JP6566464B2 - Method for producing non-human animal with acute severe hepatitis model, screening method for therapeutic drug for fulminant hepatitis, and therapeutic drug for fulminant hepatitis B - Google Patents

Description

The present invention relates to a method for manufacturing a acute severe hepatitis model non-human animal, a screening method, and B-type fulminant hepatitis therapeutic agent for fulminant hepatitis treatment.

  Among acute hepatitis, particularly severe hepatitis is called fulminant hepatitis. Fulminant hepatitis is characterized by severe liver dysfunction and impaired consciousness (hepatic encephalopathy or coma). In fulminant hepatitis, hepatocytes are destroyed rapidly and in large quantities, resulting in decreased liver function and inability to maintain. Known symptoms of fulminant hepatitis include jaundice, hepatic encephalopathy, and ascites. In the case of the acute type, the period from when the first symptom is noticed until hepatic encephalopathy appears is as short as 10 days or less, and the survival rate for fulminant hepatitis is as low as about 50%. Fulminant hepatitis is caused by hepatitis virus infection, drug allergy, autoimmune hepatitis, and the like. In particular, fulminant hepatitis is most often caused by infection with human hepatitis B virus (HBV), accounting for about 40% of the total. On the other hand, the cause of about 30% of fulminant hepatitis is unknown. Therefore, establishment of a model animal that contributes to elucidation of the pathological mechanism of fulminant hepatitis and the creation of an effective therapeutic agent are required.

  As an animal model of hepatitis B caused by HBV infection, Non-Patent Document 1 discloses an animal model established with uPA (urokinase-type plasma activator) -SCID mice transplanted with human hepatocytes and human peripheral blood mononuclear cells. It is disclosed.

Okazaki A, Hiraga N, Imamura M, Hayes CN, Tsuge M, Takahashi S, Aikata H, Abe H, Miki D, Ochi H, Tateno C, Yoshizato K, Ohdan H, Chayama K., `` Severe necroinflammatory reaction caused by natural killer cell-mediated Fas / Fas ligand interaction and dendritic cells in human hepatocyte chimeric mouse. '', Hepatology. 2012 Aug; 56 (2): 555-566. Ando K, Moriyama T, Guidotti LG, Wirth S, Schreiber RD, Schlicht HJ, Huang SN, Chisari FV., "Mechanisms of class I restricted immunopathology. A transgenic mouse model of fulminant hepatitis.", J Exp Med. 1993 Nov 1 ; 178 (5): 1541-1554 Cote PJ, Toshkov I, Bellezza C, Ascenzi M, Roneker C, Ann Graham L, Baldwin BH, Gaye K, Nakamura I, Korba BE, Tennant BC, Gerin JL., `` Temporal pathogenesis of experimental neonatal woodchuck hepatitis virus infection: increased initial viral load and decreased severity of acute hepatitis during the development of chronic viral infection. '', Hepatology. 2000 Oct; 32 (4 Pt 1): 807-817

  The rapid destruction of hepatocytes in fulminant hepatitis B is thought to be due to the activation of cytotoxic T cells against hepatocytes infected with HBV (see Non-Patent Document 2 and Non-Patent Document 3).

  On the other hand, in the animal model of hepatitis B described in Non-Patent Document 1, human peripheral blood mononuclear cells were hardly maintained in the body, and HBV-specific cytotoxic T cells were not detected. For this reason, it is difficult to say that the animal model is an animal model in which a rapid injury of human hepatocytes occurs.

The present invention has been made in view of the above circumstances, a manufacturing method of an acute severe hepatitis model non-human animal can be advanced sudden injury of human hepatocytes, fulminant hepatitis treatment screening method and B-type The purpose is to provide a drug for fulminant hepatitis.

A method for producing an acute severe hepatitis model non-human animal according to the first aspect of the present invention,
A first administration step of administering human hepatitis B virus to a hyperimmune-deficient non-human animal engrafted with human hepatocytes in the liver;
A second administration step of administering to the hyperimmune-deficient non-human animal administered with the human hepatitis B virus human peripheral blood mononuclear cells isolated from a patient after acute hepatitis cure ;
including.

In addition, before Symbol acute hepatitis,
Is acute hepatitis B,
It is good as well.

In the second administration step,
Further administering cytokines,
It is good as well.

In the second administration step,
Administering 1 × 10 ⁵ to 1 × 10 ⁷ of said human peripheral blood mononuclear cells,
It is good as well.

The hyperimmune-deficient non-human animal is
A hyperimmune deficient mouse,
It is good as well.

A screening method for a therapeutic drug for fulminant hepatitis according to the second aspect of the present invention,
A step of administering a test substance to the acute severe hepatitis model non-human animal produced by the method for producing an acute severe hepatitis model non-human animal according to the first aspect of the present invention;
Evaluating a hepatitis-related marker in the acute severe hepatitis model non-human animal;
Selecting the test substance as a therapeutic drug for fulminant hepatitis based on the result of evaluating the hepatitis-related marker;
including.

The therapeutic agent for fulminant hepatitis B according to the third aspect of the present invention is:
It contains a fusion protein of the extracellular domain of human CTLA4 and the Fc region of human immunoglobulin IgG1 .

  ADVANTAGE OF THE INVENTION According to this invention, the acute severe hepatitis model non-human animal which can advance the rapid injury of a human hepatocyte can be provided. In addition, since the test substance can be selected as a therapeutic drug for fulminant hepatitis using the non-human animal model of acute severe hepatitis, an effective therapeutic drug for fulminant hepatitis can be provided.

It is a figure which shows the time-dependent change of the human serum albumin (HSA) density | concentration in the serum of a mouse | mouth, and the titer of HBV DNA. It is a figure which shows the result of the histological examination of the liver sample extract | collected from the mouse | mouth. It is a figure which shows the result analyzed by the flow cytometry method using the antibody with respect to human CD45 and mouse CD45 about the mononuclear cell of the liver isolated from the mouse | mouth. It is a figure which shows the time-dependent change of the density | concentration of the alanine aminotransferase (ALT) in the serum of a mouse | mouth. It is a figure which shows the time-dependent change of the density | concentration of granzyme A in a mouse | mouth serum, and the density | concentration of interferon (IFN) -gamma. It is a figure which shows the result analyzed by the flow cytometry method using the antibody with respect to various human cell surface markers about the mononuclear cell of the liver isolated from the mouse | mouth. It is a figure which shows the ratio of the mononuclear cell which has various human cell surface markers about the mononuclear cell of the liver isolated from the mouse | mouth (n = 6). It is a figure which shows the density | concentration of the hepatitis B surface antigen and the hepatitis B surface antibody in a mouse | mouth serum. It is a figure which shows the time-dependent change of the density | concentration of HSA in the serum of the mouse | mouth which administered CTLA4Ig, and the titer of DNA of HBV. It is a figure which shows the result of the histological examination of the liver sample extract | collected from the mouse | mouth which administered CTLA4Ig. It is a figure which shows the time-dependent change of the density | concentration of ALT in the serum of the mouse | mouth which administered CTLA4Ig, the density | concentration of granzyme A, and the density | concentration of IFN-γ. It is a figure which shows the result analyzed by the flow cytometry method using the antibody with respect to various human cell surface markers about the mononuclear cell of the liver isolated from the mouse | mouth which administered CTLA4Ig. It is a figure which shows the time-dependent change of the density | concentration of HSA in the serum of the mouse | mouth which administered CTLA4Ig in a different dose, and the titer of HBV DNA.

Embodiments according to the present invention will be described.
(Embodiment 1)
First, the first embodiment will be described in detail. A method for producing an acute severe hepatitis model non-human animal according to the present embodiment includes a first administration step of administering an immunostimulatory substance to a hyperimmune-deficient non-human animal in which human hepatocytes are engrafted in the liver, And a second administration step of administering human peripheral blood mononuclear cells to a hyperimmune-deficient non-human animal to which a stimulating substance has been administered.

  The hyperimmune-deficient non-human animal used in the first administration step is, for example, a hyperimmune-deficient mouse. In this embodiment, a case where a mouse is used as an example of a non-human animal will be described. Here, “superimmune deficiency” means that T cells, B cells, NK cells and dendritic cells / macrophages are deficient. That is, hyperimmune-deficient mice are mice that lack T cells, B cells, NK cells, and dendritic cells / macrophages. An example of a hyperimmune deficient mouse is a NOG mouse. The NOG mouse is an IL-2 receptor γ chain (IL-2Rγ) knockout to a lean-type diabetic NOD (non-obese diabetic) mouse (NOD-scid mouse) that spontaneously develops autoimmune diabetes into which the scid gene has been introduced. It is a mouse to which an immunodeficiency trait of the mouse has been added. NOG mice have T cell and B cell deficiency due to the Prkdc gene mutation (scid), NK cells are deficient due to IL-2Rγ gene knockout, and have an IFN-γ production disorder of dendritic cells.

  Human hepatocytes can be engrafted in the liver of hyperimmune-deficient mice by known methods. In order to engraft human hepatocytes in the liver of hyperimmune-deficient mice, improved hyperimmune-deficient mice that develop liver injury may be used. By transplanting human hepatocytes into an improved hyperimmune deficient mouse, the human hepatocytes can be engrafted in the mouse liver and the liver can be reconstructed. The improved type of hyperimmune deficient mouse is, for example, a TK-NOG mouse in which a herpes simplex virus thymidine kinase (HSV-tk) is expressed. TK-NOG mice can induce liver injury by administering ganciclovir (GCV), which is an antiviral drug. In addition, as an improved hyperimmune deficient mouse, a spontaneous liver injury uPA-NOG mouse in which mouse uPA is expressed may be used. NOG mice and TK-NOG mice can be obtained, for example, from the Central Institute for Experimental Animals.

When human hepatocytes are engrafted in TK-NOG mice, 1 × 10 ⁵ to 1 × 10 ⁷ , preferably 1 × 10 ⁶ to 2 × 10 ⁶ human hepatocytes may be injected into the spleen. Human hepatocytes may be human hepatocytes isolated by a known method. For example, it is preferable to use human hepatocytes obtained by the collagenase perfusion method from a uPA-SCID chimeric mouse transplanted with human hepatocytes.

  Human hepatocytes are engrafted in the liver of hyperimmune-deficient mice by hematoxylin-eosin (HE) staining, Periodic Acid-Schiff (PAS) staining, and immunohistological staining of the liver using HSA antibody and anti-cytokeratin 8/18 antibody. It can be confirmed with.

  The immunostimulatory substance administered to the hyperimmune-deficient mouse in the first administration step is any substance that induces, induces or enhances some immune response in the body when administered to mammals including humans. Immunostimulatory substances include, for example, hepatitis viruses such as HBV, human hepatitis A virus, human hepatitis C virus, human hepatitis D virus and human hepatitis E virus, and antibiotics, antipyretics, analgesics, central nervous system drugs Drugs such as anticancer drugs, antiallergic drugs and immunosuppressive drugs. Suitably, the immunostimulant is HBV.

The case where the immunostimulatory substance administered to the hyperimmune-deficient mouse in the first administration step is HBV will be described in detail. HBV is administered by inoculating hyperimmune deficient mice. The HBV inoculated into hyperimmune-deficient mice is, for example, HBV in serum collected from hepatitis patients. In particular, HBV derived from a chronic hepatitis patient whose serum is HBeAg positive is preferable. Although the genotype of HBV is not specifically limited, For example, it is C type. The serum preferably contains 1 × 10 ⁴ to 1 × 10 ⁸ copies / mL of HBV DNA. HBV may be isolated from serum collected from hepatitis patients and used in addition to a suitable solvent. Hyperimmune deficient mice are inoculated with 1 × 10 ⁴ to 1 × 10 ⁷ copies, more preferably 1 × 10 ⁵ to 1 × 10 ⁶ copies of HBV per animal.

  HBV can be inoculated into hyperimmune-deficient mice by, for example, intraperitoneal injection of human serum containing HBV. It is preferable to inoculate HBV after a predetermined period of time has elapsed after human hepatocytes have engrafted. For example, HBV is inoculated 6 to 10 weeks after the transplantation of hepatocytes, preferably 8 weeks after the transplantation.

  The dose when the immunostimulatory substance is the above drug is appropriately set so as to induce, induce or enhance an immune response, and is, for example, 1 μg to 100 mg per 1 g body weight of the mouse.

  Next, the second administration process will be described. Human peripheral blood mononuclear cells (PBMC) can be isolated from human blood using, for example, Ficoll-Paque density gradient centrifugation. Preferably, the human PBMC is isolated from a patient after acute hepatitis cures. In this case, human PBMC isolated from a patient after healing of acute hepatitis caused by an immunostimulatory substance administered to a hyperimmune-deficient mouse in the first administration step is preferable. For example, when the immunostimulatory substance is human hepatitis B virus, human PBMC is preferably isolated from a patient after acute hepatitis B has been cured.

The dose of human PBMC is, for example, 1 × 10 ⁵ to 1 × 10 ⁷ per mouse. Preferably 5 × 10 ⁵ to 8 × 10 ⁶ , more preferably 4 × 10 ^{6 to} 6 × 10 ⁶ human PBMCs may be administered to the mice. Human PBMC may be administered to mice by injection into the peritoneal cavity of mice with a suitable solvent such as saline.

  Human PBMC is administered to the mice after 6 to 12 weeks, preferably after 8, 9 or 10 weeks from the administration of the immunostimulatory substance in the first administration step.

  In order to efficiently induce an immune response in the mouse, it is preferable that the HLA type of human hepatocytes engrafted in the mouse and the HLA type of human PBMC are the same. For example, when the HLA type of human hepatocytes engrafted in mice is HLA-A24, human PBMC whose HLA type is HLA-A24 may be used. The HLA type can be identified by a known DNA typing method.

  In the second administration step, cytokines may be further administered to enhance the immune response. As the cytokine, for example, one or a plurality of types may be selected and administered from IL-1, IL-6, IL-8, IL-12, TNF-α and the like. The dose of the cytokine is, for example, 1 to 100 mg or 1 to 100 μg per 1 g body weight of the mouse.

  In an acute severe hepatitis model mouse produced by the method according to the present embodiment using HBMC as an immunostimulant, the serum HSA concentration and HBV DNA concentration decrease after administration of human PBMC. This indicates a rapid injury of HBV infected hepatocytes. Damage to hepatocytes infected with HBV can also be confirmed by HE staining and immunohistochemical staining. In the liver of an acute severe hepatitis model mouse, human hepatocytes disappear extensively. In addition, many mononuclear cell clusters are found in the area of human hepatocytes.

  In acute severe hepatitis model mice, the concentration of ALT in serum significantly increased after administration of human PBMC. ALT is an enzyme mainly present in hepatocytes and is released into the blood when hepatocytes are destroyed. For this reason, the concentration of ALT in serum is a marker for hepatitis or liver injury. Therefore, the acute severe hepatitis model mouse exhibits liver injury.

When HBV is used as an immunostimulant, hepatitis B surface antibody (HBsAb) involved in immune response in serum of acute severe hepatitis model mice and human granzyme A mainly contained in IFN-γ and cytotoxic T cells Increases after administration of human PBMC. Further, when human cell surface markers of hepatocytes infiltrated into the liver of the acute severe hepatitis model mouse were analyzed by flow cytometry, the proportion of CD4 ⁺ CD8 ⁻ cells decreased, and regulatory T cells that are CD25 ⁺ FOXP3 ⁺ Also decreases. On the other hand, the proportion of CD4 ^- CD8 ⁺ cells increases. For this reason, in the acute severe hepatitis model mouse, the administration of human PBMC induces an immune response to HBV, and the activation of cytotoxic T cells against hepatocytes infected with HBMC causes acute severe hepatitis. .

  As described above in detail, since the acute severe hepatitis model mouse according to the present embodiment induces an immune response to an immunostimulatory substance by administering human PBMC, human hepatocytes by cytotoxic T cells are induced. A sudden injury can progress.

  In the present embodiment, the immunostimulatory substance may be HBV. By doing so, the acute severe hepatitis model mouse can more faithfully reproduce the rapid human hepatocyte injury caused by HBV infection, which accounts for about 40% of fulminant hepatitis.

  In the present embodiment, human PBMC isolated from a patient after acute hepatitis has been cured may be used. The human PBMC is considered to include T cells and memory T cells involved in acute hepatitis, and can more reliably induce an immune response of cytotoxic T cells to an immunostimulatory substance. In particular, when HBV is used as an immunostimulator, the immune response of cytotoxic T cells against HBV infection is very reliably induced by using human PBMC isolated from a patient after acute hepatitis B healing. be able to.

  In the second administration step, a cytokine may be further administered. By doing so, the immune response to the immunostimulatory substance can be enhanced, so that acute severe hepatitis can be efficiently developed.

  In this embodiment, a hyperimmune-deficient mouse is used as the hyperimmune-deficient non-human animal. Mice are easy to breed and various mice lacking immune function have been developed, so hyperimmune-deficient mice are easily available. For this reason, an acute severe hepatitis model non-human animal can be produced more easily. Non-human animals are not limited to mice. Non-human animals are preferably mammals other than humans, and may be rats, dogs, guinea pigs and the like.

  In another embodiment, an acute severe hepatitis model non-human animal is provided. The acute severe hepatitis model non-human animal is induced with an immune response of cytotoxic T cells to an immunostimulant. That is, the acute severe hepatitis model non-human animal has cytotoxic T cells specific for immunostimulants. For example, when HBV is used as an immunostimulating substance, the acute severe hepatitis model non-human animal has cytotoxic T cells specific to HBV infection, and therefore cytotoxic T cells against HBV infection. An immune response is induced. The acute severe hepatitis model non-human animal is produced, for example, by the method for producing an acute severe hepatitis model non-human animal described above.

(Embodiment 2)
Next, a second embodiment will be described. The method for screening a therapeutic drug for fulminant hepatitis according to the present embodiment uses an acute severe hepatitis model non-human animal produced by the method for producing an acute severe hepatitis model non-human animal described in the first embodiment. More specifically, the screening method for the therapeutic agent for fulminant hepatitis comprises the steps of administering a test substance to the above-mentioned acute severe hepatitis model non-human animal, and evaluating a hepatitis-related marker in the acute severe hepatitis model non-human animal And a step of selecting the test substance as a therapeutic drug for fulminant hepatitis based on the result of evaluating the hepatitis related marker.

  The test substance is a compound, antibody, fusion protein, peptide, DNA aptamer, RNA aptamer, siRNA, miRNA, antisense nucleic acid or the like. The method of administration to the non-human animal with acute severe hepatitis model is not particularly limited, and an appropriate method may be selected for the test substance. For example, when the test substance is a compound, it may be oral administration, intraperitoneal administration, or intravenous administration. When the test substance is an antibody, peptide, fusion protein, DNA aptamer, RNA aptamer, siRNA, miRNA, antisense nucleic acid or the like, intravenous injection, subcutaneous injection, or the like may be used. The test substance may be administered once or multiple times. What is necessary is just to determine the density | concentration of a test substance suitably with the kind of test substance. Moreover, you may administer the test substance of the several density | concentration which serially diluted the density | concentration of the test substance.

  The hepatitis related marker is, for example, the concentration of HSA, the concentration of ALT or aspartate aminotransferase (AST) in the serum of an acute severe hepatitis model non-human animal. In addition, as a hepatitis related marker, results such as HE staining and immunohistochemical staining using an antibody against HSA may be used. In particular, when HBV is used as an immunostimulatory substance, hepatitis-related markers include the results of immunohistochemical staining using antibodies against HBV DNA concentration and hepatitis B core antigen (HBc-Ag) in addition to the above. is there.

  In order to evaluate the hepatitis-related marker, for example, based on the result of a preliminary experiment using a non-human animal that is not infected with HBV, the value of the concentration of HSA when hepatitis is not developed is set, What is necessary is just to compare with the density | concentration of HSA in the serum of the administered acute severe hepatitis model non-human animal. You may compare the result of the HE dyeing | staining of the non-human animal at the time of hepatitis non-onset, and the acute severe hepatitis model non-human animal which administered the test substance or the said immunohistochemical dyeing | staining. Also, qualitatively or quantitatively compare hepatitis-related markers between the non-human animal with the acute severe hepatitis model that was administered the test substance and the non-human animal with the acute severe hepatitis model that was not administered the test substance. May be.

  In the step of selecting a test substance as a therapeutic drug for fulminant hepatitis, a test substance that suppresses, delays, stops, or treats the pathological condition of a non-human animal with acute severe hepatitis model based on the evaluation result of a hepatitis-related marker Is selected as a treatment for fulminant hepatitis. In addition, for example, a test substance that suppresses a decrease in the concentration of HSA in serum by administration is selected as a therapeutic drug for fulminant hepatitis. Preferably, a test substance that restores the concentration of HSA that has been reduced by human hepatocyte injury may be selected. In addition, a test substance that suppresses, lowers, or recovers the ALT or AST concentration in serum that has increased due to human hepatocyte injury may be selected. In addition, a test substance that suppresses the damage of human hepatocytes and the accumulation of mononuclear cells in human liver tissue may be selected by HE staining and immunohistochemical staining.

  The acute severe hepatitis model non-human animal according to the first embodiment advances rapid injury of human hepatocytes. For this reason, the selected test substance is suitable as a therapeutic drug for fulminant hepatitis in which rapid hepatic cell injury progresses and encephalopathy appears.

  As described above in detail, the screening method for a therapeutic drug for fulminant hepatitis according to the present embodiment causes a rapid injury of human hepatocytes by the immune response of cytotoxic T cells to immune inducers such as HBV. Since non-human animals that can be used are used, the effect of the test substance on fulminant hepatitis can be evaluated.

  In another embodiment, a therapeutic drug for fulminant hepatitis selected by the screening method for a therapeutic drug for fulminant hepatitis according to the present embodiment is provided. The therapeutic agent for fulminant hepatitis is selected using a non-human animal in which a rapid injury of human hepatocytes by cytotoxic T cells, which is the main cause of fulminant hepatitis, is selected. It is valid.

(Embodiment 3)
A third embodiment will be described. The therapeutic agent for fulminant hepatitis according to the present embodiment includes a T cell selective costimulatory regulator. The T cell selective costimulatory modulator is any as long as it targets the T cell activation pathway that directly or indirectly inhibits the costimulatory signal from CD80, CD86 on antigen presenting cells. For example, a T cell selective costimulatory regulator is a fusion protein of the extracellular domain of CTLA4, which is a molecule on the surface of T cells, and the Fc region of human immunoglobulin IgG1 (hereinafter simply referred to as “CTLA4Ig”). ). CTLA4Ig is also known as avatarcept.

  The dosage form of the therapeutic drug for fulminant hepatitis according to the present embodiment is not limited, but an injection is particularly preferable. The therapeutic agent for fulminant hepatitis may be, for example, a combination with a pharmacologically acceptable carrier. The pharmacologically acceptable carriers are various organic carrier materials or inorganic carrier materials used as pharmaceutical materials. The fulminant hepatitis therapeutic agent may contain pharmacologically acceptable additives such as sucrose and a buffer. If necessary, additives such as preservatives, antioxidants, colorants, sweeteners and the like can be used.

  Although the administration method of the therapeutic agent for fulminant hepatitis is arbitrary, it is preferably subcutaneous injection or intravenous injection. As a treatment for fulminant hepatitis, when CTLA4Ig is administered, the dosage of the therapeutic drug for fulminant hepatitis is adjusted as appropriate according to the patient's condition such as the patient's weight, pathological condition, etc. 500 mg to 1 g is preferred.

  A therapeutic agent for fulminant hepatitis including CTLA4Ig is particularly suitable for the treatment of fulminant hepatitis in which liver injury is rapidly progressing. In this case, the therapeutic agent for fulminant hepatitis is preferably administered once or several times in a relatively short period of 1, 2, 3 or 4 weeks. Of course, the administration method such as the number of administrations and the administration interval can be appropriately adjusted while observing the patient's condition.

  As shown in Example 2 below, the therapeutic agent for fulminant hepatitis according to the present embodiment suppresses rapid injury of human hepatocytes due to, for example, infection with HBV. For this reason, it is preferable that the therapeutic agent for hepatitis is used for the treatment of fulminant hepatitis such as fulminant hepatitis B.

  As described above in detail, the therapeutic agent for fulminant hepatitis according to the present embodiment can treat fulminant hepatitis because it suppresses the activation of cytotoxic T cells important for the severity of hepatitis. .

  Another embodiment is a method of treating fulminant hepatitis by administering the T cell selective costimulatory agent to a patient. Yet another embodiment is a T cell selective costimulatory modulator for use as a therapeutic for fulminant hepatitis. Another embodiment is the use of a T cell selective costimulatory modulator for the manufacture of a medicament for treating fulminant hepatitis.

  The following examples further illustrate the present invention, but the present invention is not limited to the examples.

(Example 1: Preparation of type B acute hepatitis model mouse)
Using a human hepatocyte chimeric mouse, an acute severe hepatitis B model mouse was prepared as follows. First, HLA-A24 human hepatocytes were transplanted into TK-NOG mice (obtained from the Central Laboratory for Experimental Animals). Specifically, GCV was injected twice a day at 6 mg / kg into the abdominal cavity of an 8-week-old TK-NOG mouse (hereinafter also simply referred to as “mouse”). Two days after injection, mice were again injected with the same amount of GCV. Seven days after the first GCV injection, mice were transplanted by injecting 1 × 10 ⁶ or 2 × 10 ⁶ human hepatocytes into the spleen. The transplanted human hepatocytes were obtained from a uPA-SCID chimeric mouse transplanted with human hepatocytes by the collagenase perfusion method. After 8 weeks from the transplantation of hepatocytes, 5 × 10 ⁵ copies of HBV positive human serum was injected into the vein of the mouse. In addition, mouse infection, collection of serum samples, and euthanasia were performed under ether anesthesia. The human serum is collected from a patient with chronic hepatitis who has agreed to donate, and has a high titer of HBeAg-positive and genotype C HBV DNA (10.2 × 10 ⁶ copies / mL). Human serum was stored in liquid nitrogen until use.

Next, 8-10 weeks after HBV inoculation, 5 × 10 ⁶ human PBMCs were intraperitoneally injected and transplanted into mice. Human PBMC were isolated using Ficoll-Paque density gradient centrifugation from patients with HLA-A24 who had cured acute B hepatitis B. As a control, mice not inoculated with HBMC were similarly injected with human PBMC.

  Since HSA is produced in transplanted human hepatocytes, the concentration of HSA is a marker for normal human hepatocytes. A 5 μL blood sample was taken from the tail vein at regular intervals. The concentration of HSA in the blood sample was determined with a Human Albumin ELISA Quantitation kit (Bethyl Laboratories Inc.).

  The DNA concentration of HBV in the serum was quantified using COBAS AmpliPre / COBAS TaqMan HBV TEST, v2.0 (Roche Diagnostics) according to the protocol provided by the manufacturer. The lower limit of detection of HBV DNA in real-time PCR was 4.4 log copies / mL.

For histological examination, mouse liver specimens collected 2 weeks after human PBMC injection were fixed with 10% buffered paraformaldehyde and incorporated into paraffin blocks. The activity of endogenous peroxidase was blocked with 0.3% H ₂ O ₂ and methanol, and in addition to HE staining, an antibody against HSA (Bethyl Laboratories Inc.) and an antibody against HBc-Ag (DAKO Diagnostics) were used. Immunohistochemical staining was performed. The immunoreactive substance was visualized using a streptavidin-biotin staining kit (Histofine SABPO kit, manufactured by Nichirei) and diaminobenzidine.

Human PBMCs isolated from mice 2 weeks after human PBMC injection were analyzed by flow cytometry. The antibodies used were APC-H7 anti-human CD3 (clone SK7), APC-conjugated anti-CD4 (clone SK), BD Horizon ™ V450 anti-human CD8 (clone RPA-T8), HU HRZN V500 MAB conjugated anti-human. CD45 (clone H130), Alexa Fluor 488 conjugated anti-human CD56 (clone B159), PerCP-Cy5.5 anti-human FoxP3 (clone 236a), PE conjugated anti-human CD25 (clone M-A251) and PE-Cy7 conjugated It is anti-mouse CD45 (clone 30-F11) (all manufactured by BD Bioscience). PE conjugated HBV C117-126 and P756-763-induced immunodominant CTL epitopes were obtained from the Institute of Medical Biology. Dead cells identified by light scattering and propidium iodide staining were excluded from the analysis. For intracellular staining, the cells were permeabilized and fixed after surface staining using a BD Cytofix / Cytoperm kit (BD Biosciences). Flow cytometry was performed with a FACSAria ™ II flow cytometer (BD Bioscience), and the results were analyzed with FlowJo (TreeStar). Regulatory T cells and human B cells were defined as human CD45 ⁺ CD4 ⁺ CD25 ⁺ FoxP3 ⁺ cells and CD45 ⁺ CD3 ⁻ CD19 ⁺ cells, respectively.

  Hepatitis B surface antigen (HBsAg) and HBsAb in the serum of mice before and 2 weeks after the injection of human PBMC were quantified using Abott Architect platform (Abbott). Serum ALT concentration was measured using Fuji DRI-CHEM (Fuji Film) according to the instructions provided by the manufacturer. In addition, the concentrations of human IFN-γ and granzyme A in the serum were measured using a chemokin cytometric bead array (CBA) kit (manufactured by BD Bioscience) according to the instructions provided by the manufacturer. The upper and lower detection limits were 10 and 2500 pg / mL, respectively.

  The concentration of HSA and HBMC DNA in mouse serum and the percentage of human PBMC in mouse liver were statistically compared by Mann-Whitney U test and unpaired t test. A p value of less than 0.05 was considered significant.

(result)
FIG. 1 shows changes over time in mouse HSA concentration and HBV DNA concentration. In HBV-infected mice transplanted with human PBMC, there was a short and significant decrease in the concentration of HSA and the DNA concentration of HBV. On the other hand, in HBV-infected mice not transplanted with human PBMC, no decrease in HSA concentration or HBV DNA concentration was observed. Thus, it was shown that human PBMC damages HBV-infected hepatocytes.

  FIG. 2 shows images obtained by HE staining and immunohistochemical staining. “H” indicates human hepatocytes, and “M” indicates mouse hepatocytes. In the livers of HBV-infected mice transplanted with human PBMC, human hepatocytes disappeared extensively, and many mononuclear cells were concentrated in the area of human hepatocytes.

  FIG. 3 shows the results of the flow cytometry method. In the HBV-infected mice transplanted with human PBMC, the proportion of human CD45-positive mononuclear cells was significantly increased compared to non-HBV-infected mice transplanted with human PBMC. Therefore, it was shown that the engraftment rate of human PBMC was increased in HBV-infected mice transplanted with human PBMC.

  As shown in FIG. 4, the concentration of ALT in only HBV-infected mice transplanted with human PBMC was significantly increased 2 weeks after the injection of human PBMC. For this reason, it was shown that the HBV-infected mouse transplanted with human PBMC exhibits hepatitis.

Further, as shown in FIG. 5, the concentrations of IFN-γ and human granzyme A were significantly increased only in the HBV-infected mice transplanted with human PBMC two weeks after human PBMC injection. FIG. 6 and FIG. 7 show the results of a flow cytometry method for analyzing human cell surface markers of hepatocytes infiltrating the liver in mice injected with human PBMC. Two weeks after injection of human PBMC, the percentage of CD4 ⁺ CD8 ⁻ cells was significantly reduced in HBV infected mice. In CD4 ⁺ CD8 ⁻ cells, regulatory T cells that are CD25 ⁺ FOXP3 ⁺ were significantly reduced in HBV-infected mice, consistent with the development of acute hepatitis. On the other hand, the percentage of CD4 ^- CD8 ⁺ cells was significantly increased in HBV-infected mice. CD8 positive and tetramer positive HBV specific cytotoxic T cells were detected only in HBV infected mice injected with human PBMC.

  The concentrations of HBsAg and HBsAb in the serum of HBV-infected mice injected with human PBMC are shown in FIG. The concentration of HBsAg before human PBMC injection was as high as in human HBV infected patients. On the other hand, the concentration of HBsAg decreased to an undetectable level two weeks after human PBMC injection. The concentration of HBsAb was increased 2 weeks after human PBMC injection.

  As described above, hepatic injury could be caused by inoculating HTKV into a TK-NOG mouse engrafted with human hepatocytes and injecting human PBMC derived from a patient who was cured of acute severe hepatitis B. In the mice, it was confirmed that an immune response of cytotoxic T cells to HBV infection was induced. Thus, the HBV-infected mouse transplanted with the human PBMC of this example exhibits acute liver injury due to HBV-specific cytotoxic T cells, and thus exhibits acute B hepatitis.

(Example 2: Administration of T cell selective costimulatory agent)
In this example, CTLA4Ig, a T cell-selective costimulatory regulator, was administered to the B-type acute severe hepatitis model mouse prepared in Example 1 above, and the effect on liver injury was examined. Note that the methods for measuring mouse HSA concentration, HBV DNA concentration, and ALT concentration, histological examination, and flow cytometry were the same as in Example 1.

  One day before the injection of human PBMC and seven days after the injection of human PBMC, mice were injected with 1.5 mg, 1.2 mg or 0.2 mg of CTLA4Ig (Bristol-Meyer's Squibb). Saline was used as a solvent for CTLA4Ig.

(result)
As shown in FIG. 9, CTLA4Ig (1.5 mg administered twice) markedly inhibited the decrease in HSA concentration and HBV DNA concentration as compared to CTB4Ig non-administered HBV-infected mice (control). Also in HE staining and immunohistochemical staining, in the livers of HBV-infected mice administered with CTLA4Ig, as in non-HBV-infected mice, damage to human hepatocytes and aggregation of mononuclear cells into human liver tissues were observed. (FIG. 10). That is, it was shown that hepatitis was suppressed by administration of CTLA4Ig.

  The result that the hepatitis was suppressed in the histological examination is reflected in each concentration of ALT, IFN-γ and human granzyme A in the serum as shown in FIG. By administering CTLA4Ig, the increase in the concentrations of ALT, IFN-γ and human granzyme A by injection of human PBMC was suppressed to the same extent as in HBV-uninfected mice. In addition, as shown in FIG. 12, the proportions of human CD45-positive mononuclear cells, CD4-positive, CD8-positive and regulatory T cells in the liver of HBV-infected mice are comparable to those in uninfected mice by administration of CTLA4Ig. It was.

  The results of administering different doses of CTLA4Ig are shown in FIG. In this experiment, CTLA4Ig was further administered 14 days after human PBMC injection. CTLA4Ig suppressed the decrease in the concentration of HSA in HBV-infected mice injected with human PBMC in a concentration-dependent manner. CTLA4Ig inhibited the decrease in HBV DNA concentration compared to the CTLA4Ig non-administered group (0 mg).

  From the above results, it was shown that CTLA4Ig is effective in treating fulminant hepatitis B.

  The above-described embodiments are for explaining the present invention and do not limit the scope of the present invention. In other words, the scope of the present invention is shown not by the embodiments but by the claims. Various modifications within the scope of the claims and within the scope of the equivalent invention are considered to be within the scope of the present invention.

  The present invention is suitable for producing non-human animals that develop acute severe hepatitis. The acute severe model non-human animal according to the present invention can also be used for screening for therapeutic agents for fulminant hepatitis. In addition, the therapeutic agent for fulminant hepatitis according to the present invention contributes to the treatment of fulminant hepatitis patients and thus to the improvement of quality of life.

Claims (7)

A first administration step of administering human hepatitis B virus to a hyperimmune-deficient non-human animal engrafted with human hepatocytes in the liver;
A second administration step of administering to the hyperimmune-deficient non-human animal administered with the human hepatitis B virus human peripheral blood mononuclear cells isolated from a patient after acute hepatitis cure;
A method for producing an acute severe hepatitis model non-human animal comprising: The acute hepatitis is
Is acute hepatitis B,
The method for producing an acute severe hepatitis model non-human animal according to claim 1. In the second administration step,
Further administering cytokines,
The method for producing an acute severe hepatitis model non-human animal according to claim 1 or 2. In the second administration step,
Administering 1 × 10 ⁵ to 1 × 10 ⁷ of said human peripheral blood mononuclear cells,
The method for producing an acute severe hepatitis model non-human animal according to any one of claims 1 to 3. The hyperimmune deficient non-human animal is
A hyperimmune deficient mouse,
The method for producing an acute severe hepatitis model non-human animal according to any one of claims 1 to 4. A step of administering a test substance to an acute severe hepatitis model non-human animal produced by the method for producing an acute severe hepatitis model non-human animal according to any one of claims 1 to 5;
Evaluating a hepatitis-related marker in the acute severe hepatitis model non-human animal;
Selecting the test substance as a therapeutic drug for fulminant hepatitis based on the result of evaluating the hepatitis-related marker;
Screening method for fulminant hepatitis drug. A fusion protein of the extracellular domain of human CTLA4 and the Fc region of human immunoglobulin IgG1 ;
A therapeutic agent for hepatitis B fulminant.