphocytic leukemia (ALL) (DSMZ, ACC 128); ⁇ KMS-26 cells: derived from human multiple myeloma (MM) (JCRB1187); ⁇ KG-1 cells: derived from human acute myeloblastic leukemia (AML) (ATCC CCL-246); -Rmos cells: derived from human Burkitt lymphoma (BL) (DSMZ, ACC-603); ⁇ KMS-11 cells: derived from human multiple myeloma (MM) (JCRB1179); ⁇ JJN-3 cells: derived from human multiple myeloma (MM) (DSMZ, ACC
  • Each cell was cultured by the method specified by the supplier.
  • the cells were suspended in PBS, 5 ⁇ 10 5 cells were collected in a PCR tube, BP1206 antibody was added to a final concentration of 10 ⁇ g/ml, and the mixture was incubated at 37° C. for 2 hours in a thermal cycler.
  • the cells were washed with PBS, stained with 7-AAD (Biolegend, 400625), and subjected to FACS analysis.
  • the ratio of 7-AAD unstained cells was calculated as the ratio of viable cells. The results are shown as a bar graph in FIG.
  • the target HLA-DR antigen expression level of the cell line was analyzed by FACS (BD Verse) after labeling each cell with a labeled anti-human HLA-DR antibody (Biolegend, 307615).
  • the expression level of the HLA-DR antigen is shown by dots in FIG.
  • the HLA-DR antigen-binding substance of the present invention is used to treat HLA-DR antigen-expressing cancer cells, to inhibit the growth of cancer cells, and thus to reduce or eliminate tumors. Can be made.
  • this binding substance particularly an antibody or a human antibody derivative can bind to the HLA-DR antigen, immunization of the HLA-DR antigen in vivo as a test application of cancer cells expressing the HLA-DR antigen. It can be used for biological detection (ELISA, Western blotting, flow cytometry, etc.).
  • the cell viability of the cancer cells is decreased by bringing the cancer cells collected from the subject into contact with the binding substance of the present invention, particularly an antibody or a humanized antibody derivative under culture conditions, or immunity
  • the binding substance of the present invention particularly the antibody or the humanized antibody derivative is administered to a subject having cancer cells
  • the cancer cells are treated in the subject. It is possible to investigate the degree of cytotoxicity against the.

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Abstract

La présente invention vise à procurer une substance qui se lie à l'antigène HLA-DR et qui peut exercer une activité cytotoxique sur des cellules cancéreuses exprimant l'antigène HLA-DR, ainsi q'un agent thérapeutique antitumoral contenant la substance qui se lie à l'antigène HLA-DR.　À cet effet, la solution de la présente invention porte sur une substance qui se lie à un antigène HLA-DR, ladite substance comportant des régions déterminant la complémentarité (CDR) de chaîne lourde 1 à 3 et des CDR de chaîne légère 1 à 3 avec des séquences d'acides aminés spécifiques, en particulier, un anticorps ou un dérivé d'anticorps humanisé contre l'antigène HLA-DR.　Selected drawing: none
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WO2009066655A1 (fr) * 2007-11-19 2009-05-28 Kagoshima University Anticorps humain capable d'induire une apoptose

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WO2009066655A1 (fr) * 2007-11-19 2009-05-28 Kagoshima University Anticorps humain capable d'induire une apoptose

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Title
ANONYMOUS: "HLA- DRB1 monoclonal antibody, clone LN-3", LABOME, 17 December 2018 (2018-12-17), pages 1 - 2, XP055735526, Retrieved from the Internet <URL:https://www.labome.com/product/Abnova/MAB13305.html> [retrieved on 20200416] *
KOSTELNY, S. A. ET AL.: "Humanization and characterization of the anti-HLA-DR antibody 1D10.", INT. J. CANCER, vol. 93, 2001, pages 556 - 565, XP002962301, DOI: 10.1002/ijc.1366 *
NAGY, Z. A . ET AL.: "Fully human, HLA-DR-specific monoclonal antibodies efficiently induce programmed death of malignant lymphoid cells", NATURE MEDICINE, vol. 8, no. 8, 2002, pages 801 - 807, XP002432676 *

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