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Definitions

• the present invention relates to an immunostimulation characterized by inhibiting an immunosuppressive signal induced by PD-1, PD-Ll, or PD-L2, and a thread for the treatment of cancer or infection. And therapeutic methods using them. More specifically, a composition for treating cancer or treating infectious diseases via immunostimulation caused by inhibiting an immunosuppressive signal induced by PD-1, PD-Ll, or PD-L2. , Therapeutic methods using them, immunostimulatory substances contained in the composition as active ingredients, screening methods for cancer therapeutic substances or infectious disease therapeutic substances, cell lines used in these screening methods, evaluation for screening cancer therapeutic substances The present invention relates to a method and a cancer cell transplantation mammal used for the evaluation. Background art
• Immunotherapy is expected to be an extremely specific treatment method because it reduces the unavoidable side effects of most drug therapies.
• drug therapy in cancer treatment and infectious disease treatment is a treatment method that places a heavy burden on patients, recovery of patients' quality of life (QOL) is regarded as important.
• Immunotherapy is based on the objective of restoring the patient's QOL by stimulating the immune response inherent in humans by exogenous methods and by taking part of the burden of drug administration. Can be done.
• Stimulation of immunity can be performed by a method of activating the immune response of T lymphocyte cells.
• Activation of T cells involves antigen receptor (TCR) '-mediated stimulation.
• TCR antigen receptor
• conjugated stimulating molecule groups eg CD 28
• CTLA-4 and PD-1 molecular groups having a structure homologous to the conjugate stimulating molecule group, have been discovered and reported to emit a signal that suppresses the antigen receptor (TCR) signal.
• PD-1 was cloned as a 55 kD type I membrane protein belonging to the immune globulin family (The EMBO Journal, 1992, No. 11, No. 11, p. 3887-3895, JP-A-5-336973 JP-A-7-291996).
• Human PD 1-1c DNA is composed of the nucleotide sequence shown in EMBL I GenBank Acc. No. M-005018, and mouse PD_lc DNA is composed of the nucleotide sequence shown in Acc. No. X67914, and their expression. Is observed in thymocytes upon differentiation from CD 4—CD 8— to CD 4 + CD 8 + cells (International Immunology, 1996, No. 18, Vol.
• PD-1 expression in the periphery is expressed by T cells and B cells activated by stimulation from antigen receptors (see International Immunology, 1996, Vol. 18, No. 5, p.765-772) or activity It has been reported to be found in bone marrow cells containing modified phages.
• ITIM motif Immunoreceptor Tyrosine-based Inhibitory Motif
• PD-1-deficient mice are affected by lupus-like autoimmune diseases such as glomerulonephritis and arthritis (in the case of C 5 7 B LZ 6 gene background) (International Immunology, 1998, 10, 10, ⁇ ⁇ 1563 ⁇ 1572, Immunity, 1999, 11th, 2nd, ⁇ .141 ⁇ 151) and dilated cardiomyopathy-like disease (in the case of BALBZc gene background) (Science, 2001, 291 ⁇ , 5502) , P.319-332), PD-1 is associated with the development of autoimmune diseases, especially the control of peripheral autoimmunity tolerance. It is also suggested that it is a factor.
• PD-L 1 which is a ligand of PD-1 (human PD-LI cDNA is EMBL / GenBank Acc. No. AF233516, mouse PD-L 1 cDNA is composed of the base sequence shown by M-0221893.) Is expressed in so-called antigen-presenting cells such as activated monocytes and dendritic cells (Journal of Experimental Medicine, 2000, Vol. 19, No. 7, ⁇ .1027-1034). These cells present interacting molecules that induce various immune-inducing signals to T lymphocytes, and PD — L 1 is one of the molecules that induces PD-1 suppressive signals. .
• antigen-presenting cells such as activated monocytes and dendritic cells
• PD-L1 ligand stimulation has been shown to inhibit the activity of T lymphocyte cells expressing PD-1 (cell proliferation, induction of various cyto force in production). Furthermore, PD-L1 is expressed not only in immunocompetent cells but also in certain tumor cell lines (monocytic leukemia-derived cell lines, mast cell type-derived cell lines, liver cancer-derived cell lines, neuroblastoma types). (See Nature Immunology, 2001, Vol. 2, No. 3, p.261-267).
• PD-L 2 Heat PD—L 2 c DNA is composed of EMBL I GenBank Acc. No. NM— 025239, and mouse PD—L 2 c DNA is composed of the base sequence represented by M—021896 (Nature Immunology, 2001, Vol. 2, No. 3, p.261-267)) was identified as the second ligand of PD-1, but its expression and function are almost the same as PDL 1. It has been reported.
• Inhibition signals from conjugation-inhibiting molecules are linked to immune tolerance and self-expression during lymphocyte development or maturation by mechanisms that adequately control the positive signals of antigen receptor (TCR) and conjugation stimulating molecules. It is thought to control abnormal immune responses to antigens. Some tumors and viruses also block T cell activation and proliferation by direct or indirect mechanisms, and use these coupled inhibitory molecules to attenuate host immune responses against themselves. (Cell, 1992, No. 1 71, No. 7, ⁇ .1093-1102, Science, 1993, Vol. 259, No. 5093, .368-370). Furthermore, in some of the diseases thought to be caused by T cell dysfunction, abnormalities in these conjugation-inhibiting molecules are thought to cause T cell dysfunction. Disclosure of the invention
• An object of the present invention is to inhibit a suppressive signal by PD-1, PD-Ll, or PD-L2, and to immunostimulate the composition, and to treat cancer or infection through this mechanism. In providing a composition.
• the present inventors focused on PD-1, PD-Ll, or PD-L2 as a new target in cancer treatment or infectious disease treatment, and PD-1, PD-Ll, or PD- It was found that a substance that inhibits the suppression signal by L2 inhibits cancer growth through recovery of immune function and activation mechanism. Furthermore, we found that PD-1 signal, specifically PD_1 and PD-L1 or PD-1 and PD-L2 interaction, was involved in eliminating infected viruses. Based on these facts, the present inventors have found that a substance that inhibits a suppression signal by PD-1, PD-Ll, or PD-L2 has a therapeutic effect on cancer or infectious diseases, and completed.
• an immunostimulatory composition comprising an immunosuppressive signal inhibitor of PD-1, PD—L1, or PD—L2,
• a cancer therapeutic composition comprising an immunosuppressive signal inhibitor of PD-1, PD-Ll, or PD-L2,
• the cancer treatment composition according to item 2 which is a composition that suppresses cancer metastasis
• a composition for treating infectious disease comprising an immunosuppressive signal inhibitor of PD-1, PD-Ll, or PD-L2, 5.
• composition according to the above item 7 which is an interaction inhibitor of one or more PD-1 and PD-L1 selected from 0—1 antibody, solubilized PD-1, and solubilized PD—L1;
• L 2 production inhibitor is selected from proteins, polypeptides or peptides, polynucleotides or polynucleosides, antibodies or their derivatives, organic synthetic compounds, inorganic compounds, and natural products- The composition described,
• An immunostimulatory method comprising administering an immunosuppressive signal inhibitor substance of PD-1, PD—L1, or PD—L2,
• PD—1, PD—L 1, or PD—L 2 immunosuppressive signal inhibitors A method for treating cancer comprising administering a substance,
• a method for treating an infectious disease comprising administering an immunosuppressive signal inhibitor substance of PD-1, PD-Ll, or PD-L2,
• PD-1 and PD-L 1 or PD-1 and PD-L2 interaction inhibitor PD-1 intracellular signal inhibitor, and PD-1, PD-L 1 or PD-L 2 18.
• PD-1 antibody, PD-L1 antibody, solubilized PD-1, and solubilized PD — L-1 is one or more PD-1 and PD—L1 interaction inhibitors 18 method,
• lymphocyte cell whose PD-1 expression is inhibited by genetic modification is an immunosuppressive signal inhibitor, 22.
• PD-1 and PD-L 1 or PD Inhibitor of interaction between PD-1 and PD—L2, Inhibitor of intracellular signal of PD-1, or PD-1.
• 0—1 or PD—L 2 production inhibitor is selected from proteins, polypeptides or peptides, polynucleotides or polynucleosides, antibodies or derivatives thereof, organic synthetic compounds, inorganic compounds, and natural products.
• cancer treatment composition is a cancer metastasis-suppressing composition
• PD—1, PD—L 1, or PD—L 2 for producing an infection treatment composition
• immunosuppressive signal inhibitors Use of immunosuppressive signal inhibitors
• a cancer cell line for screening that is transformed to express PD-L 1 or PD—L 2,
• An immunostimulatory substance characterized by contacting the cell, lymphocyte cell and test substance according to item 27 above, and evaluating the enhancing effect of the test substance on the immune response of the lymphocyte cell to the cell according to item 27 above SCREENING method,
• the cell according to the preceding paragraph 27 infected with a pathogen or a cell that expresses PD-L1 or PD-L2, a lymphocyte cell, and a test substance are contacted, and the immune response of the lymphocyte cell to the infected cell
• a screening method for a therapeutic substance for infectious diseases characterized by evaluating a potentiating action of a test substance or an inhibitory action on pathogen growth,
• a method for selecting a cancer therapeutic substance comprising administering a test substance to the mammal described in the preceding paragraph 31, and evaluating the inhibition rate of the transplanted cancer cell proliferation by the test substance or the survival rate of the recipient mammal.
• PD-1, PD-L1 or PD-L2 in the present invention includes those derived from primate mammals including mice, rats, hamsters, guinea pigs, dogs, pigs, monkeys or humans, respectively. .
• the immunosuppressive signal by PD-1, PD-L1, or PD-L2 is at least an interaction between PD-1 and PD-L1 or PD-1 and PD-L2. Consists of PD-1 intracellular signals. In addition, this includes the production of PD_1, PD-L1, or PD-L2 molecules themselves.
• the immunosuppressive signal by PD-1, PD-Ll, or PD-L2 in the present invention is the interaction between PD-1 and PD-L1 or PD-1 and PD-L2, or PD — Inhibited by direct or indirect inhibition of 1 intracellular signal.
• Substances having these inhibitory activities include substances that selectively bind to PD-1, PD-Ll, or PD-L2, respectively.
• protein, polypeptide or peptide, polynucleotide or polynucleoside, antibody or derivative thereof, organic synthetic compound, organic compound, or natural product can be used.
• antibodies with high specificity include antibodies to PD-1, PD-Ll, or PD-L2.
• the immunosuppressive signal is also inhibited by inhibiting the production of PD-1, PD-Ll, or PD-L2 molecules themselves.
• Antibodies to PD-1, PD-Ll, or PD-L2 are human-derived antibodies or mouse-derived antibodies that inhibit the immunosuppressive signal by PD-1, PD-L1, or PD-L2. These antibodies may be antibodies, rat-derived antibodies, rabbit-derived antibodies or goat-derived antibodies, and their polyclonal or monoclonal antibodies, complete or truncated (for example, F (a b ') 2 , (F ab ⁇ Fab or F v fragment) Any antibody, chimerized antibody, humanized antibody or fully human antibody may be used.
• Such an antibody can be produced according to a known antibody or antiserum production method using a partial protein in the extracellular region of PD-1, PD-Ll, or PD-L2 as an antigen.
• a partial protein in the extracellular region can be prepared by known protein expression and purification methods.
• Polyclonal antibodies can be produced by known methods. For example, it can be produced by immunizing a suitable animal with an antigen protein or a mixture of it and a carrier protein, collecting an antibody-containing substance against the antigen protein from the immunized animal, and performing separation and purification of the antibody. . Commonly used animals include mice, rats, hidges, goats, rabbits and guinea pigs. In order to increase antibody production, complete Freund adjuvant or incomplete Freund's adjuvant can be administered with the antigen protein. The administration is usually about once every 2 weeks, about 3 to 10 times in total. Polyclonal antibodies can be collected from blood, ascites, etc. of animals immunized by the above method.
• the polyclonal antibody titer in the antiserum can be measured by the ELISA method.
• Separation and purification of polyclonal antibodies include, for example, purification methods using an antigen-binding solid phase or an active adsorbent such as protein A or protein G, salting-out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, It can be carried out according to the separation and purification method of immunoglobulin such as adsorption / desorption method by ion exchanger, ultracentrifugation method, gel filtration method.
• monoclonal antibodies or their modifications are more preferred.
• Monoclonal antibody-producing cells are prepared by selecting individuals with antibody titers from animals immunized with antigen, collecting spleen or lymph nodes 2 to 5 days after the final immunization, and collecting antibody-producing cells contained in them. By fusing with myeloma cells of the same or different species, a monoclonal antibody-producing hybridoma that can be subcultured can be produced.
• Administration of antigen protein It is carried out at the site where antibody production is possible, or with the carrier and diluent. For administration, in general, Freund's adjuvant or incomplete Freund's adjuvant is administered in order to enhance antibody production ability. Animals can also be immunized by a method called “DNA immunization”.
• cardiotoxin is treated in the hind paw tibialis muscle of an immunized animal, a vector that expresses an antigen protein is introduced, and then the vector is taken into muscle cells during the tissue repair process to express the protein.
• the animal to be immunized can be mouse, rat, hidge, goat, rabbit or guinea pig, preferably mouse or rat.
• the fusion operation can be performed by the method of Kohler and Milstein (Nature, 1975, No. 256, No. 5517, ⁇ ⁇ 495-497). Polyethylene Darikonore (PEG) and Sendai virus are used.
• Examples of myeloma cells include myeloma cells such as P 3 U 1, NS 1, SP 2 Z 0, and AP 1, but usually P 3 U 1 is often used.
• Monoclonal antibody-producing cells can be selected by, for example, detection by the ELISA method or the like by adding a hybridoma culture supernatant to a solid phase on which an antigen protein is adsorbed directly or together with a carrier. Furthermore, the antibody titer of the hybridoma culture supernatant can be measured by the ELISA method.
• the separation and purification of the monoclonal antibody can be carried out according to the same immunoglobulin separation and purification method as the above-described separation and purification of the polyclonal antibody.
• High Pri Doroma identified by the international accession number FERM BP-8392, is located on the 19th of December 1st, 1st East, Tsukuba City, Ibaraki, Japan. 305-8566), deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary under the deposit number FERM P-19162, and transferred to an international deposit on June 5, 2003.
• Hypridoma identified by international deposit number FERM BP-8396 was deposited with the center under deposit number FERM P-18908 on June 25, 2002, and transferred to international deposit on June 11, 2003. Yes.
• Antibody fragments are F (ab ′) 2 , Fab ′, Fab or scFv antibody fragments, which can be obtained by treatment with a protease enzyme and optionally reduction.
• F (ab ') 2 antibody fragments are either fully digested with pepsin of purified monoclonal antibodies, and can be either ion exchange chromatography, gel filtration, or affinity chromatography such as protein A or protein G columns. It can be purified by the method. Since pepsin digestion time varies depending on the I g subtype, it must be prepared appropriately.
• the Fab ′ antibody fragment can be prepared by partially reducing the prepared F (ab ′) 2 with 2_mercaptoethylamine.
• Fab antibody fragments can be prepared by direct digestion with the digestive enzyme papain in the presence of cysteine and purification.
• Monoclonal antibodies can be modified by gene recombination techniques to determine the amino acid sequence of the antibody or use DNA sequences that encode antibodies that can be isolated from hybridomas to produce modified antibodies and hybrid antibodies. It is also possible. For example, it can be prepared as a single chain antibody instead of a normal complete antibody.
• the sc Fv antibody (Single Chain Fv) can be obtained by the method of Jost et al. (Journal of Biological Chemistry, 1994, No. 269, No. 42, p.26267-26273). DNA fragments encoding the heavy and light chain variable regions are ligated with a spacer encoding a neutral amino acid (glycine or serine), and the expression vector containing this fusion DNA is expressed in an appropriate host cell. This makes it possible to produce a single-chain antibody that retains the properties and affinity of the original antibody.
• a humanized antibody that has been modified so that it can be administered to humans is modified to reduce antigenicity or improve blood kinetics to an extent that is pharmacologically acceptable when the antibody is administered to humans.
• Antibody is essential to reduce the antigenicity of the antibody.
• the patient's immune response to the antibody often shortens the effective treatment period, and requires steps to reduce the antigenic individualization of the antibody by humanizing or fully humanizing the antibody.
• a humanized antibody that has been modified so that it can be administered to humans is modified to reduce antigenicity or improve blood kinetics to an extent that is pharmacologically acceptable when the antibody is administered to humans.
• Antibody is formulated to reduce antigenicity or improve blood kinetics to an extent that is pharmacologically acceptable when the antibody is administered to humans.
• the human PD-1 antibody or the human PD-1 L1 antibody in the specification of the present invention includes an antibody obtained by humanizing or fully humanizing it.
• a humanized antibody can be produced by substituting a part of a non-human antibody produced by immunizing a mammal other than human with a part of human antibody. Specifically, it is known that it can be produced by constructing a chimera with a gene encoding the constant region of a human antibody (Proc. Natl. Acad. Sci. (USA), 1987, 84th, p. .3439-3443, Journal of Immunology, 1987, Vol. 139, No. 1, ⁇ ⁇ 3521). DNA sequences for human constant regions are described in the literature, and the constant region genes are readily available from known clones. Subsequently, the DN sequence encoding the variable region of the antibody is fused to the human constant region sequence.
• Human constant region isotypes can be selected according to the desired effector function or activity in antibody-dependent cellular cytotoxicity. Preferred isotypes are I g G l, I g G 3 and I g G 4. In addition, any of human light chain constant region, ⁇ chain, or ⁇ chain can be used. This humanized chimeric antibody can be expressed by a conventional method.
• Fully human antibodies include mice that have been introduced with human immunoglobulin constant region genes (Xenomouse (Chemical Biology, 2000, No.7, No.8, p.R185-6), Hyuumab mice (infection and immunity, 2002). Year 70, No. 2, p.612-9), TC mice (Biotechnology and Genetics Enginnering Revew, 2002, Vol. 19, p.73-82), KM mice (Cloning Stem Cells, 2002, vol. 4, No. 1, p.91-102)) Furthermore, the antibody-producing lymphocytes isolated from these mice can be made into a hybridoma, and the desired antibody can be mass-produced. It can also be prepared by the phage display method (FEBS Letter, 1998, 441, pp. 20-24).
• This method utilizes a phage in which a human antibody gene is incorporated into a circular single-stranded DNA, and the human antibody is expressed on the surface of the phage in a form fused with the outer shell protein constituting the phage. Can be done.
• Polypeptides or their derivatives that bind to PD-1, PD-L1, or PD-L2 are partial proteins of PD-1, PD_L1, or PD-L2, and induce immunosuppressive signals.
• the thing which does not do is mentioned.
• PD-L1 or PD-L present in antigen-presenting cells, tumors or cancer cells Constraints due to interaction with 2 are required. Therefore, solubilized PD-L 1 or solubilized PD-L 2 having only the extracellular domain and interacting with PD-1 can inhibit the immunosuppressive signal of PD-1.
• solubilized PD-1 which has a similar structure and can bind to PD-L1 or PD-L2, can also block immunosuppressive signals.
• solubilized proteins may contain any extracellular region necessary and sufficient for binding to PD-1, PD-Ll, or PD-L2, and can be used for known protein expression and purification methods. Therefore, it can be prepared.
• PD-1 and PD—L 1 or PD-1 and PD—L 2 interaction inhibitors are proteins or polypeptides, and the region essential for their interaction is composed only of continuous polypeptides.
• Such polypeptide fragments can be antagonists of each other.
• this polypeptide fragment It is possible to identify antagonists having a more potent activity from molecular groups that have been chemically modified or designed by a computer based on the three-dimensional structure of the polypeptide fragment.
• the optimal antagonist molecule can be selected more efficiently from a group of molecules designed by a computer based on protein tertiary structure analysis data in the interaction region.
• substances that inhibit the interaction between PD-1 and PD-L1 or PD-1 and PD-L2 can be directly screened.
• substances include proteins, polypeptides or peptides, polynucleotides or polynucleosides, non-peptide 'I "biocompounds, organic synthetic compounds, or natural products (eg, fermentation products, cell extracts, It can be identified from libraries of plant extracts and animal tissue extracts.
• the inhibitory signal of the PD-1 intracellular domain is the dephosphorylation enzyme bound to ITIM, a PD-1 intracellular domain (eg, SHP-1, 2 (SathishJG, Journal of Immunology), 2001, vol. 166 , No. 3, p.1763-70) Force It is caused by contact with the intracellular complex of the antigen receptor complex, and is generally inhibited by inhibition of the contact between the antigen receptor complex and PD_1.
• a substance that inhibits the inhibitory signal a substance that inhibits phosphorylation of tyrosine residues of ITIM, a substance that inhibits binding of dephosphorylation enzyme to ITIM, and directly inhibits the activity of the dephosphorylation enzyme Examples include substances.
• the antigen receptor complex include a T cell receptor complex and a B cell receptor complex.
• Production of PD-1, PD-Ll, or PD-L2 can be inhibited by specific polynucleotides or polynucleotides, organic synthetic compounds, inorganic compounds, or natural products.
• suitable polynucleotides or polynucleotides include antisense nucleotide derivatives called ribozymes. This is because a polynucleotide derivative complementary to the mRNA of PD-1, PD-L1, or PD-L2 is introduced into the target expression cells. Therefore, it utilizes the mechanism by which the expressed mRNA is destroyed.
• the vector can be used for genetic manipulations that inhibit PD-1 expression on lymphocyte progenitor cells collected from patients, and the manipulated cells are proliferated, differentiated, and activated. It can be used for cell therapy to be administered to the patient again.
• a specific lymphocyte cell that is specific to the target cell is added by adding a specific antigen of the target cell during maturation and activation of lymphocyte progenitor cells. Can be prepared.
• the screening method of the present invention can be performed by a method for measuring cell function.
• Screening cancer cell lines that have been transformed to express PD-L1 or PD-L2 used in the same method are those that are constructed to express PD-L1 or PD-L2.
• a vector is introduced into a cell by a known method and includes a transient or stably transformed cancer cell line.
• Cancer cell lines used include monkey COS-1 cell, COS-7 cell, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dh fr gene-deficient Chinese hamster cell CHO (hereinafter referred to as CHO cell) Abbreviated as CHO (dh fr—) cells) mouse L cells, mouse At T-20, mouse myeloma cells, rat GH3, HEK293 T cells, human FL cells and the like.
• CHO dh fr—
• Such cells include white blood cells, preferably monocytes, macrophages or antigen presenting cells, epithelial cells, tumor cells, cancer cells or cell lines thereof.
• white blood cells preferably monocytes, macrophages or antigen presenting cells, epithelial cells, tumor cells, cancer cells or cell lines thereof.
• Examples of cells that can be used as tumor cells or cancer cells include P 38 D 1 cells, P 815 cells, NB 41 A 3 cells, MD A—231 cells, SKBR—3 cells, MCF—7 cells, BT474 cells, J 558 L thin Cells, P3U1 cells, PAI cells, X63 cells or SP2Z0 cells can be used.
• Infectious agents include human hepatitis virus (hepatitis B, hepatitis C, hepatitis A) or hepatitis E, human retrovirus, human immunodeficiency virus (HIV 1, HIV 2), human T cell leukemia Virus, human T-lymphotropic Winores (HT LV 1, HTLV 2), simple herpes virus type 1 or type 2, Epstein-in.Berwinores, cytomegaloinoles, chickenpox herpes zoster winores, human herpesvirus 6
• herpes simplex virus poliovirus, measles virus, rubella virus, Japanese encephalitis virus, mumps virus, influenza virus, adenovirus, enterovirus, rhinovirus, severe acute respiratory syndrome (SARS) Virus, Ebola virus, West Nile virus, or artificially modified them
• the lymphocyte cells used in the screening method of the present invention are T cells or B cells, preferably cytotoxic T lymphocyte cells (CTL).
• CTL cytotoxic T lymphocyte cells
• the immune reaction of lymphocyte cells in the screening method of the present invention includes cytotoxic reaction (for example, tumor immune reaction), mixed lymphocyte reaction, production of cytokine, antibody, complement or other cell surface antigen, or cell proliferation. Can be mentioned.
• the screening method of the active ingredient of the immunostimulatory or cancer therapeutic composition of the present invention specifically measures the cytotoxic activity of cytotoxic T lymphocytes against target cells.
• This method is based on cytotoxic T lymphocyte cells (CTLs) or cell lines (eg 2C cells) that naturally express PD-1 and syngeneic mice, and naturally produce PD-L1 or PD-L2. It is used to quantify the recovery or enhancement of cytotoxic activity by adding a test substance to a mixed culture of expressed or forcibly expressed cells.
• CTLs cytotoxic T lymphocyte cells
• cell lines eg 2C cells
• This method is characterized by low cytotoxic activity against cells expressing PD-L1 or PD-L2 compared to cytotoxicity against cells that do not express PD-L1 or PD-L2.
• the recovery (increase) of the cytotoxic activity by the test substance can be measured more clearly.
• Recovery of cytotoxicity by the test substance can be evaluated as corresponding to inhibition of suppression of cytotoxicity by PD-L 1 or PD-L 2 which is a feature of the present invention.
• the cells used for this purpose include tumor cell lines or cancer cell lines that naturally express PD-L1 or PD-L2 (Nature Immunology, 2001, No. 2, No. 3, p.261-267).
• P 38 D 1 cell, P 815 cell, NB 41 83 cell, MDA-231 cell, SKBR-3 cell, MCF-7 cell, BT474 cell, J 558 L cell, P 3 U 1 cell, PAI cells, X63 cells, or SP 2 cells can be used, but tumor cell lines transformed to stably or transiently express PD-L1 or PD-L2 Alternatively, cancer cell lines can be used.
• the cytotoxic lymphocytes that can be used are those that express PD-1 and are preferably cells derived from animals that are syngeneic with the target cells.
• the screening of the active ingredient of the composition for treating infectious diseases of the present invention comprises cytotoxic T lymphocyte cells (CTL) or cell lines (for example, 2C cells), pathogens or viruses that naturally express PD-1.
• CTL cytotoxic T lymphocyte cells
• 2C cells for example, 2C cells
• Infected PD-L 1 from the same strain Shows the effect of enhancing the immune response of lymphocyte cells to infected cells by adding a test substance to a mixed culture of cells that naturally express or forcibly express PD-L2, or pathogens or viruses This is a method for quantifying the inhibitory effect on the growth activity of sucrose.
• a screening cancer cell line transformed to express PD-L1 or PD-1L2 or naturally PD-L1 or PD-L2 It is possible to use a mammal produced by transplanting a cell expressing the gene into a syngeneic mammal.
• a process of transplanting cells and a process of breeding the mammal until the evaluation target is appropriate are indispensable.
• This evaluation method is used for the growth of transplanted cells, the production of various site force-in or cell surface antigens, especially if the cells are cancer cells, the histological analysis of the proliferation, invasion or metastasis It is characterized by evaluating the survival rate of transplanted mammals.
• Cell proliferation can be evaluated by the number of cancer cells per unit volume in the case of ascites cancer or blood cancer, and by the size or weight after removal in the case of solid cancer.
• the cancer therapeutic effect of the test substance in this method can be evaluated as equivalent to the effect derived from inhibition of cytotoxicity suppression by PD-L1 or PD-L2.
• the cancer cell line to be transplanted can be a cancer cell line that can be used in an in vitro screening method, but is preferably derived from the same strain as the transplanted mammal and has good growth ability. Mammals include primates excluding humans, mice, rats, hamsters, guinea pigs, innu, pigs, and sal. Industrial applicability
• the present inventors inhibit each of the functions of PD-1 and PD-L1.
• Specific antibody anti-PD-1 antibody, anti-PD-L1 antibody
• These antibodies showed the effect of restoring or enhancing the cytotoxic activity that is relatively decreased by the presentation of PD-L1 ligand to CTL (cytotoxic T lymphocyte cells) expressing PD-1. (See Example 1, Figure 1). This suggests that the cytotoxic activity of CTLs against cancer cells can be enhanced by administration of these antibodies.
• anti-PD-L1 antibody was achieved by using a cancer cell transplanted animal model (protein / nucleic acid / enzyme, 1981) using syngeneic mice transfected with a mastocytoma-derived cell line that artificially expressed PD-L1 Year 26, No. 3, ⁇ ⁇ 208-224), the growth of cancer cells and invasion were suppressed, and the survival effect of the individual was shown (see Fig. 2 and Fig. 3). Furthermore, it was suggested that the same effect as that obtained by the inhibition of PD-L1 function by this antibody can be obtained by inhibiting the function or production of PD-1. This is because in the cancer transfer model using PD-1-deficient mice, the growth of the transferred cancer cells was not observed at all, and PD-1 function inhibition or production inhibition would be an effective cancer treatment method. (See Example 5, FIG. 5).
• any substance capable of inhibiting the immunosuppressive signal from L2 has almost the same effect.
• substances having such effects include anti-PD-L 2 antibody, solubilized PD_1, solubilized PD-L l, solubilized PD-L 2, PD-1 antagonist, PD-L 1 antagonist, P DL 2 antagonist, PD-1 and PD-LI or PD-1 and PD-L2 interaction inhibitor, PD-1 production inhibitor, 0-1 production inhibitor, PD-L2 production inhibitor Or intracellular inhibitory signal inhibitors by PD-1.
• carcinomas eg, cervix, sputum, conjunctiva, vaginal lung, oral cavity, skin, bladder, Tongue, larynx, esophagus
• adenocarcinoma e
• the expression of PD—L 1 or PD—L 2 can be identified by examination using a surgically excised cancer or tumor mass or a lesion taken outside the body as a sample. It is an efficient and effective method for post-surgical treatment of patients with tumors or cancers that significantly express 1 or PD-L2. Identification of PD—L 1 or PD—L 2 expression can be achieved by, for example, immunochemical methods using PD—L 1 antibody or PD—L 2 antibody, RT—PCR method or D NAT ray method. Can be examined.
• Chemotherapy and radiation therapy for cancer inevitably have the side effect of dramatically reducing lymphocyte proliferation.
• the administration of the composition of the present invention exhibits the effect of stimulating and proliferating the reduced lymphocyte cells, and can suppress the severe side effects associated with usual chemotherapy to a minimum. The same applies to radiation therapy.
• the combined use with the composition of the present invention can greatly reduce the dose of chemotherapeutic agent or radiation dose from the dose or radiation dose normally used.
• the cancer therapeutic composition of the present invention can be used in combination with or combined with an existing chemotherapeutic agent.
• chemotherapeutic agents include alkylating agents, nitrosourea agents, antimetabolites, anticancer antibiotics, plant-derived alkaloids, toposomerase inhibitors, hormone therapy agents, hormone antagonists, aromatase inhibitors, P Examples include glycoprotein inhibitors, platinum complex derivatives, other immunotherapeutic agents, and other anticancer agents.
• it can be used in combination with or combined with white blood cell (neutrophil) depletion treatment, thrombocytopenia treatment, antiemetics, and cancer pain treatment, which are cancer treatment aids for patients' QOL recovery. .
• the cancer therapeutic composition of the present invention can be used in combination with or combined with other immunostimulatory substances.
• immunostimulatory substance include various site forces and tumor antigens.
• cytokines that stimulate the immune response include GM-CSF, M-CSF, G-CSF, interferon- ⁇ , ⁇ , yIL-1, IL-2, IL-3, IL-12, and the like.
• B7 ligand derivatives, anti-CD3 antibodies, anti-CD28 antibodies, and anti-CTL A-4 antibodies can also enhance the immune response.
• Cancer antigens can be prepared as purified proteins for those whose genes are known, and as keratosomes for cancer cells themselves if they are unknown.
• cancer antigens examples include HLA-A1 and HLA-A2-restricted peptide MART-1, gp100 derived from MAGE-1, MAGE-3 of malignant melanoma.
• HER2 / neu peptide for breast cancer and ovarian cancer MUC-1 peptide for adenocarcinoma
• NY-ESO-1 for metastatic cancer can also be mentioned.
• Viruses are one way to escape the immune defenses of infected hosts. It is thought to use a lymphocyte-coupled inhibitor (Journal Experimental Medicine, 2000, 191st, 11th, ⁇ .1987-1997). Virus infection is partly due to such an escape function of the virus, and the administration of the composition of the present invention is thought to enhance the immune response of T lymphocyte cells to the virus.
• human hepatitis virus hepatitis B, hepatitis C, hepatitis A or hepatitis E
• human retrovirus human immunodeficiency virus (HI VI, HI) V2)
• HI VI, HI human immunodeficiency virus
• HTLV1, HTLV2 human T lymphotropic virus
• herpes simplex virus types 1 and 2 Epstein-Barr virus, cytomegalovirus, chickenpox-herpes zoster winores, human herpes peinovirus 6 and poliovirus, measles virus, Rubella virus, Japanese encephalitis virus, mumps virus, influenza virus, adenoviruses listed as cold viruses, enteroviruses or rhinoviruses, viruses that develop severe acute respiratory syndrome (SARS), Ebola virus, or West It is thought to be effective in treating Nile virus infection.
• herpes simplex virus types 1 and 2 Epstein-Barr virus, cytomegalovirus, chickenpox-herpes zoster winores, human herpes peinovirus 6 and poliovirus, measles virus, Rubella virus, Japanese encephalitis virus, mumps virus, influenza virus, adenoviruses listed as cold viruses, enteroviruses or rhinoviruses, viruses that develop severe acute respiratory syndrome (SARS), Ebola virus,
• pathogens are also effective against infection by eg pathogenic protozoa (eg trypanosomes, malaria and toxoplasma), bacteria (eg mycobacteria, salmonella and listeria) or fungi (eg candida) It is thought that.
• pathogenic protozoa eg trypanosomes, malaria and toxoplasma
• bacteria eg mycobacteria, salmonella and listeria
• fungi eg candida
• the infectious disease treatment composition of the present invention can be used in combination with or combined with existing anti-HIV agents, antiviral agents, antibiotic preparations, antibacterial agents, and visceral mycosis treatment agents.
• anti-HIV agents include reverse transcriptase inhibitors (eg, AZT, dd I, 3 TC, d4T), protease inhibitors (eg, saquinavir mesilate, ritonavir, nelfinavir mesylate, amprenavir, delavirdi mesylate) , Saquinavir, oral pinavir ritonavir), or a CCR 5 receptor antagonist.
• antiviral agents include antiherpes virus agents, anti-influenza virus agents, interferon a; and j3, or various immune globulins.
• composition for treating infectious diseases of the present invention can be used together with a virus or pathogen vaccine or can be formulated together.
• vaccines include polio vaccine, measles vaccine, Japanese encephalitis vaccine, BCG vaccine, triple vaccine, nerf vaccine, varicella vaccine, influenza vaccine, hepatitis A vaccine, hepatitis B vaccine, or cholera Vaccines.
• composition of the present invention is usually administered systemically or locally in an oral or parenteral form.
• the dose varies depending on the drug used in the present invention, and also varies depending on the age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc.
• one dose per adult is usually 1 In the range of ⁇ g to 10 O mg, 1 ⁇ is administered several times from one force.
• parenteral administration it is administered once or several times a day in the range of 0.1 ng to 1 O mg per adult per person, and preferably the parenteral dosage form is intravenous administration Yes, administered continuously in the vein for 1 hour to 24 hours per day.
• the dose varies depending on various conditions, and therefore, a dose smaller than the above dose may be sufficient, or administration beyond the range may be necessary.
• a solid preparation for internal use for oral administration a liquid preparation for internal use, an injection for parenteral administration, an external preparation, a suppository, Used as inhaled IJ, nasal agent, etc.
• Oral solid preparations for oral administration include tablets, pills, capsules, powders, condyles Granules etc. are included.
• Capsules include hard capsules and soft capsules.
• Tablets include sublingual tablets, intraoral adhesive tablets, and intraoral quick disintegrating tablets.
• one or more active substances are left as they are or excipients (latatose, mannitol, glucose, microcrystalline cellulose, starch, etc.), binders (hydroxypropylcellulose, polypyrrole pyrrole). Dong, magnesium metasilicate aluminate, etc.), disintegrating agents (fibrous calcium glycolate, etc.), lubricants (magnesium stearate, etc.), stabilizers, dissolution aids (glutamic acid, aspartic acid, etc.), etc. It is formulated and used according to a conventional method.
• a coating agent sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylenosulose phthalate, etc.
• a coating agent such as gelatin.
• the sublingual tablet is produced according to a known method.
• one or more active substances with excipients (latatose, mannitol, glucose, microcrystalline cellulose, colloidal silica, starch, etc.), binders (hydroxypropylcellulose, polyvinylpyrrolidone, aluminate metasilicate Magnesium), disintegrating agents (starch, L-hydroxypropylcellulose, carboxymethyl cenololose, croscanolemellose sodium, fibrinoglycolate canoleum, etc.), lubricants (magnesium stearate, etc.), swelling agents (hydroxy) Propinoresenorelose, Hydroxypropinoremethinoresenorelose, Carboponole, Strength Rubixymethylcellulose, Polybulal alcohol, Xanthan gum, Gua gum etc.), Swelling aid (glucose, fructose) Scan, mannitol, carboxymethyl Lithol, erythritol Tor
• Oral shell-holding tablets are produced according to known methods.
• one or more active substances with excipients (latatose, mannitol, glucose, microcrystalline cellulose, colloidal silica, starch, etc.), binders (hydroxypropyl cellulose, polyvinylpyrrolidone, aluminate metasilicate) Magnesium oxide, etc.), disintegrating agents (starch, L-hydroxypropylcellulose, carboxymethylcellulose, croscarmellose sodium, calcium fibrin glycolate, etc.), lubricants (magnesium stearate, etc.), adhesives (hydroxypropylene) Norecenorelose, Hydroxypropinoremethinolecellulose, Carbopol, Force Noboxymethylcellulose, Polybulal alcohol, Xanthan gum, Guar gum, etc.) Adhesion scavenger (glucose, fructose, ma Nititol, Xylitol, Erythritol, Maltose, Trehalose, Phosphate, Cen
• Stabilizer Polyethylene glycol, Propylene glycol, Glutamic acid, Asparagine) Acid, etc.
• flavoring agents range, strawberry, mint, lemon, vanilla, etc.
• a coating agent sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc.
• additives such as preservatives, antioxidants, colorants, sweeteners and the like that are commonly used can be added as necessary.
• the intraoral quick disintegrating tablet is produced according to a known method.
• one or more active substances as they are, or coating agents suitable for raw powder or granulated powder (eg ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, acrylic acid methacrylic acid copolymer), plastic Active substances coated with agents (polyethylene glycol, triethyl citrate, etc.), excipients (lactose, mannitol, glucose, microcrystalline cellulose, colloidal silica, starch, etc.), binders (hydroxypropylcellulose, poly Bullpyrrolidone, magnesium aluminate metasilicate, etc.), disintegrating agent (starch, L-hydroxypropyl cellulose, carboxymethylenosenolellose, croscanolemellose sodium, silk silicon glycolate Lucium, etc.), Lubricants (magnesium stearate, etc.), Dispersion aids (Glucose, Funolectose, Mannito Nore, Xyli Tonole, Erythoxyl
• a coating agent sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc.
• additives such as preservatives, antioxidants, colorants, sweeteners and the like that are commonly used can be added as necessary.
• Liquid preparations for internal use for oral administration include pharmaceutically acceptable solutions, suspensions, emulsions, syrups, elixirs and the like.
• a solution one or more active substances are dissolved, suspended or emulsified in a commonly used diluent (purified water, ethanol or a mixture thereof).
• this solution is a wetting agent, suspending agent, emulsifier, sweetener, flavoring agent, fragrance, preservative, buffering agent. Etc. may be contained.
• Examples of external dosage forms for parenteral administration include ointments, gels, creams, poultices, patches, liniments, sprays, inhalants, sprays, aerosols, eye drops , And nasal drops. These contain one or more active substances and are produced by known methods or commonly used formulations.
• the ointment is produced by a known or commonly used formulation. For example, it is prepared by grinding or melting one or more active substances into a base.
• the ointment base is selected from known or commonly used ones.
• higher fatty acids or higher fatty acid esters such as adipic acid, myristic acid, palmitic acid, stearic acid, oleic acid, adipic acid ester, myristic acid ester, haremitic acid ester, stearic acid ester, oleic acid ester
• wax Beeswax, whale wax, ceresin, etc.
• surfactant polyoxyethylene alkyl ether phosphate, etc.
• higher alcohols cetanol, stearyl alcohol, cetostearyl alcohol, etc.
• silicone oil dimethylpolysiloxane, etc.
• Hydrocarbons hydrophilic petrolatum, white petrolatum, purified lanolin, liquid paraffin, etc.
• glycols ethylene
• the gel is produced by a known or commonly used formulation. For example, it is prepared by melting one or more active substances in a base.
• the gel base is selected from known or commonly used ones.
• Lower Arco E.g., ethanol, isopropyl alcohol
• gelling agents carboxymethylenosylellose, hydroxyxetyl cellulose, hydroxypropenosenoleose, ethyl cellulose, etc.
• neutralizing agents triethanola
• mines diisopropanolamines, etc.
• surfactants polyethylene glycol monostearate, etc.
• gums water, absorption promoters, anti-rash agents, or a mixture of two or more.
• preservatives, antioxidants, flavoring agents, and the like may be included.
• the cream is produced by a known or commonly used formulation. For example, it is prepared by melting or emulsifying one or more active substances in a base.
• the cream base is selected from known or commonly used ones. For example, higher fatty acid esters, lower alcohols, hydrocarbons, polyhydric alcohols (propylene dallicol, 1,3-butylene glycol, etc.), higher alcohols (2-hexyldecanol, cetanol, etc.), emulsifiers (polyoxyethylene) Lenalkyl ethers, fatty acid esters, etc.), water, absorption accelerators, anti-rash agents, or a mixture of two or more. Further, it may contain a preservative, an antioxidant, a flavoring agent and the like.
• the poultice is produced by a known or commonly used formulation. For example, it is produced by melting one or more active substances in a base material, and spreading and applying the mixture on a support.
• the compress base is selected from known or commonly used ones. For example, thickeners (polyacrylic acid, polybulurpyrrolidone, gum arabic, starch, gelatin, methylcellulose, etc.), wetting agents (urea, glycerin, propylene glycol, etc.), fillers (kaolin, zinc oxide, tark, etc.) Calcium, magnesium, etc.), water, solubilizers, tackifiers, anti-rash agents, or a mixture of two or more. In addition, it may contain preservatives, antioxidants, flavoring agents and the like.
• the patch is produced by a known or commonly used formulation. For example, Manufactured by melting one or more active substances in a base and spreading them on a support.
• the patch base is selected from known or commonly used ones. For example, one selected from polymer bases, fats and oils, higher fatty acids, tackifiers and anti-rash agents may be used alone or in admixture of two or more. Further, it may contain a preservative, an antioxidant, a flavoring agent and the like.
• the liniment is produced by a known or commonly used formulation.
• one or more active substances are dissolved in water, alcohol (ethanol, polyethylene glycol, etc.), higher fatty acid, glycerin, soap, emulsifier, suspending agent, etc. Prepared by turbidity or emulsification. Further, it may contain a preservative, an antioxidant, a flavoring agent and the like.
• Sprays, inhalants, and sprays are commonly used diluents as well as buffers that provide isotonicity with stabilizers such as sodium bisulfite, such as sodium chloride, sodium citrate, or citrate.
• An isotonic agent such as
• injections for parenteral administration include solid injections used by dissolving or suspending in solutions, suspensions, emulsions, and solutions for use.
• injectables are used by dissolving, suspending or emulsifying one or more active substances in a solvent.
• the solvent include distilled water for injection, physiological saline, vegetable oil, propylene glycol, polyethylene glycol, alcohols such as ethanol, and combinations thereof.
• this injection contains a stabilizer, a dissolution aid (glutamic acid, aspartic acid, polysorbate 80 (registered trademark), etc.), a suspending agent, an emulsifier, a soothing agent, a buffering agent, a preservative, and the like. Also good. They are sterilized in the final process or manufactured by aseptic manipulation.
• aseptic solid preparations such as lyophilized products can be produced and used by dissolving them in sterilized or sterile distilled water for injection or other solvents before use.
• Inhalants for parenteral administration include aerosols, inhalable powders or inhalers.
• An infusion solution may be included, and the inhalation solution may be used in the form of being dissolved or suspended in water or other suitable medium at the time of use.
• preservatives salts, benzalkonium, parabens, etc.
• coloring agents for example, in the case of inhalation solutions, preservatives (salts, benzalkonium, parabens, etc.), coloring agents, buffering agents (sodium phosphate, sodium acetate, etc.), isotonic agents (sodium chloride) , Concentrated glycerin, etc.), thickeners (calipoxybule polymer, etc.), absorption promoters, etc., as necessary.
• lubricants stearic acid and its salts, etc.
• binders starch, dextrin, etc.
• excipients lactose, cellulose, etc.
• coloring agents preservatives (benzalkcoyl chloride, parabens) Etc.) It is prepared by appropriately selecting an absorption accelerator or the like as necessary.
• a nebulizer (atomizer or nebulizer) is usually used to administer an inhalation solution, and an inhaler for powder medicine is usually used to administer a powder for inhalation.
• compositions for parenteral administration include suppositories for rectal administration and pessaries for intravaginal administration, which contain one or more active substances and are prescribed by conventional methods.
• sprays contain a buffer that provides isotonicity with stabilizers such as sodium bisulfite, for example, isotonic agents such as sodium chloride, sodium citrate, or citrate. You may contain.
• stabilizers such as sodium bisulfite
• isotonic agents such as sodium chloride, sodium citrate, or citrate. You may contain.
• the method for producing the spray agent is described in detail, for example, in US Pat. No. 2,868,691 and US Pat. No. 3,095,355. Brief Description of Drawings
• Figure 1 is, H- 2 L d-specific 2C CTL and PD- 1 expression clone, P 8 1 5 PD in PD- L 1 expression stable transformants in (mastocytoma-derived cell line) — Flow cytometry showing L 1 expression, (B) shows cytotoxicity of 2C CTL cell line against PD—L 1 expressing P81 5 cell line and anti-PD—L 1 antibody (anti—PD-L 1 F (a b ') shows the effect of 2 I gG) on the cytotoxic activity.
• FIG. 2 shows tumor growth and invasiveness of transplanted PD-L1-expressing P815 cell line in syngeneic mice.
• A Tumor volume of transplanted PD—L 1 expression P 815 tumor (top) and survival after transplantation (bottom),
• B Transplanted PD—L 1 expression P in syngeneic DBA / 2 mice 815 Tumor staining image of a tumor mass (a is a 40-fold image showing infiltration of tumor cells into the abdominal wall and peritoneum, b is a 400-fold image, c is metastasis to the spleen, d is metastasis to the liver )
• FIG. 3 shows the in vivo effect of anti-PD-L1 antibody on tumor growth of transplanted PD-L1-expressing P815 cell line in syngeneic mice.
• A In vivo effect of anti-PD-L1 antibody on IFN- ⁇ production from tumor-specific CD 8 + T cells in the mouse
• B Transplanted PD-L1 expressing P815 tumor in the mouse Shows the in vivo effect of anti-PD-L1 antibody on tumor volume (top) and survival rate (bottom) of the mouse (in the figure, the mouth is the control group (rat I gG administration group), and ⁇ is the anti-PD-L1 antibody ( anti—PD—L 1 F (a b ′) 2 I g G) administration group).
• FIG. 4 shows inhibition of proliferation of PD-L1-expressing B16 melanoma in PD-1 gene homo-deficient syngeneic mice (PD-1).
• FIG. 5 shows the in vivo effect of anti-PD-L1 antibody and the involvement of PD-1 in the tumor growth of transplanted myeloma cell lines in syngeneic mice (BALBZc).
• FIG. 6 shows PD-L1 expression in the vascular endothelium.
• A is mouse heart blood The expression of PD-L1 and PD-L2 in vascular endothelial cells is shown.
• B shows tissue staining to confirm the expression of PD-L1 in each mouse tissue.
• A Eyeball
• Ch is the choroid
• CV is the central vein
• G 1 is the glomerulus
• Re is the retina.
• Each arrow indicates a vascular endothelial cell.
• Each stained image is a 40x magnified image.
• FIG. 7 shows PD-L 1 expression in liver non-parenchymal cells.
• A shows ICAM-1 in mouse liver
• B shows PD-L1 expression (CV: central vein).
• FIG. 8 shows the cell surface molecule funotypes in Tapfah cells and peritoneal sinusoidal endothelial cells (LSEC).
• Fig. 9 shows the phenotype of cell surface molecules in PD-1 gene homo-deficient mice (PD-1) or wild-type mice (wt) in CD4 positive I "live T cells.
• PD— 1— Phenotypes of cell surface molecules in CD8 positive T cells from 1-gene homozygous mice (PD— 1—) or wild-type mice (wt)
• Figure 1 shows LNPC PD- L for T cell proliferation
• A shows cell proliferation upon stimulation of naive T cells in PD-1— and wt mice, and
• B is already activated in PD-1— ⁇ and wt mice. Shows the effect of anti-PD — L 1 antibody on cell proliferation in coculture of cultured T cells and LNPC.
• Figure 12 shows the effect of LNPC PD-L 1 on site force-in production.
• A shows cytokine production upon stimulation of na ⁇ ve T cells in PD-l-mouse oppy wt mice.
• B shows the effect of anti-PD-L1 antibody on the production of cytokines in the coculture of T-1 cells already derived from PD-1-tomouse mouse wt mouse and LNPC.
• C shows cell division of activated T cells from PD-1 and wt mice in co-culture with LNPC.
• FIG. 13 shows the involvement of PD-1 in the proliferation of T lymphocyte cells in virus-infected mouse liver.
• A Day 0 after infection with adenovirus
• B Day 7 of PD-1 mouse growth of CD 19 positive and CD 3 positive lymphocyte cells in liver and spleen of wt mouse. Show.
• FIG. 14 shows the involvement of PD-1 in the proliferation of T lymphocyte cells in virus-infected mouse liver.
• A shows the proliferation of CD4-positive and CD8-positive lymphocyte cells in the liver of PD-1-mouse mice after 7 days after adenovirus infection.
• B shows the proportion of various proliferating lymphocytes 7 days after infection.
• Figure 15 shows the involvement of PD-1 in viral infection.
• (a) to (d) are tissue staining images showing cell proliferation in the livers of PD-I mice and WT mice on day 0 and day 7 after adenovirus infection.
• (E) is tissue staining images showing cell proliferation in the livers of PD-I mice and WT mice on day 0 and day 7 after adenovirus infection.
• Figure 16 shows the involvement of PD-1 in viral infection.
• (g) to (n) are hematoxylin and eosin tissue stained images of the livers of PD-1- ⁇ mice and wt mice on days 0 and 7 after adenovirus infection.
• (o) to (r) are X-Ga1-stained images of the livers of PD-1 and wt mice on day 0 and day 7 after adenovirus infection.
• FIG. 17 shows the enhancing effect of each substance on the cytotoxic activity.
• A Enhancement of anti-mouse PD-1 antibody
• B Enhancement of anti-mouse PD-L1 antibody
• c Enhancement of mouse PD_1Fc
• d Human PD-lFc This shows the enhanced action.
• the mouse PD-L 1 expression vector was prepared by digesting mouse PD-LlcDNA (Journal of Experimental Medicine, 2000, Vol. 19, No. 7, ⁇ .1027-1034) with the restriction enzyme EcoRI. This was done by joining and connecting pApuroXS (The EMBO Journal, 1994, Vol. 13, No. 6, ⁇ .1341-1349).
• the prepared expression vector pApuroXS-PD-Ll was introduced into P815 cells by the electoporation method (360 V, 500 F).
• ⁇ 81 5 cells can be cultured in FCS (10%), 2-mercaptoethanol (10 _5 ⁇ ), RPMI-1640 medium containing various antibiotics, and antibiotics such as Puromycin;
• a transformed P815 cell line stably expressing mouse PD-L1 was obtained by subculturing the same cell line resistant to culture of medium containing 3 ⁇ g / m1) .
• the expression of PD-L1 was confirmed by flow cytometry analysis.
• Fig. 1 (A) shows the PD-1 expression of H-2L d- specific 2CCTL clone (i) and PD-L1 stable transformant of P815 (mast cell type-derived cell line). 1 shows flow cytometry showing expression (ii).
• a transformed B 16 cell line (B 16 / PD-L 1) that stably expresses PD-L 1 was obtained (see FIGS. 1 (A) (iii) to (V)).
• pEFBOSneo-PD-Ll Nucleic Acid Research, 1990, No.18, No.17, p.5322
• G 418 for selective culture of cell lines (0.5 mg / m 1) was used.
• the dialyzed PD-L1 protein was immunized with complete Freund's adjuvant to 8-week-old female Whister rats (purchased from SLC Japan), and a few days later, 2 x 10 8 cells recovered from the peripheral lymph node were treated with PEG1500 ( The cells were fused with the same number of SP 2 cells using Amersham. Furthermore, RPMI1640 medium (purchased from HA T (Sigma), Origen ( 10%, purchased from Igen), FCS (10%) , 2- mercaptoethanol (10- 5 M), various antibiotics) depending on culturing in The presence of the produced antibody was confirmed by flow cytometric analysis.
• the hybridoma established in this way (international accession number: hybridoma recognized by FERMBP-8396) was transferred to Balb / Cnu / nu mice, and the recovered solution from ascites was later purified by protein G Sepharose column chromatography. By purification, a monoclonal antibody (1-1 1 1) against PD-L1 was obtained.
• the antibody used in flow cytometry and the like used was piotinized using Sulfo-NHS-LC-biotin (trade name: purchased from Pierce).
• an anti-human PD-1 antibody (international accession number: monoclonal antibody produced from a hybridoma recognized by FERM BP-8392) was prepared.
• 2C cells ( ⁇ : effector) are labeled with 5 X Cr labeled P 815 cells (T: target) ( ⁇ ) or 3 PD-L1 expressing P 8 15 cell lines ( P 81 5 / PD— L 1) with ( ⁇ , ⁇ , ⁇ ) or even 10 mg / m 1 in the presence of rat anti-PD-LlF (ab ') 2 IgG ( ⁇ ) at various E / T ratios
• P 81 5 / PD— L 1 3 PD-L1 expressing P 8 15 cell lines ( P 81 5 / PD— L 1) with ( ⁇ , ⁇ , ⁇ ) or even 10 mg / m 1 in the presence of rat anti-PD-LlF (ab ') 2 IgG ( ⁇ ) at various E / T ratios
• the result of measuring 51 Cr released by mixing in 4 hours is shown.
• Anti-PD-L 1 antibody restored the reduced cytotoxic activity of cytotoxic T lymphocyte cells. From these results, it is considered that the inhibition of PD-1 and PD-L1 signanole by inhibiting the function of PD-L1 can enhance the cytotoxic activity against cancer cells.
• Example 2
• a is a 40-fold image showing tumor cell infiltration into the abdominal wall and peritoneum
• b is also a 400-fold image
• c is metastasis to the spleen
• d is metastasis to the liver.
• cytotoxic T cell CTL from mice immunized with P 815 cells, and prepare 2 X 10 6 CTL cells and 5 X 10 6 P 815 cells or P 815 / PD-L 1 cells only, Alternatively, P815 PD-L 1 cells are mixed and cultured in the presence of anti-PD-LlF (ab ') 2 IgG (1 Omg / m 1), and IFN-V in the culture supernatant after 24 hours is ELISA kit (Purchased from Bioscience). The results are shown in Fig. 3 (A).
• the P 815 / PD-L 1 cells 3X 10 6 number in syngeneic DBA / 2 mice subcutaneously transfected (n 10), is had in rats I gG ( ⁇ ) anti- PD-LlF (ab ') 2 IgG (0.1 mg Z—animal) ( ⁇ ) was administered intraperitoneally 1, 3, 5 and 7 days after cell transfer, respectively, and tumor growth and mouse survival rate were evaluated. Indicates.
• the anti-PD-L 1 antibody restored I FN- ⁇ production from cytotoxic T lymphocyte cells suppressed by P 815 / PD-L 1 (FIG. 3 ( ⁇ )).
• Administration of anti-D-L1 antibody suppressed cancer cell growth and showed a clear survival effect (Fig. 3 ( ⁇ )). This result shows that administration of anti-PD-L1 antibody is effective for cancer treatment.
• Example 4
• vascular endothelial cells (hereinafter abbreviated as EC s) were obtained from the mouse heart by the method of Malery-Pag (Journal Immunology Methods, 2000, No. 244, No. 1-2, p.205-215). Specifically, heart tissue was digested with collagenase, pre-cultured with mouse Ig, and FI TC-modified anti-CD31 antibody, the same modified anti-CD 105 antibody, the same modified anti-isolectinB4 antibody, and anti-FI TC bee. was added and cultured. The vascular endothelial cells were purified by positive selection using Magnetic-activated cell-sorting separation columns (trade name: purchased from Miltenyi Biotec).
• PD-L1 and PD-L2 in the obtained vascular endothelial cells were confirmed by flow cytometry.
• the cells were labeled with anti-PD-L1 antibody (antibody name: 1.-11 1), anti-PD-L2 antibody (antibody name: # 122) and a fluorescently labeled secondary antibody ( Fig. 6 (A)).
• Analysis was performed at 10,000 events using Facscalibur (device name: purchased from Becton Dickinson) using CellQuest software (purchased from Dickinson).
• Facscalibur device name: purchased from Becton Dickinson
• CellQuest software purchased from Dickinson.
• the expression of PD—L 1 or PD—L 2 is indicated by the open curve, and the control Ig is indicated by the filling curve.
• LNPCs liver non-parenchymal cells
• LNPCs were isolated from mouse liver by the pronaseE method (Experimental Cell Research, 1976, 99th, p.444-449). Specifically, LNPCs were separated by density gradient centrifugation after refluxing and culturing the liver in a pronaseE solution (Merck). The relative distribution of Kupffer Cells (CD 54 +, CD 1 1 b hish ) in the cell suspension is 20-25%, and hepatic sinusoidal endothelial cells (hereinafter abbreviated as LSEC s) (CD54 +, CD 1 1 b high ) is 75-80%.
• LSEC s hepatic sinusoidal endothelial cells
• Kupfer cells and LS EC s were isolated from FI TC-labeled anti-CD11b antibody and ICAM-1 and PD-Ll, B7-1 and B7-2, respectively. Subsequent staining was performed with PE-labeled Streptavidin. Tappha cells and LSECs are gated as CD 1 1 b high and CD 11 b li) W cells, respectively ( Figure 8).
• PD-1 gene homo-deficient mice PD-1— ⁇
• wild-type C 57 BL / 6 mice from spleen and lymphoid tissue, T-cell enrichment column (trade name: purchased from Genzyme)
• Naive T cells purified by negative selection, and the cells were activated with 10 ⁇ ⁇ / 1! 11 monoclonal antibody (2C 11) for 48 hours to activate.
• the naive T cells activated by the above method are treated with FI TC-labeled anti-CD4.
• the high-pridoma identified by the international accession number FERM BP-8118 is 1st 1st East, Tsukuba City, Ibaraki Prefecture, Japan, as of May 30, 2001, 1st Chuo 6 (postal code 305-8566), an independent administrative agency. Deposited at the National Institute of Advanced Industrial Science and Technology Patent Biodeposition Center under the deposit number FERMP-18356 and transferred to an international deposit on July 16, 2002.
• Activation of naive T cells in PD-1 gene homo-deficient mice (PD-1) or wild-type mice (w t) was performed by the method described in Example 8 above. Proliferation of the same cells after activation was measured by Br dU incorporation (Fig. 11 (A)). BrdU was added to the last 6 hours of 48 hours to label the cells, and cell proliferation was determined by measurement using Proliferation ELISA kit (trade name: purchased from Roche). In addition, the amount of IFN- ⁇ produced at this time was measured with ELISA Kit (trade name: purchased from Genzyme) (FIG. 12 (A)).
• PD-1 gene homo-deficient mice (PD-1) or wild-type mice (wt) -derived T cells were activated in advance by the method described in Example 8 above. Subsequently, in the presence or absence of mitomycin C-treated LNPCs from wild-type mice, 30 / zg / ml anti-PD-L 1 antibody (1 1 1 1) (rat I g G as control), 20 ⁇ g / m 1 CTLA4-Ig (Genzyme) (Heat IgG) was cultured in the presence or absence of each for 60 hours, and the growth of the same cells for the last 12 hours It was measured by the dU incorporation method (Fig. 11 (B)). The amount of IFN-y produced at 48 hours was also measured (Fig. 12 (B)).
• Activated sputum cells derived from wild-type mice showed no significant change in the amount of I FN— ⁇ produced by sputum cell growth when cocultured with LNPC s.
• the activated sputum cells derived from the cells showed a significant increase in their cell proliferation when co-cultured with LNPCs (Fig. 11 ( ⁇ ), Fig. 12 ( ⁇ )).
• the addition of anti-PD-L1 antibody to the coculture of activated sputum cells derived from wild-type mice and LNPCs showed an increase in T cell proliferation (Fig. 11 (B )).
• PD- 1 gene homo-deficient mice PD- 1- / _
• activated T cells in wild type mice wt
• 5 CFSE 5- (6) -carboxy-fluorescein diacetate succinimidyl diester
• co-cultured with LNPCs for 48 hours.
• Cell division at this time was determined by measuring CFSE activity using FACS (Fig. 12 (C)).
• Ad-lacZ was administered intravenously Infected mouse with adenovirus.
• Ad-lacZ used here is a type 5 adenovirus that has deletions in the E1 and E3 regions and has the 1 ac Z gene. After growth in 293 cells, the density of cesium chloride Purified according to the description of gradient centrifugation (Nucleic Acid Research, 1995, No. 234, No. 19, p.3816 to 3821).
• B r dU (trade name: purchased from Sigma) 1 hour before sacrifice, and the collected spleen cells and moon spleen Endolymphocytes were double-labeled with anti-B r dU antibody and anti-CD 19 antibody or anti-CD 3 antibody (FIG. 13).
• PD-1 gene homo-deficient mice (PD-1) or wild-type mice (wt) (3 animals / group) were administered 10 9 -10 1C) PFU Ad-lacZ intravenously to infect mice with adenovirus, and 0.5 hours before sacrifice at 0 or 7 days after infection.
• mg B r dU (trade name: purchased from Sigma) was intravenously administered, and the collected liver sections were labeled with an anti-B r dU antibody (Fig. 15 (a) to (d)), 20x magnified image) .
• PD-1 gene homo-deficient mice (PD-1—) liver slices were double-labeled with anti-Br dU antibody, anti-CD4 antibody or anti-CD8 antibody. (Fig. 15 (e), (f), 40x magnified image).
• liver of wild-type mice 30 days after infection showed moderate local cell infiltration into sinusoidal capillaries and non-parenchymal areas, but PD-1 mice showed symptoms of hepatitis. (Fig. 16 (h), (i) and (j), (n)).
• P- 81 5 / PD- L 1 cells in which mouse PD—L 1 is forcibly expressed are in a normal medium containing 5 g / mL puromycin (purchased from Sigma). The ) was used to inoculate a culture flask and cultured under conditions of 37 ° C. and 5% CO 2 Z9 5% air until 50% to 90% confluent.
• Mouse cytotoxic T lymphocyte 2C cells were subcultured in normal medium for several days together with PMC 8 M cells treated with MMC (Mitomycin C).
• BATDA Reagent of DELFIA EuTDA Cytotoxicity Reagents purchased from PerkinElmer was added to the recovered P-8 15 / PD-L 1 cells and incubated for 15 minutes. Further, it was washed with PBS. As the 2 C cells, fine J3 sachets were used on the 5th to 8th day after addition of P-8 15 cells.
• Anti-mouse PD-1 antibody in Fig. 17, anti-mPD-lAb (J 4 3)), anti-mouse PD-L1 antibody (in Fig. 17, anti-mPD-LlAb (1— 1 1 1)), mouse PD-1 F c (mPD— l F c), human PD— l F c (h PD-1 F c), mouse I g G 2 a ⁇ (Controllg in the figure) or PBS was dispensed into 96-well plates in 20 L (10 ng L) increments, and P— 8 1 5 / PD— L 1 cells or normal medium was added in 50 L portions. . Furthermore, 50 ⁇ L of 2 C cells, normal medium or normal medium containing 1% TritonX100 was added.
• the composition of the evaluation group consists of the test substance, P-8 15 ZPD—L 1 cell and 2 C cell
• the composition of the high control group consists of PBS, P-8 15 / PD-L 1 cell and 1% TritonXlOO.
• Normal medium containing, low control group composition is PBS, P—8 1 5 / PD-L 1 cell and normal medium
• 2 C cell control group composition is PBS, normal medium and 2 C cell
• background composition composition is PBS, P-815 / PD—L 1 Cells and normal media.
• CTL activity (%) was calculated by the following formula. All values were calculated by subtracting the average value obtained in the background.
• CTL activity (%) ([Evaluation group measurement value] — [2C cell control group measurement value] — [Low control group measurement value]) ⁇ ([High control group measurement value] — [Low control group measurement value) ]) X100 anti-PD-1 antibody, anti-PD-L1 antibody and PD-1Fc significantly enhanced CTL activity ( Figures 17 (a) to (d), where E: Tmtio The mixing ratio of 2C cells and PD-L1 / P815 cells is shown.) Example 13
• mice with 16 melanoma cells transferred to the spleen were administered anti-mouse PD-1 monoclonal antibody intraperitoneally every 2 days, and liver weights were measured on the 18th day after transfer. The inhibitory effect of the anti-PD-1 antibody on the anti-PD-1 was evaluated.
• liver weight / cancer cell non-transfer group 1.3 g
• control group 6.8 g
• To anti-PD-1 antibody administration group decreased to 3.5 g. This suppression of the increase in weight indicates that the metastasis of B 16 melanoma cells is suppressed.

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