WO2022089576A1

WO2022089576A1 - Application of reagent for inhibiting mhc-i and/or ii signaling pathways in treatment of biliary atresia

Info

Publication number: WO2022089576A1
Application number: PCT/CN2021/127347
Authority: WO
Inventors: 张玉霞; 徐艳慧; 陈章华; 王俊; 林泽锋; 夏慧敏
Original assignee: 广州市妇女儿童医疗中心
Priority date: 2020-10-30
Filing date: 2021-10-29
Publication date: 2022-05-05
Also published as: CN114432449A

Abstract

Disclosed in the present invention is a use of a reagent for inhibiting MHC-I and/or II signal pathways in the treatment of biliary atresia. In the present invention, it is found by means of a model of BA disease induced by Rhesus monkey rotavirus (RRV) that the reagent for inhibiting MHC-I and/or II signaling pathways can significantly reduce the degree of liver injury and inhibit BA disease progression.

Description

Use of agents for inhibiting MHC-I and/or II signaling pathways in the treatment of biliary atresia

technical field

The invention belongs to the field of biomedicine, in particular to the application of an agent for inhibiting MHC-I and/or II signaling pathway in the treatment of biliary atresia.

Background technique

Biliary atresia (BA) occurs in infants and young children during the perinatal period (eg, 28 weeks of gestation to 4 weeks after birth), and the incidence varies from 1/5000 to 1/18000 in different countries and regions. Its etiology and pathogenesis are still unknown, and it is generally believed to be jointly determined by developmental and/or environmental factors (such as viral infection, etc.) , the fatality rate is high. The basic pathological changes of biliary atresia are, for example, progressive inflammation of intrahepatic and extrahepatic bile ducts, liver fibrosis and liver cirrhosis, and the development of liver fibrosis/cirrhosis is faster and more aggressive than other adult diseases. Although extrahepatic bile duct obstruction can partially relieve symptoms and delay the progression of the disease through Kasai's operation, most children still develop progressively due to postoperative intrahepatic bile duct inflammation, eventually leading to liver cirrhosis, portal hypertension, and even liver disease. failure, becoming a serious disease that threatens the life of children. At present, there is no effective method for prevention, diagnosis and/or treatment of BA. After the diagnosis of BA, Kasai surgery should be performed immediately to remove the remaining blocked bile ducts, and porto-enteric anastomosis should be performed to clear the bile flow. Without Kasai surgery, end-stage cirrhosis and liver failure will develop within a year. After Kasai surgery, about 50% of children require liver transplantation within two years of age, and the remaining patients suffer from long-term poor prognosis such as recurrent bile duct inflammation, portal hypertension, esophageal varices bleeding, and hepatic osteodystrophy. Wait. Therefore, preventive intervention for pregnant women and infants during pregnancy and perinatal period, as well as screening of infants during neonatal jaundice, early differential diagnosis of BA, and early intervention, are effective in the prevention, treatment and prognosis of BA. significant.

The study found that the autoimmune damage of bile duct caused by viral infection is an important factor in the occurrence and development of BA. Among them, the document "Hepatic overexpression of MHC Class II antigens and macrophage-associated antigens (CD68) in patients with biliary atresia of poor prognosis", Hiroyuki Kobayashi et al., Journal of Pdidatric Surgery, 1997, discloses MHC class II antigens and CD68 antigens. Liver expression strongly correlates with the severity of the clinical course of BA patients and can serve as a prognostic factor in these patients. The document "HLA and cytokine gene polymorphisms in biliary atresia", Howard EHadzic NClare M et al., Liver, 2002, discloses earlier studies showing that BA is weakly associated with HLA, but these have not been confirmed, and this study further compares BA Compared with healthy controls, biliary atresia was not an HLA-related disease. The document "Type-I but not type-II interferon receptor knockout mice are susceptible to biliary atresia", Joachim F Kubler et al., Pediatr Res., 2006, discloses that inactivation of type I IFN receptor significantly increases postpartum rotavirus Incidence of BA after infection. However, none of the above-mentioned documents have disclosed or verified: which indicators can be used to find out early and accurately whether there is BA-related immune damage and respond to it; Safe and accurate purpose.

SUMMARY OF THE INVENTION

The purpose of the first aspect of the present invention is to provide the application of reagents for MHC-I and/or MHC-II signaling pathways.

The purpose of the second aspect of the present invention is to provide the use of a reagent for detecting the ratio of IgM to IgG4 in the preparation of a product for prognostic analysis of biliary atresia.

The purpose of the third aspect of the present invention is to provide a kit for prognostic analysis of biliary atresia.

The purpose of the fourth aspect of the present invention is to provide the use of the ratio of IgM and IgG4 as a marker for prognostic analysis of biliary atresia.

The purpose of the fifth aspect of the present invention is to provide the use of a reagent for detecting IgG in the preparation of biliary atresia diagnosis or auxiliary diagnosis products.

The purpose of the sixth aspect of the present invention is to provide diagnostic or auxiliary diagnostic markers for biliary atresia.

The purpose of the seventh aspect of the present invention is to provide a diagnostic or auxiliary diagnostic kit for biliary atresia.

The purpose of the eighth aspect of the present invention is to provide the use of IgG antibody as a diagnostic or auxiliary diagnostic marker for biliary atresia.

The purpose of the ninth aspect of the present invention is to provide the use of a reagent for detecting biochemical indicators of liver function in the preparation of biliary atresia diagnosis or auxiliary diagnosis products.

The tenth aspect of the present invention aims to provide diagnostic or auxiliary diagnostic markers for biliary atresia.

The object of the eleventh aspect of the present invention is to provide the use of an IFNAR blocking agent.

The purpose of the twelfth aspect of the present invention is to provide the use of a reagent for detecting BAFF in the preparation of a disease diagnosis product.

The object of the thirteenth aspect of the present invention is to provide a diagnostic kit for the degree of biliary atresia or fibrosis of biliary atresia.

The object of the fourteenth aspect of the present invention is to provide the use of a specific blocking agent for BAFF.

The object of the fifteenth aspect of the present invention is to provide a pharmaceutical composition.

The first aspect of the present invention provides the use of an agent for inhibiting MHC-I and/or MHC-II signaling pathway in any one of (a1) to (a8);

(a1) preparing a medicine for preventing and treating biliary atresia;

(a2) preparing a product that reduces the rate of jaundice;

(a3) preparing a product that reduces the level of pro-inflammatory factors;

(a4) preparing a product that inhibits RRV virus gene expression;

(a5) preparing a product that increases the levels of Cx3cl1 and C1q;

(a6) preparing a product that inhibits the activation of autoreactive CD8 ⁺ T cells;

(a7) preparing a product that increases the proportion of Kupffer cells;

(a8) Preparation of a product that increases the phagocytic ability of Kupffer cells.

Preferably, the pro-inflammatory factor is IFN-γ.

Preferably, the RRV viral gene includes at least one of NSP3 and VP6.

Preferably, the reagent is a blocking antibody for MHC-I and/or MHC-II.

Preferably, the reagent is a bispecific blocking antibody for MHC-I and MHC-II.

Preferably, the blocking antibody is a monoclonal antibody.

Preferably, the agent is a blocking antibody for HLA-DR.

Preferably, the HLA-DR blocking antibody is selected from at least one of IMMU-114, Hu1D10 and Lym-1.

Preferably, the reagent is a chimeric antibody, modified antibody, humanized antibody or fully human antibody of MHC-I and/or MHC-II.

Preferably, the agent is an antibody fragment that blocks MHC-I and/or MHC-II.

Preferably, the antibody fragment comprises Fab, F(ab')2, Fd, Fv, scFv, scFv-Fc, diabodies and antibody minimal recognition units.

Preferably, the medicament includes at least one of pharmaceutically acceptable excipients, carriers, buffers and stabilizers.

Preferably, the drug is lyophilized powder or injection.

Preferably, the medicament is prepared in a dosage form suitable for adults or children; further preferably, the medicament is prepared in a dosage form suitable for children.

The second aspect of the present invention provides the use of a reagent for detecting the ratio of IgM to IgG4 in the preparation of a product for prognostic analysis of biliary atresia; preferably, the product is selected from detection reagents, detection test strips or strips, detection chips or a kit; preferably, the ratio is a content ratio or a concentration ratio.

Preferably, the prognostic analysis of biliary atresia is prognostic analysis after Kasai surgery for biliary atresia.

Preferably, the prognostic analysis comprises step 1) of determining the ratio of IgM to IgG4 in the sample from the subject.

Preferably, the prognostic analysis further comprises step 2) of comparing the ratio described in 1) with a reference level.

Preferably, when the prognostic analysis includes step 1), a ratio of IgM to IgG4 > 0.01, preferably > 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05 or 0.055, indicates a good prognosis for biliary atresia.

Preferably, when step 1) and step 2) are included, the ratio of IgM to IgG4 is increased relative to the reference level, indicating a good prognosis for biliary atresia.

Preferably, the reference level is the ratio of IgM to IgG4 in a sample from a patient with a poor prognosis for biliary atresia.

Preferably, the good prognosis of the biliary atresia includes one or more of weight gain, reduction or disappearance of jaundice, prolongation of survival time, and reduction in the degree of liver fibrosis.

Preferably, the sample of the subject is one or more of whole blood, serum, plasma, cerebrospinal fluid, tissue or tissue lysate, cell culture supernatant, semen, saliva sample, amniotic fluid, and villi; Preferably, one or more of whole blood, serum, plasma, tissue or tissue lysate, and cell culture supernatant; preferably, the tissue or tissue lysate is liver tissue or liver tissue lysate.

Preferably, the prognostic analysis product for biliary atresia is suitable for performing one or more of the following detection methods: indirect immunofluorescence method, particle agglutination method, antibody confirmation experimental detection method, rapid diagnostic reagent method, enzyme-linked immunosorbent assay , radioimmunoassay, immunodiffusion method, colloidal gold method, flow multi-factor detection method, biological mass spectrometry, electrophoresis, chromatography, immunofluorescence, immunochemiluminescence, immunoturbidimetry, western blotting and spotting blot.

Preferably, the product for prognostic analysis of biliary atresia includes anti-human IgM and anti-human IgG4.

Preferably, the subject of the product is a child or an adult, preferably a child.

The object of the third aspect of the present invention is to provide a kit for prognostic analysis of biliary atresia, the kit includes a reagent for detecting the ratio of IgM to IgG4, preferably, the ratio is a content ratio or a concentration ratio.

Preferably, the prognostic analysis comprises the following steps 1), or comprises the following steps 1) and 2):

1) determining the ratio of IgM to IgG4 in a sample from the subject;

2) Compare the ratio described in 1) with the reference level.

Preferably, when step 1) is included, the ratio of IgM to IgG4>0.01, preferably ≥0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05 or 0.055, it indicates a good prognosis for biliary atresia; when steps 1) and 2 are included ), the ratio of IgM to IgG4 increased relative to the reference level, indicating a good prognosis for biliary atresia.

Preferably, the kit for prognostic analysis of biliary atresia is suitable for performing one or more of the following detection methods: indirect immunofluorescence method, particle agglutination method, antibody confirmation experimental detection method, rapid diagnostic reagent method, enzyme-linked immunosorbent assay test, radioimmunoassay, immunodiffusion method, colloidal gold method, flow multi-factor detection method, biological mass spectrometry, electrophoresis, chromatography, immunofluorescence, immunochemiluminescence, immunoturbidimetry, western blotting and Dot blot.

Preferably, the kit includes anti-human IgM and anti-human IgG4.

Preferably, the kit further includes sample treatment reagents, the sample treatment reagents include at least one of sample lysis reagents, sample purification reagents and protease inhibitors.

Preferably, the subject of the kit is a child or an adult, preferably a child.

The purpose of the fourth aspect of the present invention is to provide the use of the ratio of IgM and IgG4 as a marker for prognostic analysis of biliary atresia, preferably, the ratio is a content ratio or a concentration ratio.

Preferably, when the ratio of IgM and IgG4 is increased relative to the reference level, the diagnosis or auxiliary diagnosis of biliary atresia is favorable.

Preferably, the reference level is determined from the ratio of IgM to IgG4 in a sample from a patient with a poor prognosis for biliary atresia.

Preferably, the ratio of IgM to IgG4 at the reference level is 0-0.01.

Preferably, the marker is derived from one or more of whole blood, serum, plasma, cerebrospinal fluid, tissue or tissue lysate, cell culture supernatant, semen, saliva sample, amniotic fluid, and villi; One or more of blood, serum, plasma, tissue or tissue lysate, and cell culture supernatant; preferably, the tissue or tissue lysate is liver tissue or liver tissue lysate.

Preferably, the ratio of IgM to IgG4 is obtained by one or more of the following detection methods: indirect immunofluorescence method, particle agglutination method, antibody confirmation experimental detection method, rapid diagnostic reagent method, enzyme-linked immunosorbent assay , radioimmunoassay, immunodiffusion method, colloidal gold method, flow multi-factor detection method, biological mass spectrometry, electrophoresis, chromatography, immunofluorescence, immunochemiluminescence, immunoturbidimetry, western blotting and spotting blot.

Preferably, the marker is detected from children or adults, preferably from children.

Preferably, the product includes detection reagents, detection test strips or strips, detection chips or kits.

Preferably, the IgG is a Ro/SSA specific IgG.

Preferably, when the IgG antibody increases relative to a reference level, the diagnosis or auxiliary diagnosis is biliary atresia, and the reference level represents the level of the corresponding index from subjects of the same age group without biliary atresia; preferably, the non-biliary atresia age-matched subjects were healthy, physiologic jaundice, choledochal cyst, or liver-cancer age-matched subjects.

Preferably, the samples tested for the product include at least one of whole blood, serum, plasma, cerebrospinal fluid, tissue/tissue lysate, cell culture supernatant, semen, saliva samples, amniotic fluid, and villi from the subject. kind.

Preferably, the sample detected by the product is selected from at least one of tissue or tissue lysate, whole blood, serum, and plasma. Preferably, the tissue/tissue lysate is liver tissue or liver tissue lysate.

Preferably, the product further includes a sample treatment reagent, the sample treatment reagent includes at least one of a sample lysis reagent, a sample purification reagent, and a protease inhibitor.

Preferably, the product is suitable for performing one or more of the following detection methods: indirect immunofluorescence method, particle agglutination method, antibody confirmation experimental detection method, rapid diagnostic reagent method, enzyme-linked immunosorbent assay, radioimmunoassay, Double immunodiffusion method, colloidal gold method, flow multi-factor detection method, biological mass spectrometry, electrophoresis, chromatography, immunofluorescence, immunochemiluminescence, immunoturbidimetry, western blotting and dot blotting.

Preferably, the product comprises anti-human IgG antibodies, preferably anti-human Ro/SSA-specific IgG autoantibodies.

Preferably, the product further includes a reagent for detecting IgM antibodies; preferably, the IgM antibodies are natural IgM antibodies; further preferably, the IgM antibodies are anti-double-stranded DNA IgM antibodies, anti-nucleosome IgM antibodies , one or more of the IgM antibody of anti-ribonucleoprotein and the IgM antibody of anti-Ro/SSA; preferably, when the IgM antibody is reduced relative to the reference level, then further diagnosis or auxiliary diagnosis is biliary atresia, the said The reference level represents the level of the corresponding index from non-biliary atresia subjects in the same age group; preferably, the non-biliary atresia subjects in the same age group are healthy, physiological jaundice, choledochal cyst or liver cancer in the same age group subject.

Preferably, the product further includes a reagent for detecting BAFF; preferably, when BAFF is elevated relative to a reference level, biliary atresia is further diagnosed or aided in diagnosis, and the reference level represents subjects of the same age from non-biliary atresia The level of the corresponding index; preferably, the non-biliary atresia subjects in the same age group are healthy, physiological jaundice, choledochal cyst or liver cancer subjects in the same age group.

The purpose of the sixth aspect of the present invention is to provide diagnostic or auxiliary diagnostic markers for biliary atresia, and the markers include:

a) IgG antibodies; and/or

b) IgM antibodies; and/or

c) BAFF.

Preferably, the IgG is a Ro/SSA specific IgG.

Preferably, the IgM antibody is a natural IgM antibody; preferably, the natural IgM antibody is an anti-double-stranded DNA IgM antibody, an anti-nucleosome IgM antibody, an anti-ribonucleoprotein IgM antibody, an anti-Ro/SSA one or more of the IgM antibodies.

Preferably, biliary atresia is diagnosed or aided in diagnosis when IgG autoantibodies are increased relative to a reference level, and/or IgM antibodies are decreased relative to a reference level, and/or BAFF is increased relative to a reference level, said reference level representing a diagnosis of biliary atresia. The level of the corresponding index of the subjects in the same age group without biliary atresia; preferably, the subjects in the same age group without biliary atresia are healthy, physiological jaundice, choledochal cyst or liver cancer subjects in the same age group.

The purpose of the seventh aspect of the present invention is to provide a diagnostic or auxiliary diagnostic kit for biliary atresia, the kit comprising:

a) Reagents for the detection of IgG antibodies; and/or

b) Reagents for the detection of IgM antibodies; and/or

c) Reagents for detecting BAFF.

Preferably, the IgG is a Ro/SSA specific IgG; preferably, when an increase in IgG antibody relative to a reference level is detected, and/or an increase in BAFF relative to a reference level, and/or a decrease in IgM antibody relative to the reference level, Then the diagnosis or auxiliary diagnosis is biliary atresia, and the reference level represents the level of the corresponding index from the subjects of the same age group without biliary atresia; preferably, the subjects of the same age group without biliary atresia are healthy, physiological Age-matched subjects with jaundice, choledochal cyst, or liver cancer.

Preferably, the IgM antibody is a natural IgM antibody; preferably, the IgM antibody is an anti-double-stranded DNA IgM antibody, an anti-nucleosome IgM antibody, an anti-ribonucleoprotein IgM antibody, and an anti-Ro/SSA IgM antibody one or more of the antibodies.

Preferably, the kit is suitable for performing one or more of the following detection methods: indirect immunofluorescence method, particle agglutination method, antibody confirmation experimental detection method, rapid diagnostic reagent method, enzyme-linked immunosorbent assay, radioimmunoassay method , immunodiffusion method, colloidal gold method, flow multi-factor detection method, biological mass spectrometry, electrophoresis, chromatography, immunofluorescence, immunochemiluminescence, immunoturbidimetry, western blotting and dot blotting.

Preferably, the sample of the kit comprises at least one of whole blood, serum, plasma, cerebrospinal fluid, tissue or tissue lysate, cell culture supernatant, semen, saliva sample, amniotic fluid, villus from the subject kind.

Preferably, the kit further includes sample treatment reagents, the sample treatment reagents include at least one of sample lysis reagents, sample purification reagents, and protease inhibitors.

Preferably, the subject of the kit is a child or an adult, preferably a child.

Preferably, the IgG is a Ro/SSA specific IgG.

Preferably, the marker further comprises IgM antibody and/or BAFF.

Preferably, the marker is sampled from whole blood, serum or plasma.

The purpose of the ninth aspect of the present invention is to provide the use of a reagent for detecting biochemical indicators of liver function in the preparation of biliary atresia diagnosis or auxiliary diagnosis products, the biochemical indicators of liver function include r-GT, ALT, AST, TBA, DBIL and IBIL; preferably, the product includes detection reagents, detection test strips or strips, detection chips or kits.

Preferably, the increase of r-GT, ALT, AST, TBA, DBIL relative to the reference level combined with the decrease of IBIL relative to the reference level will diagnose or assist in the diagnosis of biliary atresia, wherein the reference level represents non-biliary atresia from The level of the corresponding index of the subjects of the same age; preferably, the subjects of the same age without biliary atresia are healthy, choledochal cyst or physiological jaundice subjects of the same age.

Preferably, the product further includes one or more of the following detection reagents: a reagent for detecting GGT, a reagent for detecting IgG4, a reagent for detecting TBIL, and a reagent for detecting IFN-β.

Preferably, an increase in one or more of GGT, IgG4, TBIL, and IFN-β relative to a reference level further diagnoses or assists in diagnosing biliary atresia, wherein the reference level represents the source from the same group representing non-biliary atresia. The level of the corresponding index of the subjects in the age group; preferably, the subjects in the same age group without biliary atresia are healthy, choledochal cyst or physiological jaundice subjects in the same age group.

Preferably, the test sample of the biliary atresia diagnosis or auxiliary diagnosis kit includes whole blood, serum, plasma, cerebrospinal fluid, tissue/tissue lysate, cell culture supernatant, semen, saliva from the subject One or more of sample, amniotic fluid, villi; preferably whole blood, serum or plasma.

Preferably, the biliary atresia diagnosis or auxiliary diagnosis product is suitable for performing one or more of the following detection methods: biological mass spectrometry, electrophoresis, chromatography, immunofluorescence, immunochemiluminescence, immunoturbidimetry, Western blot and dot blot.

The tenth aspect of the present invention aims to provide diagnostic or auxiliary diagnostic markers for biliary atresia, wherein the diagnostic or auxiliary diagnostic markers include r-GT, ALT, AST, TBA, DBIL and IBIL.

Preferably, when the increase of r-GT, ALT, AST, TBA, DBIL relative to the reference level is combined with the decrease of IBIL relative to the reference level, the diagnosis or auxiliary diagnosis is biliary atresia, wherein the reference level represents the non-biliary tract The level of the corresponding index in subjects of the same age group with atresia; preferably, the subjects of the same age group without biliary atresia are healthy, choledochal cyst or physiological jaundice subjects in the same age group.

Preferably, the diagnostic or auxiliary diagnostic markers also include one or more of GGT, IgG4, TBIL, and IFN-β; preferably, when one or more of GGT, IgG4, TBIL, and IFN-β With respect to the increase of the reference level, the further diagnosis or auxiliary diagnosis is biliary atresia, wherein the reference level represents the level of the corresponding index from the subjects of the same age group representing non-biliary atresia; preferably, the non-biliary atresia Subjects in the same age group were healthy, choledochal cyst or physiological jaundice.

The object of the eleventh aspect of the present invention is to provide the use of an IFNAR blocker in any one of (b1) to (b8);

(b1) Preparation of medicines for the treatment of diseases

(b2) preparing a product that reduces the jaundice rate;

(b3) preparing a product that reduces the level of pro-inflammatory factors;

(b4) preparing a product that increases the level of IL-17a;

(b5) preparing a product that reduces the level of inflammatory monocytes;

(b6) preparing products that inhibit the activation of autoreactive CD4 ⁺ T cells and CD8 ⁺ T cells;

(b7) preparing a product that increases the proportion of Kupffer cells;

(b8) Preparation of a product that increases the phagocytic ability of Kupffer cells.

Preferably, the IFNAR blocking agent is an IFNAR blocking antibody.

Preferably, the blocking antibody of IFNAR is a neutralizing antibody or a functional antibody.

Preferably, the blocking antibody for IFNAR is a monoclonal antibody, preferably a humanized monoclonal antibody against IFNAR.

Preferably, the antibody is a chimeric antibody, a remodeled antibody, a humanized antibody, a fully human antibody, a bispecific antibody, a multispecific antibody or an antibody fragment.

Preferably, the humanized antibody is selected from one or more of Anifrolumab, Faralimomab, MEDI-545, Rontalizumab, Sifalimumab, AGS-009, IFNαKinoid, NI-0101.

Preferably, the medicament includes a pharmaceutically acceptable carrier.

Preferably, the medicine is lyophilized powder, injection or oral liquid injection capsule.

Preferably, the medicament is prepared in a dosage form suitable for use in children or adults, preferably a dosage form for children.

Preferably, the biliary atresia is biliary atresia, liver fibrosis caused by biliary atresia or liver cirrhosis caused by biliary atresia.

Preferably, the pro-inflammatory factor is IFN-γ.

Preferably, the inflammatory monocytes are Ly6G ⁺ Ly6C ⁺ CD11b ⁺ cells.

The purpose of the twelfth aspect of the present invention is to provide the use of a reagent for detecting BAFF in the preparation of a disease diagnosis product, which is a biliary atresia disease diagnosis product or a biliary atresia disease diagnosis product for the degree of liver fibrosis.

Preferably, the reagent for detecting BAFF is a reagent for detecting the protein level or mRNA level of BAFF.

Preferably, biliary atresia is diagnosed when the BAFF increases relative to a reference level representing a level of a corresponding index from a subject representing the same age group without biliary atresia; preferably, the same age group without biliary atresia The subjects were healthy, physiological jaundice, choledochal cyst or liver cancer subjects of the same age; BAFF level was positively correlated with liver fibrosis, when the subject was diagnosed with biliary atresia, the higher the BAFF level, The more severe the degree of hepatic fibrosis was diagnosed as biliary atresia disease.

Preferably, the product also includes one or more combinations of the following detection reagents: a reagent for detecting GGT, a reagent for detecting IgG4, a reagent for detecting ALT, a reagent for detecting AST, a reagent for detecting TBIL, a reagent for detecting DBIL Reagents, reagents for detecting IBIL, reagents for detecting TBA, reagents for detecting IFN-β.

Preferably, the test sample of the product includes at least one of whole blood, serum, plasma, cerebrospinal fluid, tissue or tissue lysate, semen, saliva sample, amniotic fluid, and villi from the subject to be tested.

Preferably, the test sample of the product is selected from at least one of tissue or tissue lysate, whole blood, plasma, and serum; preferably, the tissue or tissue lysate is liver tissue or liver tissue lysate.

Preferably, the product further includes a sample treatment reagent, and the sample treatment reagent includes at least one of a sample lysis reagent, a sample purification reagent, and a protease inhibitor.

Preferably, the product is suitable for performing one or more of the following detection methods: indirect immunofluorescence method, particle agglutination method, antibody confirmation experimental detection method, rapid diagnostic reagent method, enzyme-linked immunosorbent assay, radioimmunoassay, Double immunodiffusion, colloidal gold, biological mass spectrometry, electrophoresis, chromatography, immunofluorescence, immunochemiluminescence, immunoturbidimetry, western blotting, western blotting, flow cytometry, and immunohistochemical analysis .

Preferably, the product further comprises a detection reagent for detecting MMP7; preferably, the reagent for detecting MMP7 is its protein or mRNA level; preferably, when MMP7 increases relative to a reference level, biliary atresia is further diagnosed.

The purpose of the thirteenth aspect of the present invention is to provide a diagnostic kit for the degree of biliary atresia or biliary atresia fibrosis, the kit includes: a detection reagent for BAFF and a detection reagent for MMP7; preferably, the detection The reagent detects the protein or mRNA level; preferably, BAFF and/or MMP7 are increased relative to a reference level, then biliary atresia is diagnosed, and the reference level represents the level of the corresponding index from subjects of the same age group representing non-biliary atresia; preferably Specifically, the subjects of the same age group without biliary atresia are healthy, physiological jaundice, choledochal cyst or liver cancer subjects of the same age group; BAFF level is positively correlated with liver fibrosis, when the subject is diagnosed with After biliary atresia, the higher the level of BAFF, the more severe the degree of liver fibrosis diagnosed as biliary atresia.

Preferably, the sample source suitable for the kit is at least one of whole blood, serum, plasma, cerebrospinal fluid, tissue or tissue lysate, semen, saliva sample, amniotic fluid, and villi; preferably, whole blood, Serum or plasma.

Preferably, the kit includes an anti-BAFF antibody and an anti-human MMP-7 antibody; preferably, the antibody is a monoclonal antibody.

Preferably, the kit is suitable for performing one or more of the following detection methods: indirect immunofluorescence method, particle agglutination method, antibody confirmation experimental detection method, rapid diagnostic reagent method, enzyme-linked immunosorbent assay, radioimmunoassay method , double immunodiffusion, colloidal gold, biological mass spectrometry, electrophoresis, chromatography, immunofluorescence, immunochemiluminescence, immunoturbidimetry, western blotting, western blotting, flow cytometry, and immunohistochemistry analyze.

Preferably, the kit further includes one or more of sample processing reagents, sample dilution reagents, positive or negative control solutions, washing solutions, stop solutions, and preservatives.

Preferably, the subject for which the kit is suitable is a child or an adult, preferably a child.

The purpose of the fourteenth aspect of the present invention is to provide the use of a specific blocking agent for BAFF in any one of (c1) to (c7);

(c1) Preparation of medicines for preventing and treating diseases

(c2) preparing a product that reduces the rate of jaundice;

(c3) preparing a product that reduces IgG4 levels;

(c4) preparing a product that reduces liver function indexes;

(c5) preparing a product that reduces the level of pro-inflammatory factors;

(c6) preparing a product that increases the proportion of Kupffer cells;

(c7) preparing a product that improves the phagocytic ability of Kupffer cells;

Preferably, the disease is biliary atresia, liver fibrosis caused by biliary atresia or cirrhosis caused by biliary atresia.

Preferably, the specific blocker of BAFF blocks the binding of BAFF to at least one BAFF receptor selected from the group consisting of BCMA, TACI and BAFF-R; alternatively, the specific blocker of BAFF enables the function of BAFF weaken or fail.

Preferably, the specific blocking agent for BAFF is a blocking antibody for BAFF.

Preferably, the antibody is a neutralizing antibody or a functional antibody.

Preferably, the neutralizing antibody neutralizes human B cells in at least one form of membrane-bound B cell activating factor (mbBAFF), soluble trimeric B cell activating factor (sBAFF), and soluble 60-mer B cell activating factor activating factor.

Preferably, the antibody is an anti-BAFF monoclonal antibody, preferably an anti-BAFF humanized monoclonal antibody.

Preferably, the antibody fragment comprises Fab, F(ab')2, Fd, Fv, scFv, scFv-Fc, bispecific antibodies and antibody minimal recognition units.

Preferably, the blocking antibody is selected from Tabalumab, Lymphostat B, anti BAFF scFv, anti BAFF scFv Fc.

Preferably, the medicine also includes other active ingredients; preferably, the active ingredient is selected from at least one of MHC-I blocking antibodies, MHC-II blocking antibodies, and IFNAR blocking antibodies.

Preferably, the medicament further includes a pharmaceutically acceptable carrier.

Preferably, the applicable object of the medicament is children or adults, preferably children.

The object of the fifteenth aspect of the present invention is to provide a pharmaceutical composition for preventing and/or treating biliary atresia, liver fibrosis caused by biliary atresia, or liver cirrhosis caused by biliary atresia, the The pharmaceutical composition comprises: the pharmaceutical composition comprises at least one of a), b) and c); further, at least two of a), b) and c) are included; further, a), b) and c);

a) Agents that inhibit MHC-I and/or II signaling pathways;

b) specific blockers of IFNAR;

c) Specific blockers of BAFF.

Preferably, the specific blocking agent for BAFF is a blocking antibody for AFF; preferably, the agent for inhibiting MHC-I and/or II signaling pathway is a blocking antibody for MHC-I and/or MHC-II Preferably, the specific blocking agent of IFNAR is a blocking antibody of IFNAR; preferably, the blocking antibody is a humanized antibody or a fully human antibody.

Preferably, the blocking antibody is a monoclonal antibody; or the blocking antibody is a bispecific antibody, a trispecific antibody or a multispecific antibody.

Preferably, the blocking antibody of BAFF is selected from Tabalumab, Lymphostat B, anti-BAFF scFv, anti-BAFF scFv Fc; the blocking antibody of MHC-I or MHC The blocking antibody of -II is selected from IMMU-114, Hu1D10 and Lym-1; the blocking antibody of IFNAR is selected from Anifrolumab, Faralimomab, MEDI-545, Rontalizumab, Sifalimumab, AGS-009, IFNαKinoid, NI-0101.

Preferably, the dosage form of the pharmaceutical composition is lyophilized powder, injection or oral liquid injection capsule.

The beneficial effects of the present invention are:

Through single-cell RNA sequencing and bioinformatics analysis, flow cytometry, immunofluorescence and other technologies, the present invention surprisingly finds that human beings still have a complete B cell development process in the liver after birth, and the imbalance of B cells leads to Accumulation of autoantibodies and liver failure in children with BA, thus inferring that B cell modification therapy may be an effective prevention and treatment method for BA disease.

Further, by collecting a large number of clinical samples and combining a variety of immunological analysis methods and bioinformatics analysis methods, on the one hand, a variety of convenient, non-invasive, economical and effective biomarkers that can be used for BA diagnosis, auxiliary diagnosis or prognostic diagnosis have been found. Including: IgM/IgG4 ratio (such as the ratio in whole blood/serum/plasma) to evaluate the prognosis of BA patients after Kasai surgery, a high IgM/IgG4 ratio indicates a good prognosis; for example, in serum/plasma/liver, High Ro/SSA-specific IgG, and/or low native IgM (including DsDNA-specific IgM, Nucleosome-specific IgM, RNP-specific IgM, and Ro/SSA-specific IgM) are indicative of biliary atresia; r-GT, The increase of ALT, AST, TBA, and DBIL combined with the decrease of IBIL is an effective indication of biliary atresia; the level of BAFF in serum/plasma and liver of BA patients is positively correlated, and BAFF is also related to the process of BA liver fibrosis. And BAFF is also positively correlated with GGT, Ro/SSA-IgG autoantibodies, and IgG4, so the increased level of BAFF and BAFF combined with IgG4, GGT, Ro/SSA-IgG autoantibodies, etc., can be used as signs of BA and its fibrosis progression. thing. Therefore, the markers alone or in combination can be developed into products related to biliary atresia and/or fibrosis process diagnosis or auxiliary diagnosis or prognostic diagnosis, such as detection chips or detection kits.

On the other hand, through human specimen experiments sampled from liver and blood (including whole blood, serum, and plasma), in vitro and in vivo cell experiments, and BA disease model animal experiments induced by rhesus rotavirus (RRV) infection in newborn mice, A variety of convenient, cost-effective and effective drugs or drug targets for BA prevention and/or treatment have been discovered and validated from multiple perspectives, including: agents that inhibit MHC-I and/or II signaling pathways (such as MHC-I/ Blocking antibodies of II, their bispecific antibodies, monoclonal antibodies, etc.), specific blocking agents of IFNAR and/or BAFF (eg blocking antibodies, neutralizing antibodies). They can all be achieved through a variety of mechanisms found in the present invention (including improving body weight loss, reducing jaundice rate, reducing the level of inflammatory factors IFN-α/β/γ, reducing biochemical indicators of liver injury, IFN-inhibiting viral proliferation, increasing the number of Kupffer cells) and enhance its scavenger function, reduce lymphocyte infiltration, and reduce the degree of liver fibrosis), to achieve the purpose of definite prevention and/or treatment of the occurrence and development of biliary atresia diseases (such as the development of liver fibrosis/cirrhosis). Therefore, they can be used alone and/or in combination in the preparation of medicines or pharmaceutical compositions for preventing and/or treating biliary atresia, hepatic fibrosis or cirrhosis caused by biliary atresia, providing new and various clinically effective prevention and treatment of BA. Favorable options for reducing drug resistance and toxicity.

Description of drawings

Figure 1 shows the results of B cell development and autoreactive B cell expansion in the liver of children with BA: A is the distribution of cell subsets at different stages of B cell development on t-SNE; B is Violin plot showing the expression of marker genes of hematopoietic stem cells (HSC), pre-pro-B, pro-B, pre-B and immature B cell subsets; C is a heat map showing the expression of marker genes at different stages of B cell development; D is a bar graph showing the distribution of B cell subsets among samples within different groups; E is a graph showing the results of flow cytometry detection of B cell precursors in the liver and bone marrow; F is a graph showing the results from liver biopsies, bone marrow and peripheral blood Heat map of marker gene expression in B cells at different stages isolated in ; G is a two-dimensional principal component analysis (PC) plot showing the development of B cells on different samples; H is the expression of RAG1/2 and CD10 in liver samples Immunofluorescence results of the expression in precursor B cells; I is the dot plot showing the abundance of precursor B cells in the liver of BA patients; J is the scatter of the abundance of precursor B cells in the liver of different children Figure; K is the result of the number of mutated nucleotides in the Fv region of the heavy chain and light chain of the intrahepatic B cell subsets of different children; L is the Ig of memory B cell (CSR) subsets of different children The bar graph of class composition; M is the t-SNE graph showing the content of TNFSF8 in the liver of children with BA in different children's livers; N is the t-SNE graph of the expression of TNFSF8 in T cells and NK cells.

Figure 2 is a graph showing the survival curve of children with BA with high IgM/IgG4 ratio and low IgM/IgG4 ratio in serum after Kasai surgery.

Figure 3 is a graph showing the concentrations of IgM and IgG in serum and liver of children with BA, CC control group and other control groups.

Figure 4 is a Receiver Operating Characteristic (ROC) plot of the predictive performance of Ro/SSA specific IgG in distinguishing BA from non-BA (CC) subjects.

Figure 5 is a graph showing the effects of different treatments on the body weight, jaundice rate, inflammatory factors, intrahepatic B1-a, B cell precursors and mature B cells of mice: A is the control group, the RRV group, the RRV- Body weight gain of anti-MHC I mice and RRV-anti-MHC II mice; B is the control group, RRV group, RRV-anti-MHC I and RRV-anti-MHC II mice The graph of the jaundice-free rate; C is the intrahepatic IFN-γ ⁺ CD4 ⁺ T cells, IFN-γ ⁺ CD8 ⁺ T cell abundance map; D is the result of flow cytometry detection of B1-a, B cell precursors and mature B cells in the liver of mice in the control group and RRV group at 11 days; E is the control group mice , RRV group mice, RRV-anti-MHC I mice and RRV-anti-MHC II mice intrahepatic B1-a, B cell precursor, the ratio of mature B cells.

Figure 6 shows the effects of different treatments on the expression of RRV virus genes, the concentrations of Cx3cr1 and C1q in the liver, Kupffer cells in the liver, and the liver: A is the control group mice, the RRV group mice, the RRV-anti-MHC I small mice The relative expression levels of RRV virus genes in mice and RRV-anti-MHC II mice; B is the liver of control mice, RRV mice, RRV-anti-MHC I mice and RRV-anti-MHC II mice Cx3cr1 and C1q concentration map; C is the result of phagocytosis of fluorescently labeled E. coli by Kupffer cells in the liver of different groups of mice; D is the phagocytic ability and proportion of Kupffer cells in the liver of different groups of mice; E is the control group, HE staining images of liver sections of mice in RRV group and anti-MHC-I/II group; F is the Masson trichrome staining image of liver sections of mice in control group, RRV group and anti-MHC-I/II group.

Figure 7 is a heatmap showing 58 significant clinical indicators identified by regression models in 12943 electronic medical records including BA and non-BA subjects.

Figure 8 is a scatter plot showing TBA, DBIL, IBIL concentrations along the time axis.

Figure 9 is a Receiver Operating Characteristic (ROC) plot of the combined values of reduced IBIL and increased r-GT, ALT, AST, TBA and DBIL in predicting performance in distinguishing BA from non-BA subjects.

Figure 10 is a graph of IFN-[beta] levels in the liver of differently treated mice.

Figure 11 is a visual graph of the size and jaundice rate of mice with different treatments.

Figure 12 is a graph showing the proportions of CD4 ⁺ T cells and CD8 ⁺ T cells in the liver and spleen of mice with different treatments.

Figure 13 is a graph of the abundance of IFN-γ ⁺ CD4T, IFN-γ ⁺ CD8T, IL-17a ⁺ CD4 T cells in the livers of differently treated mice.

Figure 14 is a graph showing the results of phagocytosis of inflammatory monocytes, Kupffer cells, and Kupffer in mice treated with different treatments.

Figure 15 is a graph of IFN-[beta] levels in plasma of different patients.

Figure 16 shows the correlation between the levels of BAFF, the concentration of BAFF in the liver and serum, the correlation between the concentration of BAFF in serum and the concentration of γ-glutamyltransferase (GGT), the level of BAFF in the liver and the serum and the concentration of BAFF in the liver and serum of different children. The results of the correlation between lymphocyte infiltration and fibrosis degree: A is the level of BAFF in serum and liver of different children (BA and CC); B is the correlation between the concentration of BAFF in liver and serum; C is a graph of the levels of BAFF in serum and liver of different children (BA, CC controls, other controls); D is the receiver operating characteristic (ROC) of the predictive performance of BAFF in distinguishing BA from non-BA (CC) subjects ) curve diagram; E is the correlation diagram between BAFF in serum and γ-glutamyltransferase (GGT) concentration; F is the correlation between the level of BAFF in liver and serum and the degree of lymphocyte infiltration and fibrosis in liver Result graph.

Figure 17 shows the results of the correlation between BAFF gene expression, CD14 ⁺ and CD68 ⁺ expression, IgG4, CD20, IgG4 and BAFF levels in different children: A is a t- SNE plot; B is a violin plot showing expression levels of genes encoding BAFF receptors in B cell subsets and myeloid cells encoding receptors that bind to the constant region of IgG; C is a graph showing CD14 ⁺ and CD68 ⁺ in liver DAPI immunofluorescence images co-stained with BAFF; D is the number of cells co-stained with CD14 ^{+ and} CD68 ⁺ in the liver of different children; E is the level of immunoglobulin IgG4 in plasma and liver of different children; Correlation diagram of BAFF levels; G is the DAPI immunofluorescence results of CD20 ⁺ B cells and IgG4 ⁺ cells in liver samples of BA patients; H is the correlation between the concentration of BAFF in the liver and the level of Ro/SSA-IgG autoantibodies picture.

Figure 18 is a graph of the results of BAFF-related in vitro cell experiments: wherein, A is the expression level of IgG4 in natural B cells with different treatments; B is the fibroblast-related genes COL1A2, The expression level of ACTA1; C is the expression level of BAFF after the pathogen toxin component LPS and the inflammatory factor IFN-β stimulated the THP1 cell line; D is the effect of BAFF antibody (anti-BAFF) on the expression level of IgG4 induced by IFN-β picture.

Figure 19 is a graph of the results of in vivo animal experiments related to BAFF and its blocking antibody: wherein, A is the BAFF level graph of the different treated mice; B is the jaundice rate graph of the different treated mice; C is the body weight of the different treated mice Variation chart; D is the HE staining chart of hepatocyte necrosis foci and lymphocyte infiltration in mice with different treatments; E is the level chart of liver function indexes ALT, AST, DBIL, and TBA of mice with different treatments.

Figure 20 is a graph of the results of in vivo animal experiments related to BAFF blocking antibodies: wherein, A is a graph of the expression levels of IFN-γ in CD4 ⁺ T cells and CD8 ⁺ T cells in the liver and spleen of mice with different treatments; B is a graph of different treatments The results of the number of Kupffer cells and phagocytosis in mice; C is the concentration of BAFF in the plasma of mice with different treatments; D is the levels of cytokines TNF-α, IFN-β, and IFN-γ in the plasma of mice with different treatments Panel; E is a graph of the expression level of IgG1 in the spleen of mice with different treatments.

Detailed ways

The content of the present invention will be described in further detail below with reference to specific embodiments and accompanying drawings.

It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.

The first aspect of the present invention relates to the use of an agent for inhibiting MHC-I and/or MHC-II signaling pathway in any one of (a1) to (a8);

(a1) preparation of medicines for the prevention and treatment of diseases;

(a2) preparing a product that reduces the rate of jaundice;

(a3) preparing a product that reduces the level of pro-inflammatory factors;

(a4) preparing a product that inhibits RRV virus gene expression;

(a5) preparing a product that increases the levels of Cx3cl1 and C1q;

(a7) preparing a product that increases the proportion of Kupffer cells;

In some embodiments, the proinflammatory factor is IFN-γ.

In some embodiments, the RRV viral genes include at least one of NSP3 and VP6.

The term "MHC" refers to major histocompatibility complex (MHC), and human MHC is called HLA (human leukocyte antigen, HLA), that is, human leukocyte antigen. MHC can be divided into two categories: classical MHC and non-classical MHC. Classical MHC includes MHC I, MHC II, and MHC III genes, which encode MHC I molecules, MHC II molecules, and MHC III molecules, respectively. In an adaptive immune response, foreign antigens are recognized by receptor molecules such as immunoglobulins and T cell receptors or TCRs on B and T lymphocytes. These foreign antigens are presented on the cell surface as peptide fragments by specialized proteins collectively referred to as major histocompatibility complex (MHC) molecules.

In the present invention, "an agent that inhibits MHC-I and/or II signaling pathway" means an agent that partially inhibits or blocks each key node of MHC-I and/or II signaling pathway, such as MHC-I blocking agent, MHC-II blocker, TCR blocker, CD4 blocker, CD8 blocker. Such agents may be chemical agents (eg compounds described in patent EP1605963B1 or statins described in patent WO03061702A1) or biological agents (eg antibodies).

In some embodiments, the agent is a blocking antibody for MHC-I and/or MHC-II, eg, a bispecific blocking antibody for MHC-I and MHC-II.

The term "antibody" includes both polyclonal and monoclonal antibodies. In some embodiments, the blocking antibody is a monoclonal antibody. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD. In addition, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric, bifunctional, humanized and fully human antibodies, and related Synthetic isoforms. The term "antibody" is used interchangeably with "immunoglobulin".

In some embodiments, the agent is a humanized antibody or a fully human antibody.

In some embodiments, the agent is a humanized antibody to MHC-I and/or MHC-II. "Humanized antibody" refers to an antibody in which the antigen binding site is derived from a non-human species and the variable region framework is derived from human immunoglobulin sequences. Humanized antibodies may contain substitutions in framework regions such that the framework may not be an exact copy of the expressed human immunoglobulin sequences or germline gene sequences. Humanized antibodies include chimeric antibodies, remodeled antibodies, or fully humanized antibodies.

In some embodiments, the agent is a fully human antibody to MHC-I and/or MHC-II. A "fully human antibody" refers to an antibody having a heavy chain variable region and a light chain variable region in which the framework and antigen binding site regions are derived from sequences of human origin. If the antibody comprises a constant region, the constant region is also derived from sequences of human origin.

In some embodiments, the agent is an HLA-DR blocking antibody.

"HLA-DR" refers to human leukocyte class II antigen, which is an MHC class II molecule containing two subunits (α subunit and β subunit) with molecular weights of 36kD and 27kD, respectively. HLA-DR is expressed on B lymphocytes, monocytes, macrophages, activated T lymphocytes, activated NK lymphocytes and human progenitor cells.

In some embodiments, the HLA-DR blocking antibody is selected from at least one of IMMU-114, Hu1D10, and Lym-1.

Developed by the Garden State Cancer Center in the United States and promoted by Immunomedics, IMMU-114 is an anti-HLA-DR antibody drug. Due to the substitution of the antibody type to IgG4, IMMU-114 does not produce complement-dependent cytotoxicity and antibodies. cytotoxicity-dependent side effects. IMMU-14 can effectively eliminate all antigen presenting cells including B cells, monocytes, mDC1, mDC2, pDC. IMMU-114 can selectively inhibit the proliferation of HLA-DR+ T cells. IMMU-114 is currently in phase I as monotherapy for the treatment of non-Hodgkin lymphoma or chronic lymphocytic leukemia. IMMU-114 also has the potential to treat GVHD.

Lym-1 is a mouse IgG2a monoclonal antibody directed against the HLA-DR10 protein, a cell surface marker present on more than 80% of lymphoma cells. When combined with a radioisotope, the Lym-1 monoclonal antibody selectively transports the cytotoxic radioisotope to tumor cells expressing HLA-DR10, thereby protecting healthy B cells and normal tissues from killing. The drug also mediates ADCC, thereby promoting Raji B lymphoid lysis of human neutrophils.

Humanized monoclonal antibody (RemitogenTM3; Protein design Labs, Fremont, CA) to Hu1D10 (Apolizumab), a polymorphic determinant on the HLA-DR beta chain, on normal and tumor B cells Express. Apolizumab induces CDC and ADCC in vitro and promotes caspase-independent apoptosis of 1D10 antigen-positive B cells.

In some embodiments, the agent is an antibody fragment that blocks MHC-I and/or MHC-II. The term "antibody fragment" includes antigenic compound-binding fragments of these antibodies, including Fab, F(ab')2, Fd, Fv, scFv, diabodies, and antibody minimal recognition units, as well as single-chain derivatives of these antibodies and fragments , such as scFv-Fc and so on.

The second aspect of the present invention also relates to the use of a specific blocker of IFNAR in any one of (b1) to (b8);

(b1) Preparation of medicines for preventing and treating diseases

(b2) preparing a product that reduces the rate of jaundice;

(b3) preparing a product that reduces the level of pro-inflammatory factors;

(b4) preparing a product that increases the level of IL-17a;

(b5) preparing a product that reduces the level of inflammatory monocytes;

(b7) preparing a product that increases the proportion of Kupffer cells;

In some embodiments, the proinflammatory factor is IFN-γ.

In some embodiments, the inflammatory monocytes are Ly6G ⁺ Ly6C ⁺ CD11b ⁺ cells.

"IFNAR" is a receptor that binds type I interferons, including interferon-alpha and -beta. A "specific blocker of IFNAR" can be a chemical or biological agent that acts to specifically block the specific binding of IFN-α/β to IFNAR (eg, compete with IFN-α/β for binding to IFNAR, or Block IFN-α/β and IFNAR binding sites), or can weaken or disable IFNAR, making it unable to effectively activate downstream signaling pathway proteins.

In some embodiments, the specific blocking agent for IFNAR is an IFNAR blocking antibody. Such antibodies may also be referred to as neutralizing antibodies or functional antibodies.

In some embodiments, the specific blocker of IFNAR is selected from one or more of Anifrolumab, Faralimomab, MEDI-545, Rontalizumab, Sifalimumab, AGS-009, IFNαKinoid, NI-0101.

MEDI-545 is a fully human 147,000 Dalton IgG monoclonal antibody (Mab) that binds most interferon-alpha (IFN-alpha) subtypes. MEDI-545 is made from 100% human protein sequences, making it a fully human monoclonal antibody. Fully human monoclonal antibodies may have advantages over other forms of monoclonal antibodies, such as chimeric and humanized antibodies, because they may have a more favorable safety profile and may be less rapidly eliminated from the body, potentially reducing Frequency of medication. MEDI-545 was derived from an IgG4ic antibody (13H5), which was selected based on functional analysis as it had the most desirable properties for a potential therapeutic. 13H5 was subsequently switched to the IgG1 antibody isotype, produced in CHO cells (Chinese Hamster Ovary cells), and selected for further characterization and preclinical development, initially designated as MDX-1103 and now known as MEDI-545.

"Anifrolumab" is an anti-interferon alpha receptor monoclonal antibody developed by AstraZeneca for the treatment of systemic lupus erythematosus, lupus nephritis and other diseases.

"Faralimomab" refers to faralimomab, an anti-interferon alpha monoclonal antibody.

"Rontalizumab" refers to an anti-interferon alpha monoclonal antibody, also known as rolizumab, CAS: 948570-30-7.

"Sifalimumab" refers to sifalimumab, also known as forgelimumab, an anti-interferon alpha monoclonal antibody, CAS: 1006877-41-3.

"AGS-009" refers to an anti-interferon alpha monoclonal antibody that reduces IFN-alpha levels, developed by Argos.

"IFNαKinoid" can induce anti-interferon alpha (IFNα) antibody response, thereby reducing IFN-α levels, also known as IFN-K-001, developed by Neovacs.

"NI-0101" is a first-generation human monoclonal antibody that blocks TLR4.

The third aspect of the present invention also relates to the use of the specific blocking agent of BAFF in any one of (c1) to (c7);

(c1) Preparation of medicines for preventing and treating diseases

(c2) preparing a product that reduces the rate of jaundice;

(c3) preparing a product that reduces IgG4 levels;

(c4) preparing a product that reduces liver function indexes;

(c5) preparing a product that reduces the level of pro-inflammatory factors;

(c6) preparing a product that increases the proportion of Kupffer cells;

(c7) Preparation of a product that enhances the phagocytic ability of Kupffer cells.

In some embodiments, the pro-inflammatory factor is at least one of TNF-α, IFN-β, and IFN-γ.

"BAFF" refers to BAFF (B cell activating factor or B lymphocyte activating factor, B lymphocyte activating factor, B cell-activating factor, BAFF), BLyS (Blymphocyte stimulator), THANK (TNF homology that activates apptosis, nuclear factor-κB) and c-Jun NH2-terminal kinase) or TALL-1 (TNF-and apoptosis ligand-related leukocyte-expressed ligand 1), which was discovered in 1999 and belongs to the 17th member of the tumor necrosis factor superfamily (TNF), is A transmembrane protein containing 285 amino acids, which plays an important role in B cell differentiation, immunoglobulin class switching, maintaining B cell survival, and inhibiting apoptosis. BAFF acts by binding to its receptors, which are B cell maturation antigen (BCMA), transmembrane activator (transmembrane activator and CAML interactor, TACI), B cell activating factor receptor (BA) FF receptor, BAFF-R). Among the three receptors, BAFF-R binds to BAFF with specificity, and plays a very important role in the process of BAFF functioning.

In some embodiments, a "specific blocker of BAFF" may be a chemical or biological agent (eg, an antibody) that acts to specifically block at least one of BAFF and its receptors (BCMA, TACI, and BAFF-R) Species) specific binding, or can weaken or disable the function of BAFF, making it unable to effectively activate downstream signaling pathway proteins.

In some embodiments, the specific blocking agent for BAFF is a blocking antibody for BAFF. Such antibodies may also be referred to as neutralizing antibodies or functional antibodies.

In some embodiments, the specific blocker of BAFF is selected from one or more of taberolumab, belimumab, anti-BAFF scFv, and anti-BAFF scFv Fc.

"Tabelumab" refers to Tabalumab, a human monoclonal antibody that neutralizes soluble and membrane-bound B cell activating factor.

"Benlimumab (lymphostat B)" refers to Benlysta, also known as LymphoStat-B, belimphos A monoclonal antibody to IgG2λ that binds to soluble BAFF with high affinity and inhibits its activity.

scFv, single-chain variable region, Single-chain variable fragment, is composed of antibody heavy chain variable domain (heavy chain variable domain, VH) and light chain variable domain (light chain variable domain, VL) through a 10-25 It is formed by connecting flexible short peptides (linkers) composed of amino acids, and is the smallest recombinant antibody form (about 27kDa). Essentially, an scFv is a fusion protein and retains the antigen specificity of the original immunoglobulin. The small molecular size of scFv brings the advantages of strong intratumoral penetration, rapid degradation in blood, and small negative feedback in the human body.

"anti BAFF scFv" refers to an anti-BAFF single chain variable region antibody.

"anti BAFF scFv Fc" refers to a single chain variable region antibody against the receptor Fc region of BAFF.

In the first, second and third aspects of the present invention, the "disease" includes an infectious disease, an immune disease or a tumor.

In some embodiments, the infectious disease is a viral infection, bacterial infection, fungal infection, parasitic infection, mycoplasma or chlamydia infection.

In some embodiments, the viruses include: adenoviridae, arenaviridae, astroviridae, bunyaviridae, caliciviridae, Flaviviridae, hepeviridae, mononegavirales, nidovirales, picornaviridae, orthomyxoviridae, papilla papillomaviridae, parvoviridae, polyomaviridae, poxviridae, reoviridae, retroviridae, and togaviruses One or more of the family (togaviridae).

In some embodiments, the bacteria are Gram-negative bacteria and/or Gram-positive bacteria, including, for example, Staphylococcus, Streptococcus, Listeria, Erysipelas, Nephrobacterium, Bacillus Bacillus, Clostridium, Mycobacterium, Actinomyces, Nocardia, Corynebacterium, Rhodococcus, Bacillus anthracis, Erysipelas, Tetanus, Listeria, Emphysema, Mycobacterium tuberculosis, Escherichia coli, Proteus, Shigella, Pneumonia, Brucella, Clostridium aeruginosa, Haemophilus influenzae, Haemophilus parainfluenzae, Moraxella catarrhalis, Acinetobacter, Yersinia One or more of the genus, Legionella pneumophila, Bacillus pertussis, Bacillus parapertussis, Shigella, Pasteurella, Vibrio cholerae, and Bacillus parahaemolyticus.

In some embodiments, the fungi include: Coccidioides crusus, Coccidioides psedes, Histoplasma capsulatum, Histoplasma dunaliella, Bacillus lobosporum, Paracoccus brasiliensis, Dermatitis Blastomyces, Sporothrix schenckii, Penicillium marneffei, Candida albicans, Candida glabrata, Candida tropicalis, Candida portuguese, Aspergillus, P. , Verrucous color mold, dermatitis color mold, Geotrichum candidum, Podopoda bordetii, Cryptococcus neoformans, Rhizopus oryzae, Rhizopus oryzae, Mucor india, A. One or more of mold, O. coronoides, E. heterosporum, Rhinosporidium sibiricum, Mycelia hyaline and Mycetium nidulans.

In some embodiments, the parasites include: gastrointestinal parasites, liver parasites, lung parasites, brain tissue parasites, vascular parasites, lymphatic parasites, muscle tissue parasites, intracellular parasites One or more of worms, bone tissue parasites, and intraocular parasites.

In some embodiments, the immune disease is selected from the group consisting of: systemic lupus erythematosus, multiple sclerosis, type I diabetes, psoriasis, ulcerative colitis, Sjogren's syndrome, scleroderma, polymyositis, rheumatoid Arthritis, mixed connective tissue disease, primary biliary cirrhosis, autoimmune hemolytic anemia, Hashimoto's thyroiditis, Addisons disease, vitiligo, Graves disease, myasthenia gravis, ankylosing spondylitis, allergies Osteoarthritis, allergic vasculitis, autoimmune neutropenia, idiopathic thrombocytopenic purpura, lupus nephritis, chronic atrophic gastritis, autoimmune infertility, endometriosis , Pasture disease, pemphigus, discoid lupus, and dense sediment disease.

In some embodiments, the tumor is selected from the group consisting of: bone, bone junction, muscle, lung, trachea, heart, spleen, artery, vein, blood, capillaries, lymph nodes, lymphatic vessels, lymphatic fluid, oral cavity, pharynx, esophagus, Stomach, duodenum, small intestine, colon, rectum, anus, appendix, liver, gallbladder, pancreas, parotid gland, sublingual gland, urinary kidney, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina, vulva, scrotum , testis, vas deferens, penis, eye, ear, nose, tongue, skin, brain, brain stem, medulla oblongata, barren medulla, brain barren fluid, nerve, thyroid, parathyroid, adrenal, pituitary, pineal, islet, thymus Tumors arising from lesions in any of the gonads, gonads, sublingual glands, and parotid glands.

In some embodiments, the disease is biliary atresia, liver fibrosis caused by biliary atresia, or cirrhosis caused by biliary atresia.

The fourth aspect of the present invention also relates to a pharmaceutical composition for preventing and/or treating biliary atresia, the pharmaceutical composition comprising at least one of a), b) and c): further, comprising a), b) and at least two of c); further, including a), b) and c);

a) The agent for inhibiting MHC-I and/or II signaling pathway according to the first aspect of the present invention;

b) specific blockers of IFNAR according to the second aspect of the invention;

c) Specific blockers of BAFF according to the third aspect of the invention.

According to the first, second, third and fourth aspects of the present invention, the medicine or pharmaceutical composition may further comprise pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other materials known to those skilled in the art . Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. Such materials can include any of the solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents, absorption delaying agents, and materials that are physiologically compatible with the previously listed materials, and the like. The pharmaceutically acceptable carrier can be, for example, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, isotonic agents such as sugars, polyols such as mannitol, sorbitol, or sodium chloride may be included in the pharmaceutical composition. Other pharmaceutically acceptable materials can also be wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf-life or utility of the antibody. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, topical, by inhalation or by injection, eg, intravenously. The optional administration mode of the present invention is intravenous injection or intraperitoneal injection; the agent for inhibiting MHC-I and/or II signaling pathway of the present invention can also be administered by any known administration method, for example, from oral, dermal Mucosal administration (such as topical, sublingual, intranasal and rectal), parenteral administration (such as subcutaneous injection, intramuscular injection, intraarticular injection, intravenous injection, arterial injection) and inhalation administration, etc. Choose appropriately. Thus, specific modes of administration include, but are not limited to, for example, oral, transdermal, mucosal, sublingual, intramuscular, intravenous, intraperitoneal, subcutaneous, and topical.

For intravenous injection, or injection at a painful site (eg, a tumor site), the active ingredient will be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pK, isotonicity and stability. Those of relevant skill in the art will readily be able to prepare suitable solutions, eg, using isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included as desired. Drugs administered by intravenous injection are generally in the form of solid sterile compositions. These compositions may also contain additives, especially mannitol, dextran, hydrolyzed gelatin, sodium citrate, glycine, and the like. Dissolve in sterile water for injection or other sterile media for injection at the time of use.

Human urokininogenase compositions for intravenous administration may also be in the form of aqueous solutions. The composition may also contain additives, especially mannitol, sodium chloride, dextrose, and the like.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form, eg, containing an inert diluent or an assimilable edible carrier. Tablets may contain solid carriers such as gelatin or adjuvants. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oils or synthetic oils. Physiological saline solution, dextrose or other saccharide solutions or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. The specific binding member (and other ingredients, if desired) can also be encapsulated in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the active ingredient may be incorporated with excipients and in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, and the like to use. In order to administer a compound of the present invention by means other than parenteral administration, it may be necessary to coat or co-administer the compound with a material that prevents its inactivation.

The medicament or pharmaceutical composition may be prepared in the form of a formulation, which may optionally be in the form of a unit dosage. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. Tablets or capsules for oral administration and liquids for intravenous administration and continuous infusion are optional compositions.

In some embodiments, the formulation is a lyophilized powder. The preparation method of the freeze-dried powder is a conventional preparation method in the field, for example: raw material→pre-freezing→slow freezing or quick freezing→vacuum→sublimation drying→testing and packaging.

In some embodiments, the formulation is an injection.

In some embodiments, the formulation is an oral liquid injectable capsule. Oral liquid injection capsules In order to improve the use of biomacromolecular drugs such as monoclonal antibodies in recent years, a team of engineers from MIT and scientists from the pharmaceutical company Novo Nordisk have jointly developed a new drug delivery method. Patients only need to take a small "pill", and the pill will automatically insert into the stomach wall after entering the stomach to release monoclonal antibody drugs or other biological macromolecular drugs throughout the body.

In particular, for antibodies, the administered dose may be 0.1 μg/kg to 100 mg/kg of the patient's body weight. For example 0.1 mg/kg to 20 mg/kg patient body weight, 1 mg/kg to 10 mg/kg patient body weight. Generally, fully human or humanized antibodies have longer half-lives in humans than antibodies from other species due to immune responses to foreign polypeptides. Thus, lower doses of fully human or humanized antibodies and less frequent administration are generally possible. In addition, enhanced uptake and tissue penetration (eg, into the brain) of the antibody through modifications, such as lipidation, can reduce the dose and frequency of administration of the antibody. The present invention also relates to a method for treating, preventing, reducing and/or alleviating a disease such as biliary atresia, comprising administering to a subject an effective amount of an agent for inhibiting MHC-I and/or II signaling pathways as described above, Or the specific blocker for IFNAR as described above, or the steps for the drug for the treatment of biliary atresia.

The term "effective amount" as used in the present invention refers to the dose of the component corresponding to the term to achieve treatment, prevention, alleviation and/or alleviation of the disease or condition described in the present invention in a subject.

The term "subject" in the present invention may refer to a patient or other animal receiving the agent or drug of the present invention to treat, prevent, alleviate and/or alleviate the disease, disorder or symptom of the present invention, a subject Included are warm-blooded animals such as mammals, such as primates, and preferably humans. Non-human primates are also individuals. The term individual includes domestic animals, such as cats, dogs, etc., domestic animals (eg, cows, horses, pigs, sheep, goats, etc.), and laboratory animals (eg, mice, rabbits, rats, gerbils, guinea pigs, etc.) ).

In some embodiments, subjects of the present invention are children or adults in humans. Thus, the medicament or pharmaceutical composition can be prepared in a dosage form suitable for use in children or in a dosage form suitable for use in adults. The children include newborns within 28 days of birth, infants within 1 year old, infants from 1 to 6 years old, and children above 6 years old and below 18 years old; the adults can be selected from female adults during pregnancy, perinatal period of female adults or lactating female adults.

In some embodiments, the method involves a single administration or multiple spaced administrations.

The present invention also relates to markers of diseases such as biliary atresia or the resulting liver fibrosis/cirrhosis.

A "marker" or "biomarker" refers to in a sample taken from a subject in one phenotypic state (eg, having a disease) as compared to another phenotypic state (eg, not having a disease) A differentially present organic biomolecule is a molecule that is intended to be used as a target for analyzing a patient's experimental sample, and is also sometimes referred to herein as a "substance to be detected". Examples of such molecular targets may be proteins, polypeptides, genes, or fragments, variants, complexes thereof or may carry post-translational modifications. Non-limiting examples of post-translational modifications are glycosylation, acylation and/or phosphorylation.

A biomarker differs in different phenotypic states if the mean or median level, eg, expression level, of the biomarker in the different groups is calculated to be statistically significant. Common tests of statistical significance include t-test, ANOVA, Kruskal-Wallis, Wilcoxon, Mann-Whitney, and odds ratios, among others. Biomarkers, alone or in combination, provide a measure of a subject's relative risk of belonging to one phenotypic state or another. Therefore, they are used as markers, eg for diseases (prognosis and diagnosis), therapeutic efficacy of drugs (theranostics).

"Level" of a marker or "level" of a biomarker refers to the amount of marker present in the sample being tested. The level of a marker can be an absolute level or amount (eg, μg/mL) or a relative level or amount (eg, relative signal intensity). A "higher level" or "increased level" of a marker refers to a marker level in a test sample, and preferably a control sample, that is greater than the standard error of the assay used to assess marker levels. A "lower level" or "reduced level" of a marker refers to the level of the marker in a test sample, and preferably a control sample, that is less than the standard error of the assay used to assess the level of the marker. The term "known standard level" or "control level" refers to a recognized or predetermined level of a marker that is used to compare the level of the marker in a sample derived from a subject. In one embodiment, the control level of the marker is based on the level of the marker in a sample from a non-biliary atresia disease, eg, normal healthy, choledochal cyst, or physiologic jaundice subject.

The fifth aspect of the present invention is the use of a reagent for detecting the ratio of IgM to IgG4 (reagent 1 for short) in preparing a kit for prognostic analysis of biliary atresia.

Among them, an increase in the ratio of IgM/IgG4, such as an increase in the content or concentration ratio in whole blood/serum/plasma, is a sign of good prognosis in biliary atresia.

In some embodiments, the good prognosis for biliary atresia is good prognosis after Kasai surgery.

In some embodiments, the main indicator of good prognosis after Kasai surgery is at least one of weight gain, reduction or disappearance of jaundice, prolongation of survival time, reduction in the degree of liver fibrosis, and the like. For example, an increase in body weight relative to a reference level for age, a reduction in jaundice, an increase in 5- or 10-year survival, a reduction in the degree of liver fibrosis, eg, by a degree that differs from the reference level by ≥ 10%, including about 15%, 20% , 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more The degree of liver fibrosis is reduced by one grade or more according to the criteria in Table 1 of this application; to obtain the reference level of the same age, for example, select the reference level for people with poor prognosis.

The sixth aspect of the present invention also relates to the use of a reagent for detecting IgG (reagent 2 for short) and/or a reagent for detecting IgM (reagent 3 for short) in preparing a diagnostic kit for biliary atresia.

In some embodiments, the reagent for detecting IgG comprises a reagent for detecting IgG in liver.

Wherein, an increase in IgG relative to a peer reference level is indicative of biliary atresia; the peer reference level obtained is selected from subjects with non-biliary atresia diseases, such as normal healthy, choledochocele, or physiologic jaundice subjects.

Among them, a decrease in IgM relative to the age-matched reference level is an indication of biliary atresia; the age-matched reference level obtained is selected from non-biliary atresia diseases, such as normal healthy, choledochocele, or physiological jaundice subjects.

In some embodiments, the IgG is a Ro/SSA specific IgG.

In some embodiments, the IgM comprises at least one of DsDNA-specific IgM, Nucleosome-specific IgM, RNP-specific IgM, and Ro/SSA-specific IgM.

The seventh aspect of the present invention also relates to a combination of markers, including r-GT, ALT, AST, TBA, DBIL and IBIL.

Among them, the increase of r-GT, ALT, AST, TBA, DBIL relative to the reference level of the same age combined with the decrease of IBIL relative to the reference level of the same age is an indication of biliary atresia, and the reference level of the same age is obtained from non-biliary atresia diseases, such as normal Healthy, choledochal cyst or physiologic jaundice subjects.

The eighth aspect of the present invention also relates to the use of the reagent for detecting the marker combination of the seventh aspect of the present invention (reagent 4 for short) in the preparation of a diagnostic kit for biliary atresia.

The ninth aspect of the present invention also relates to the use of a reagent for detecting BAFF (reagent 5 for short) in preparing a diagnostic kit for biliary atresia.

In some embodiments, the reagent for detecting BAFF is a reagent for detecting the protein level or mRNA level of BAFF.

Biliary atresia is diagnosed when BAFF increases relative to age-matched reference levels obtained from non-biliary atresia disease, such as normal healthy, choledochocele, or physiologic jaundice subjects.

The level of BAFF is positively correlated with liver fibrosis. When the subject is diagnosed with biliary atresia, the higher the level of BAFF, the more severe the degree of liver fibrosis is diagnosed as biliary atresia disease.

As mentioned earlier, "BAFF" is a B cell activating factor and is a member of the TNF family. Further, it is known that BAFF can exist in three forms: membrane-bound (mbBAFF), soluble trimeric BAFF (sBAFF) and a multimeric form consisting of 60 BAFF monomers.

"Membrane-bound B cell activating factor (mbBAFF)" refers to membrane-bound BAFF (mbBAFF). As a typical type II transmembrane protein, membrane-bound BAFF is intracellular at the N terminus and extracellular at the C terminus.

"Soluble trimeric B cell activating factor (sBAFF)", also known as soluble BAFF, is a small molecule soluble fragment with a molecular weight of 17KD obtained by proteolytic cleavage of membrane-bound BAFF. Monomeric or homotrimeric forms enter the peripheral blood circulation.

"Soluble 60-mer B cell activating factor" refers to a crystalline 60-mer of soluble BAFF.

In the fifth, sixth, seventh, eighth and ninth aspects of the present invention, the biliary atresia is biliary atresia, liver fibrosis caused by biliary atresia, or liver cirrhosis caused by biliary atresia.

In the fifth, sixth, seventh, eighth and ninth aspects of the present invention, the "diagnosis" includes auxiliary diagnosis and/or prognostic analysis.

An ideal scenario for diagnosis is a situation where a single event or process causes various diseases, eg, in biliary atresia. In all other cases, correct diagnosis can be very difficult, especially when the etiology of the disease is not fully understood, as in the case of many cancer types. As the skilled artisan will appreciate, for a given multifactorial disease, diagnosis without biochemical markers is 100% specific and with 100% sensitivity. Conversely, biochemical markers or indicators can be used to assess the presence or severity of biliary atresia with some probability or predictive value. Therefore, in routine clinical diagnosis, various clinical symptoms and biological markers are usually considered comprehensively to diagnose, treat and control the underlying disease.

For convenience of expression, Reagent A is used to refer to any one of Reagent 1, Reagent 2, Reagent 3, Reagent 4 and Reagent 5 at the same time.

In some embodiments, Reagent A is a quantitative detection agent.

In some embodiments, the reagent A is used to perform any one or more of the following methods:

Indirect immunofluorescence method, particle agglutination method, antibody confirmation test method, rapid diagnostic reagent method, radioimmunoassay, double immunodiffusion method, counter-immunoelectrophoresis method, colloidal gold method or colloidal gold-labeled spot immunodiafiltration method, flow cytometry Technique, immunohistochemical analysis, biological mass spectrometry, electrophoresis, chromatography, enzyme-linked immunosorbent assay, immunofluorescence, immunochemiluminescence, immunoturbidimetry, western blotting and dot blotting, etc.

"Indirect immunofluorescence method" refers to indirect immunofluorescence test, which is to use a specific antibody to react with the corresponding antigen in the specimen, and then use a fluorescein-labeled secondary antibody (anti-antibody) to bind to the primary antibody in the antigen-antibody complex , and observe the specific fluorescence under a fluorescence microscope after washing to detect unknown antigens or antibodies.

The "particle agglutination method" is a method of measuring an antigen to be measured by immobilizing an antibody on an insoluble carrier, and then mixing a sample containing the antigen to be measured to induce an immunoagglutination reaction based on an antigen-antibody reaction. In the related method, in order to improve the detection sensitivity, it is preferable to use a plurality of antibodies that recognize a multipurpose epitope such as a polyclonal antibody. Agglutination occurs because one antigen molecule binds to multiple antibodies (insoluble carriers). There may also be those with low affinity among their substituted antibody molecules.

"Antibody confirmation experimental detection method" refers to confirmation based on the primary screening of antibodies. Currently, the methods that can be used for confirmation are: ① Western blotting (WB); ② Band immunoassay (LIA); ③ Immunofluorescence assay ( IFA); ④ radioimmunoprecipitation assay (RIPA);

The "rapid diagnostic reagent method" is a relatively convenient and highly sensitive experimental method, which uses the chemical reaction between the virus and the reagent to obtain the experimental results.

"Radioimmunoassay" refers to an in vitro microanalysis method using radioisotopes labeled with unlabeled antigens to competitively inhibit reactions with antibodies. Also known as competitive saturation assay (Radioimmuneassay, RIA).

"Double immunodiffusion method" refers to the method in which antigen and antibody diffuse in the same gel (double immunodiffusion assay), which is one of the most basic methods to observe the reaction between soluble antigen and corresponding antibody and to identify antigen and antibody. The principle is that the corresponding antigens and antibodies diffuse freely to the surrounding in the corresponding wells of the agar gel plate. Between the antigen and antibody wells, the diffused antigen and antibody meet and react specifically, and a white precipitation line visible to the naked eye is formed at the appropriate concentration ratio of the two, which proves that there is a reaction between the antigen and the antibody. If the antibody to be tested is serially diluted, the antibody titer can be determined according to the gradual disappearance of the white precipitation line.

"Convective immunoelectrophoresis" is one of the immunoelectrophoresis techniques, and also includes methods such as immunoelectrophoresis and rocket electrophoresis. In agarose gel with pH 8.6, antibody globulin only has a weak negative charge, and its molecule is large, so it swims slowly and is greatly affected by electroosmosis, so it is often unable to resist electroosmosis. During electrophoresis, it reverses to the negative electrode instead. The general antigenic protein often has a strong negative charge, and the molecule is small, so the swimming is fast. Although the swimming speed is slowed down due to electroosmosis, it can still migrate to the positive electrode. For example, the antigen is placed at the cathode and the antibody is placed at the anode. During electrophoresis, the two components move relative to each other. After a certain period of time, the antigen and antibody will meet between the two wells, and a visible precipitation line will be formed at the appropriate proportion. In this way, due to the effect of electrophoresis, it not only helps the directional movement of the antibody, thereby accelerating the appearance of the reaction, but also limits the tendency of the antigen-antibody to diffuse freely around when the agar diffuses, thus improving the sensitivity. This method is more sensitive than the agar diffusion method. The sensitivity is 10-16 times higher, and the reaction time is short, which can be used for qualitative and semi-quantitative determination of various proteins.

"Colloidal gold method" is a technology that combines antigen-antibody immune reaction and colloidal gold labeling and tracing technology for qualitative and quantitative detection of antigen and antibody content. At present, the commonly used detection methods of colloidal gold detection platform include double antibody sandwich method, competition method, indirect method, etc. The detection principles and suitable substances of different detection methods are different. Among them, "colloidal gold-labeled spot immunodiafiltration method" refers to the use of colloidal gold label instead of enzyme on the basis of spot immunodiafiltration assay, and the step of substrate color development is omitted. The test method uses a nitrocellulose membrane (NC membrane) as a carrier, drips reagents and specimens on the membrane, and reacts step by step through diafiltration. The entire process can be completed within minutes, and positive results appear as red spots on the membrane.

"Flow multi-factor assay" refers to cytokine microsphere detection technology. Cytometric Bead Array (CBA) is a multiplex protein quantitative detection method based on flow cytometer. The detection of multiple cytokine indicators in a single sample requires less sample volume, and has higher sensitivity, wider detection range and better repeatability.

"Biomass spectrometry" is the analysis of composition and structure by determining the mass-to-charge ratio (m/z) of sample ions. Two-dimensional gel electrophoresis separates a large number of protein spots and a small amount. The identification method is to use various attribute parameters of the protein, such as relative molecular mass, isoelectric point, sequence, amino acid composition, peptide mass fingerprint, etc. Search in the protein database, Look for proteins that match these parameters.

"Electrophoresis" means that charged test samples (proteins, nucleotides, etc.) are subjected to an electric field in an inert support medium (such as paper, cellulose acetate, agarose gel, polyacrylamide gel, etc.). Under the action of the electrophoresis, it moves toward its corresponding electrode direction at their respective speeds to separate the components into narrow bands, and use appropriate detection methods to record their electrophoretic band patterns or to calculate their content (%).

"Chromatography" is a separation and analysis method, also known as "chromatographic analysis", "chromatographic analysis", "chromatography".

"Enzyme-linked immunosorbent assay" is the most widely used technique in enzyme immunoassay technology. The basic method is to adsorb a known antigen or antibody on the surface of a solid-phase carrier (polystyrene micro-reaction plate), so that the enzyme-labeled antigen-antibody reaction is carried out on the solid-phase surface, and the free components in the liquid phase are washed by washing method. remove. Commonly used ELISA methods include double antibody sandwich method and indirect method. The former is used to detect macromolecular antigens, and the latter is used to determine specific antibodies.

"Immunofluorescence" refers to labeling an antibody (or antigen) with a fluorochrome that does not affect the activity of an antigen-antibody, and after binding to its corresponding antigen (or antibody), a specific fluorescence reaction is presented under a fluorescence microscope.

"Immunochemiluminescence method", also known as chemiluminescence labeling immunoassay, is to directly label an antigen or antibody with a chemiluminescent agent (chemiluminescent agent marker), and the corresponding antibody or antigen, magnetic particle antigen or antibody in the sample to be tested In the reaction, the combined state (precipitated part) and the free state of the chemiluminescent marker are separated by a magnetic field, and then a luminescence promoter is added to carry out a luminescence reaction, and quantitative or qualitative detection is performed by detecting the luminescence intensity. The enzyme-labeled anti-hapten antibody or avidin binds to it, and the enzyme-labeled antibody or avidin bound to the hapten can catalyze the chemiluminescence substrate to emit light. For example, the antibody molecule is labeled with acridinium ester, and it emits light after activation by a trigger, which is used to detect the immobilized antigen.

"Immunoturbidimetry" is a dynamic assay method for antigen-antibody binding. The basic principle is: when the antigen and antibody react in a special dilution system and the ratio is appropriate (generally, the antibody is excessive), the soluble immune complex formed is under the action of the polymerization promoter (polyethylene glycol, etc.) in the dilution system. , precipitated from the liquid phase to form microparticles, causing turbidity in the reaction solution. When the antibody concentration is fixed, the amount of the formed immune complex increases with the increase in the amount of antigen in the test sample, and the turbidity of the reaction solution also increases. By measuring the turbidity of the reaction solution and comparing it with a series of standard substances, the content of the antigen in the test sample can be calculated.

"Immunoblotting" refers to the transfer of proteins to a membrane, which is then detected using antibodies. For the known expressed protein, the corresponding antibody can be used as the primary antibody for detection, and the expression product of the new gene can be detected by the antibody of the fusion part.

"Dot blot" refers to a technique for the qualitative detection of nucleic acids or proteins. That is, the nucleic acid or protein to be detected is spotted on a solid-phase carrier, hybridized with an isotope or non-isotopic labeled probe, and detected by developing or developing color.

In some embodiments, reagent A is typically a specific reagent, eg, a ligand or receptor (if present) for the analyte, a lectin that binds the analyte, an aptamer that binds the analyte, or a binding agent that binds the analyte. Antibodies and antibody fragments of the substance to be detected. A specific binding agent has an affinity of at least 10 ⁷ L/mol for its corresponding target molecule. A specific binding agent may optionally have an affinity for its target molecule of 10 ⁸ L/mol, or more preferably 10 ⁹ L/mol.

In some embodiments, the reagent A is an antibody specific for the substance to be detected.

In some embodiments, the kit further includes sample processing reagents including at least one of sample lysis reagents, sample purification reagents, and protease inhibitors.

The tenth aspect of the present invention also relates to a kit, the kit comprises: at least one of d1) to d5):

d1) the reagent for detecting the ratio of IgM and IgG4 in the fifth aspect of the present invention (referred to as reagent 1);

d2) the reagent for detecting IgG in the sixth aspect of the present invention (referred to as reagent 2);

d3) the reagent for detecting IgM in the sixth aspect of the present invention (referred to as reagent 3);

d4) the reagent for detecting r-GT, ALT, AST, TBA, DBIL and IBIL in the eighth aspect of the present invention (referred to as reagent 4);

d5) The reagent for detecting BAFF in the ninth aspect of the present invention (reagent 5 for short).

In some embodiments, the kit further includes an antibody-coated ELISA plate, a negative control solution, a positive control solution, an ELISA reagent, an enzyme substrate solution, a blocking solution, a sample diluent, a washing solution, and a stop solution.

The materials described above, as well as other materials, can be packaged together in any suitable combination as a kit that can be used to perform or assist in performing the disclosed methods. It is useful to design and adapt kit compositions for use with the disclosed methods.

The eleventh aspect of the present invention also relates to a method for diagnosing biliary atresia, the method comprising: using at least one of reagents 1 to 5 or the kit of the tenth aspect of the present invention to detect the substance to be detected.

In some embodiments, the test substance is selected from at least one of whole blood, serum, plasma, cerebrospinal fluid, tissue/tissue lysate, semen, saliva sample, amniotic fluid, and villi of the subject.

As used herein, "tissue lysate" may also be used generically with the terms "lysate", "lysed sample", "tissue or cell extract", etc. to refer to a sample and/or biological sample material comprising lysed tissue or cells, i.e. where the structural integrity of the tissue or cell has been disrupted. To release the contents of a cell or tissue sample, the material is typically treated with enzymes and/or chemical agents to dissolve, degrade or disrupt the cell walls and membranes of such tissue or cells. The skilled artisan is very familiar with appropriate methods for obtaining lysates. This process is encompassed by the term "cleavage".

In particular, the tissue/tissue lysate is from the liver.

In the first to eleventh aspects of the present invention,

"Ro/SSA" refers to ribonucleoprotein antigen, which is a ribonucleoprotein containing small cytoplasmic RNA. The protein component of the Ro/SSA antigen is a 60-kD protein (60-kD Ro/SSA, Ro60) that binds to one of several small cytoplasmic RNA molecules. Another component of the Ro/SSA antigen is the 52-kD peptide (52-kD Ro/SSA, Ro52). The La/SSB antigen consists of a polypeptide containing 408 amino acids. Both the 60-kD Ro/SSA and La/SSB proteins are members of a family of RNA-binding proteins that contain sequences of 80 amino acids called RNA recognition motifs (RNPs).

"IgG" refers to the immunoglobulin class G (immunoglobulin G) of antibodies, which play a role in activating complement and neutralizing a variety of toxins in the immune response. IgG antibodies are long-lasting and are the only antibodies that cross the placenta during pregnancy to protect the fetus. They are also secreted from the mammary gland into colostrum, so that the newborn is protected by antibodies for the first time. IgG is a four-chain monomer, accounting for 75% of the total serum Ig, and is the most important antibody component in serum and extracellular fluid. Human IgG can be divided into four subclasses, according to their concentration in serum. IgG1, IgG2, IgG3, IgG4. IgG begins to be synthesized three months after birth and approaches adult levels at three to five years of age. Mainly produced by plasma cells in the spleen and lymph nodes, the serum half-life is long, about 20-23 days. It is the main antibody produced by the second humoral immune response. It has high affinity and is widely distributed in the body. The main force of infection.

Choledochal cyst, also known as choledochal cyst (CC), is the most common congenital biliary malformation in clinical practice. The lesions mainly refer to the cystic or fusiform dilation of a part of the common bile duct, sometimes accompanied by congenital malformations of intrahepatic bile duct dilatation. The incidence in women is higher than that in men, accounting for about 60% to 80% of the total incidence. Also known as congenital choledochal cyst, congenital choledochectasis, primary choledochalectasis, etc. In recent years, with the deepening of the research on this disease, it was found that in addition to the cystic dilatation of the common bile duct, about half of the patients showed only the fusiform or cylindrical dilatation of the common bile duct, rather than a huge cyst. In addition to the dilatation of the extrahepatic common bile duct, about 1/4 of the cases also have dilatation of the intrahepatic bile duct.

"Physiological jaundice" refers to neonatal physiological jaundice, which is a characteristic of neonatal bilirubin metabolism, a physiological phenomenon in the growth process of normal neonates, and is a yellow staining of skin and mucous membranes caused by excessive bilirubin concentration in the body. Phenomenon.

"Sample lysis reagent" refers to a reagent that disrupts the barrier of the cell membrane or nuclear membrane, releasing the subject to be studied into the in vitro environment.

"Protease inhibitor" refers to a substance that combines with some groups on the active center of the protease molecule to reduce the activity of the protease, or even disappear, but does not denature the enzyme protein. Leupeptin, analgesin, chymostatin, elastin aldehyde, pepsin, and phosphoramidin were isolated from the fermentation broth of actinomycetes, which can inhibit trypsin, papain, chymotrypsin, elastin, etc. Various proteases such as protease, pepsin, and metalloprotease.

"Detection reagents for biochemical indicators of liver function" refer to ALT, AST, AST/ALT, GGT, ALP, TBILI, DBILI, IBILI, TP, ALB, GLB, A/G, LDH-L, Reagents for Ch, SF, PA and other indicators.

"r-GT" refers to r-glutamyl transferase, formerly known as r-glutamyl transpeptidase, is an enzyme that catalyzes the transfer of r-glutamyl group on glutathione to another peptide or another amino acid . Mainly present on stem cell membranes and microsomes.

"ALT" refers to alanine aminotransferase, which mainly exists in various cells, especially liver cells. The content of aminotransferase in the whole liver is about 100 times that in blood.

"AST" refers to aspartate aminotransferase, which is mainly distributed in cardiac muscle, followed by liver, skeletal muscle and kidney and other tissues. Normal serum AST content is low, but when the corresponding cells are damaged, the cell membrane permeability increases, and the cytoplasmic AST is released into the blood, so its serum concentration can be increased. Inspection of.

"TBA" refers to total bile acids. The bile acids synthesized by the liver of ordinary people include cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) produced in metabolism, and a small amount of lithocholic acid (LCA) and Trace ursodeoxycholic acid (UDCA), collectively referred to as total bile acid (TBA).

"DBIL" refers to direct bilirubin, also known as conjugated bilirubin, which is formed by the combination of glucuronic acid and glucuronic acid in the liver after indirect bilirubin enters the liver.

"IBIL" refers to indirect bilirubin, also known as unconjugated bilirubin, that is, bilirubin that is not bound to glucuronic acid. Total bilirubin is composed of indirect bilirubin and direct bilirubin.

"GGT" refers to glutamyltransferase, which mainly exists on the liver cell membrane and microsomes, and is involved in the metabolism of glutathione. Kidney, liver and pancreas are abundant, but GGT in serum mainly comes from the hepatobiliary system.

"IgG4" refers to immunoglobulin G subclass 4, which belongs to one of the immunoglobulins of the human IgG class. There are four subclasses of serum immunoglobulins, namely IgG1, IgG2, IgG3, and IgG4.

"TBIL" refers to total bilirubin (TBIL), which is the sum of direct and indirect bilirubin. Indirect bilirubin refers to bilirubin that is not bound to glucuronic acid. Indirect bilirubin is insoluble in water and cannot be excreted in urine through the kidneys. Indirect bilirubin is converted in hepatocytes and combined with glucuronic acid to form direct bilirubin (conjugated bilirubin). Direct bilirubin dissolves in water and can be excreted through the kidneys with urine. The liver plays an important role in the metabolism of bilirubin, including the three processes of uptake, binding and excretion of unconjugated bilirubin in the blood by hepatocytes. Obstruction of any of these processes can cause bilirubin to accumulate in the blood. , jaundice occurs.

"IFN-β" refers to β-interferon. IFN-β up-regulates and down-regulates a variety of genes through the signaling pathways STAT1 and STAT2, most of which are involved in antiviral immune responses. IFN-β plays an important role in inducing a broad range of nonspecific antiviral infections, it also affects cell proliferation and modulates immune responses.

"IFNAR" means that the interferon-alpha/beta receptor (IFNAR) is a receptor that binds type I interferons, including interferon-alpha and -beta. It is a heteromeric cell surface receptor consisting of a chain with two subunits called IFNAR1 and IFNAR2. After binding to type I IFN, IFNAR activates the JAK-STAT signaling pathway. Interferon stimulation leads to an antiviral immune response.

"MMP7" refers to matrix metalloproteinase 7 (Matrix metalloproteinases-7, MMP-7), which has been quantitatively determined by double-antibody sandwich ELISA in human serum, plasma, tissue homogenate, cell lysate, cell culture supernatant or other related The test kit of MMP7 content in biological liquid, and the report (for example CN 108267585 A) of applying the MMP7 detection kit to the diagnosis of biliary atresia, the present invention introduces MMP7 on the basis of the BAFF discovered by the inventor as the double-check of biliary atresia, and uses In the preparation of kits and other products, in order to further improve the diagnostic accuracy of biliary atresia diseases.

"Biliary atresia" refers to biliary atresia (BA), which is one of the common serious diseases of the hepatobiliary system in infancy, characterized by progressive inflammation and fibrosis of the intrahepatic and extrahepatic bile ducts. Biliary cirrhosis, portal hypertension, and liver failure are diseases of unknown etiology involving occlusive lesions of the intrahepatic and extrahepatic bile ducts, leading to cholestasis and progressive liver fibrosis until liver cirrhosis and endangering the life of children. The clinical manifestations are: (1) Postnatal jaundice regresses delayed (more than 2 weeks in term infants and more than 3 weeks in preterm infants), or reappears after remission, and continues to aggravate. (2) The color of feces gradually becomes lighter to white clay color, and the color of urine deepens to dark brown. (3) Abdominal distention, hepatosplenomegaly, abdominal wall varicose veins, etc. (4) Malnutrition or growth retardation due to malabsorption of fat-soluble vitamins.

"Liver fibrosis" refers to the imbalance between the proliferation and degradation of extracellular matrix such as collagen between liver cells when liver cells undergo necrosis and inflammatory stimulation. A pathological process that leads to abnormal deposition of fibrous connective tissue between hepatocytes and hepatic lobules in the liver. A small amount of collagen deposition is called hepatic fibrosis. The formation of special nodules can gradually evolve into liver cirrhosis, liver cancer, etc., which seriously endanger people's health.

"Liver fibrosis/cirrhosis due to biliary atresia" refers to concurrent liver fibrosis caused by biliary atresia disease, and this type of liver fibrosis develops rapidly, usually developing into cirrhosis within a few months. The activated pathway of biliary atresia produces a large number of inflammatory mediators that damage hepatocytes and cholangiocytes, releasing various pro-inflammatory factors, oxygen metabolites and cytokines, further aggravating the damage to the liver and biliary system, resulting in abnormal and rapid activation and transformation of hepatic stellate cells For fibroblasts, it rapidly promotes the process of liver fibrosis/cirrhosis.

"Liver fibrosis degree" refers to the degree of liver fibrosis evaluated according to the liver fibrosis Ishak scoring system, with

grades

0, 1, 2, 3, 4, 5, and 6. The specific evaluation criteria are shown in Table 1 of this application.

Specific embodiments of the present invention are described in more detail below.

1. Use of agents that inhibit MHC-I and/or II signaling pathways in the treatment of biliary atresia

(1) Use of an agent for inhibiting MHC-I and/or II signaling pathway in any one of (a1) to (a8);

(a1) preparing a medicine for treating biliary atresia;

(a2) preparing a product that reduces the rate of jaundice;

(a3) preparing a product that reduces the level of pro-inflammatory factors;

(a4) preparing a product that inhibits RRV virus gene expression;

(a5) preparing a product that increases the levels of Cx3cl1 and C1q;

(a7) preparing a product that increases the proportion of Kupffer cells;

(2) The use according to the above (1), wherein the reagent is a blocking antibody of MHC-I and/or II.

(3) The use according to the above (2), wherein the reagent is a bispecific blocking antibody of MHC-I and MHC-II.

(4) The use according to (2) above, wherein the blocking antibody is a monoclonal antibody.

(5) The use according to (4) above, wherein the blocking antibody is an HLA-DR blocking antibody.

(6) The use according to the above (5), wherein the HLA-DR blocking antibody is selected from at least one of IMMU-114, Hu1D10 and Lym-1.

(7) The use according to the above (2), wherein the reagent is a chimeric antibody, modified antibody, humanized antibody or fully human antibody of MHC-I and/or II.

(8) The use according to (1) above, wherein the reagent is an antibody fragment that blocks MHC-I and/or II.

(9) The use according to the above (8), wherein the antibody fragment comprises Fab, F(ab')2, Fd, Fv, scFv, scFv-Fc, diabody and antibody minimum recognition unit.

(10) The use according to any one of (1) to (9) above, wherein the medicament comprises at least one of a pharmaceutically acceptable excipient, carrier, buffer and stabilizer.

(11) The use according to any one of the above (1)-(10), wherein the medicine is a freeze-dried powder or an injection.

(12) According to the use according to any one of the above (1)-(11), the medicament is prepared into a dosage form suitable for adults or children; preferably, the medicament is prepared into a dosage form suitable for children.

(13) The use according to any one of (1) to (12) above, wherein the pro-inflammatory factor is IFN-γ.

(14) The use according to any one of (1) to (13) above, wherein the RRV viral gene includes at least one of NSP3 and VP6.

(15) The use according to any one of (1) to (14) above, wherein the biliary atresia is biliary atresia, liver fibrosis caused by biliary atresia, or liver cirrhosis caused by biliary atresia.

2. The use of IgM/IgG4 ratio in the companion diagnosis of biliary atresia prognosis

(1) use of a reagent for detecting the ratio of IgM to IgG4 in the preparation of a product for prognostic analysis of biliary atresia; preferably, the product is selected from detection reagents, detection test strips or test strips, detection chips or kits; preferably , the ratio is the content ratio or the concentration ratio.

(2) The use according to the above (1), wherein the prognostic analysis of biliary atresia is prognostic analysis after Kasai surgery for biliary atresia.

(3) According to the use according to any one of the above (1) to (2), the prognostic analysis includes step 1) of determining the ratio of IgM and IgG4 in the sample from the subject; preferably, the prognostic analysis further Including step 2), the ratio described in 1) is compared to a reference level.

(4) According to the use according to the above (3), when the prognostic analysis includes step 1), the ratio of IgM and IgG4>0.01, preferably ≥0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05 or 0.055, it indicates The prognosis of biliary atresia is good; when steps 1) and 2) are included, the ratio of IgM to IgG4 is increased relative to the reference level, indicating a good prognosis of biliary atresia.

(5) The use according to any one of (3)-(4) above, wherein the reference level is the ratio of IgM to IgG4 in a sample from a patient with a poor prognosis for biliary atresia.

(6) The use according to the above (4), wherein the good prognosis of the biliary atresia comprises one or more of weight gain, reduction or disappearance of jaundice, prolongation of survival time, and reduction in the degree of liver fibrosis.

(7) According to the use according to the above (3), the sample of the subject is whole blood, serum, plasma, cerebrospinal fluid, tissue or tissue lysate, cell culture supernatant, semen, saliva sample, amniotic fluid , one or more of villi; preferably one or more of whole blood, serum, plasma, tissue or tissue lysate, cell culture supernatant; preferably, described tissue or tissue lysate is liver tissue or liver tissue lysate.

(8) According to the use according to any one of the above (1)-(7), the product for prognostic analysis of biliary atresia is suitable for performing one or more of the following detection methods: indirect immunofluorescence method, particle agglutination method, Antibody confirmation test method, rapid diagnostic reagent method, enzyme-linked immunosorbent assay, radioimmunoassay, immunodiffusion method, colloidal gold method, flow multi-factor detection method, biological mass spectrometry, electrophoresis, chromatography, immunofluorescence , immunochemiluminescence, immunoturbidimetry, immunoblotting, and dot blot.

(9) The use according to (1)-(8) above, wherein the product for prognostic analysis of biliary atresia includes anti-human IgM and anti-human IgG4.

(10) According to the use described in (1)-(9) above, the subject of the product is a child or an adult, preferably a child.

(11) A kit for prognostic analysis of biliary atresia, the kit includes a reagent for detecting the ratio of IgM to IgG4, preferably, the ratio is a content ratio or a concentration ratio.

(12) The kit according to (11) above, wherein the prognostic analysis of biliary atresia is prognostic analysis after Kasai surgery for biliary atresia.

(13) The kit according to any one of (11)-(12) above, wherein the prognostic analysis includes the following steps 1), or includes the following steps 1) and 2):

1) determining the ratio of IgM to IgG4 in a sample from the subject;

2) Compare the ratio described in 1) with the reference level.

(14) According to the kit according to the above (13), when step 1) is included, the ratio of IgM to IgG4>0.01, preferably ≥0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05 or 0.055, it indicates biliary tract Atresia has a good prognosis; when steps 1) and 2) are included, an increase in the ratio of IgM to IgG4 relative to the reference level indicates a good prognosis for biliary atresia.

(15) The kit according to any one of (13)-(14) above, wherein the reference level is the ratio of IgM to IgG4 in a sample from a patient with poor prognosis of biliary atresia.

(16) The kit according to the above (14), wherein the good prognosis of the biliary atresia includes one or more of weight gain, reduction or disappearance of jaundice, prolongation of survival time, and reduction in the degree of liver fibrosis.

(17) The kit according to (13) above, wherein the sample of the subject is whole blood, serum, plasma, cerebrospinal fluid, tissue or tissue lysate, cell culture supernatant, semen, saliva sample, One or more of amniotic fluid and villi; preferably one or more of whole blood, serum, plasma, tissue or tissue lysate, cell culture supernatant; preferably, the tissue or tissue lysate is liver Tissue or liver tissue lysate.

(18) The kit according to any one of (11) to (17) above, the kit for prognostic analysis of biliary atresia is suitable for performing one or more of the following detection methods: indirect immunofluorescence, particle agglutination method, antibody confirmation test method, rapid diagnostic reagent method, enzyme-linked immunosorbent assay, radioimmunoassay, immunodiffusion method, colloidal gold method, flow multi-factor detection method, biological mass spectrometry, electrophoresis, chromatography, immunoassay Fluorescence, immunochemiluminescence, immunoturbidimetry, western blot, and dot blot.

(19) The kit according to any one of (11) to (18) above, which includes anti-human IgM and anti-human IgG4.

(20) The kit according to any one of (11) to (19) above, further comprising a sample processing reagent, the sample processing reagent comprising at least one of a sample lysis reagent, a sample purification reagent and a protease inhibitor A sort of.

(21) The kit according to any one of (11)-(20) above, wherein the subject of the kit is a child or an adult, preferably a child.

(22) Use of the ratio of IgM and IgG4 as a marker for prognostic analysis of biliary atresia, preferably, the ratio is a content ratio or a concentration ratio.

(23) The use according to (22) above, wherein the prognostic analysis of biliary atresia is prognostic analysis after Kasai surgery for biliary atresia.

(24) According to the use according to any one of the above (22)-(23), when the ratio of IgM and IgG4 is increased relative to the reference level, the diagnosis or auxiliary diagnosis of biliary atresia has a good prognosis.

(25) The use according to (24) above, wherein the reference level is determined from the ratio of IgM to IgG4 in a sample from a patient with a poor prognosis for biliary atresia.

(26) The use according to (25) above, wherein the ratio of IgM to IgG4 at the reference level is 0 to 0.01.

(27) The use according to the above (24), wherein the good prognosis of the biliary atresia comprises one or more of weight gain, reduction or disappearance of jaundice, prolongation of survival time, and reduction in the degree of liver fibrosis.

(28) The use according to (22)-(27) above, wherein the marker is derived from whole blood, serum, plasma, cerebrospinal fluid, tissue or tissue lysate, cell culture supernatant, semen, saliva samples , amniotic fluid, one or more of villi; preferably one or more of whole blood, serum, plasma, tissue or tissue lysate, cell culture supernatant; preferably, described tissue or tissue lysate is Liver tissue or liver tissue lysate.

(29) The use according to any one of the above (22)-(28), wherein the ratio of IgM to IgG4 is obtained by one or more of the following detection methods: indirect immunofluorescence method, particle agglutination method, Antibody confirmation test method, rapid diagnostic reagent method, enzyme-linked immunosorbent assay, radioimmunoassay, immunodiffusion method, colloidal gold method, flow multi-factor detection method, biological mass spectrometry, electrophoresis, chromatography, immunofluorescence , immunochemiluminescence, immunoturbidimetry, immunoblotting, and dot blot.

(30) The use according to any one of the above (22)-(29), wherein the marker is detected from children or adults, preferably from children.

3. The use of Ro/SSA-specific IgG autoantibodies in the diagnosis of biliary atresia

(1) Use of a reagent for detecting IgG in the preparation of a product for biliary atresia diagnosis or auxiliary diagnosis, preferably, the product includes a detection reagent, a detection test paper or test strip, a detection chip or a kit.

(2) According to the use described in (1) above, the IgG is Ro/SSA-specific IgG; preferably, when the IgG antibody increases relative to a reference level, biliary atresia is diagnosed or aided in diagnosis, and the reference level represents Levels of corresponding indexes from subjects of the same age group without biliary atresia; preferably, the subjects of the same age group without biliary atresia are subjects of the same age group with healthy, physiological jaundice, choledochal cyst or liver cancer .

(3) According to the use according to any one of the above (1)-(2), the samples detected by the product include whole blood, serum, plasma, cerebrospinal fluid, tissue/tissue lysate, cell culture from the subject At least one of supernatant, semen, saliva sample, amniotic fluid, and villi.

(4) According to the use described in the above (3), the sample detected by the product is selected from at least one of tissue or tissue lysate, whole blood, serum, and plasma, preferably, the tissue/tissue lysate is Liver tissue or liver tissue lysate.

(5) According to the use according to any one of the above (1)-(4), the product further includes a sample treatment reagent, and the sample treatment reagent includes at least one of a sample lysis reagent, a sample purification reagent and a protease inhibitor .

(6) According to the use described in any one of the above (1)-(5), the product is suitable for performing one or more of the following detection methods: indirect immunofluorescence method, particle agglutination method, antibody confirmation experimental detection method , rapid diagnostic reagent method, enzyme-linked immunosorbent assay, radioimmunoassay, immune double diffusion method, colloidal gold method, flow multi-factor detection method, biological mass spectrometry, electrophoresis, chromatography, immunofluorescence, immunochemiluminescence , immunoturbidimetry, immunoblotting, and dot blot.

(7) According to the use according to any one of the above (1)-(6), the product comprises an anti-human IgG antibody, preferably an anti-human Ro/SSA-specific IgG autoantibody.

(8) According to the use according to any one of the above (1)-(7), the product further includes a reagent for detecting IgM antibody; preferably, the IgM antibody is a natural IgM antibody; further preferably, the IgM antibody It is one or more of anti-double-stranded DNA IgM antibodies, anti-nucleosome IgM antibodies, anti-ribonucleoprotein IgM antibodies, and anti-Ro/SSA IgM antibodies; preferably, when the IgM antibodies are relative to If the reference level decreases, then further diagnosis or auxiliary diagnosis is biliary atresia, and the reference level represents the level of the corresponding index from subjects of the same age group without biliary atresia; preferably, the subjects of the same age group without biliary atresia Age-matched subjects with healthy, physiological jaundice, choledochal cyst or liver cancer.

(9) According to the use according to any one of the above (1)-(8), the product further includes a reagent for detecting BAFF; preferably, when BAFF is elevated relative to the reference level, further diagnosis or auxiliary diagnosis is biliary atresia , the reference level represents the level of the corresponding index from the subjects of the same age group without biliary atresia; preferably, the subjects of the same age group without biliary atresia are healthy, physiological jaundice, choledochal cyst or liver cancer subjects of the same age group.

(11) Diagnostic or auxiliary diagnostic markers for biliary atresia, the markers include:

a) IgG antibodies; and/or

b) IgM antibodies; and/or

c) BAFF.

(12) The marker according to (11) above, wherein the IgG is Ro/SSA-specific IgG; preferably, the IgM antibody is a natural IgM antibody; preferably, the natural IgM antibody is anti-double-stranded DNA One or more of IgM antibodies against nucleosomes, IgM antibodies against nucleosomes, IgM antibodies against ribonucleoprotein, and IgM antibodies against Ro/SSA.

(13) The marker according to any one of the above (11)-(12), when the IgG autoantibody increases relative to the reference level, and/or the IgM antibody decreases relative to the reference level, and/or the BAFF increases relative to the reference level High, then the diagnosis or auxiliary diagnosis is biliary atresia, and the reference level represents the level of the corresponding index from the non-biliary atresia subjects in the same age group; preferably, the non-biliary atresia subjects in the same age group are healthy, Age-matched subjects with physiologic jaundice, choledochal cyst, or liver cancer.

(14) The marker according to any one of the above (11)-(13), wherein the marker is detected from children or adults, preferably from children.

(15) a diagnostic or auxiliary diagnostic kit for biliary atresia, the kit comprising:

a) Reagents for the detection of IgG antibodies; and/or

b) Reagents for the detection of IgM antibodies; and/or

c) Reagents for detecting BAFF.

(16) The kit according to (15) above, wherein the IgG is Ro/SSA-specific IgG; preferably, when it is detected that the IgG antibody is increased relative to the reference level, and/or the BAFF is increased relative to the reference level, And/or the IgM antibody is reduced relative to the reference level, the diagnosis or auxiliary diagnosis is biliary atresia, and the reference level represents the level of the corresponding index from subjects of the same age group without biliary atresia; preferably, the non-biliary atresia Subjects in the same age group were healthy, physiological jaundice, choledochal cyst or liver cancer subjects in the same age group.

(17) The kit according to any one of the above (15)-(16), wherein the IgM antibody is a natural IgM antibody; preferably, the IgM antibody is an anti-double-stranded DNA IgM antibody, an anti-nucleosome One or more of IgM antibodies, anti-ribonucleoprotein IgM antibodies, and anti-Ro/SSA IgM antibodies.

(18) The kit according to any one of the above (15)-(17), which is suitable for performing one or more of the following detection methods: indirect immunofluorescence method, particle agglutination method, antibody confirmation experiment Detection method, rapid diagnostic reagent method, enzyme-linked immunosorbent assay, radioimmunoassay, immune double diffusion method, colloidal gold method, flow multi-factor detection method, biological mass spectrometry, electrophoresis, chromatography, immunofluorescence, immunochemistry Luminescence, immunoturbidimetry, immunoblotting, and dot blot.

(19) The kit according to the above (15)-(18), wherein the samples of the kit include whole blood, serum, plasma, cerebrospinal fluid, tissue or tissue lysate, cell culture At least one of serum, semen, saliva sample, amniotic fluid, and villi.

(20) The kit according to any one of (15)-(19) above, further comprising a sample processing reagent, the sample processing reagent comprising at least one of a sample lysis reagent, a sample purification reagent and a protease inhibitor A sort of.

(21) The kit according to any one of the above (15)-(20), wherein the subjects of the kit are children or adults, preferably children.

(22) Use of IgG antibody as a diagnostic or auxiliary diagnostic marker for biliary atresia.

(23) The use according to the above (22), wherein the IgG is Ro/SSA-specific IgG; preferably, the marker further comprises IgM antibody and/or BAFF.

(24) The use according to the above (23), wherein the IgM antibody is a natural IgM antibody; preferably, the IgM antibody is an anti-double-stranded DNA IgM antibody, an anti-nucleosome IgM antibody, an anti-ribonucleoprotein One or more of the IgM antibody and anti-Ro/SSA IgM antibody.

(25) The use according to the above (22-(24), wherein the marker is sampled from whole blood, serum or plasma.

4. The use of biochemical indicators of liver function in the diagnosis of biliary atresia

(1) Use of a reagent for detecting biochemical indicators of liver function in the preparation of products for biliary atresia diagnosis or auxiliary diagnosis, the biochemical indicators of liver function include r-GT, ALT, AST, TBA, DBIL and IBIL; preferably, the product Including test reagents, test strips or strips, test chips or kits.

(2) According to the use according to the above (1), the increase of r-GT, ALT, AST, TBA, and DBIL relative to the reference level is combined with the decrease of IBIL relative to the reference level, and the diagnosis or auxiliary diagnosis is biliary atresia, wherein, The reference level represents the level of the corresponding index from non-biliary atresia subjects in the same age group; preferably, the non-biliary atresia subjects in the same age group are healthy, choledochalcyst or physiological jaundice in the same age group subject.

(3) according to the purposes described in any one of the above (1)-(2), the product also includes one or more of the following detection reagents: a reagent for detecting GGT, a reagent for detecting IgG4, a reagent for detecting TBIL, Reagents for the detection of IFN-β.

(4) According to the use described in the above (3), the increase of one or more of GGT, IgG4, TBIL, and IFN-β relative to the reference level, then further diagnosis or auxiliary diagnosis is biliary atresia, wherein the said The reference level represents the level of the corresponding index from the same age group representing non-biliary atresia; preferably, the non-biliary atresia age group subject is healthy, choledochal cyst or physiological jaundice. tester.

(5) The use according to any one of the above (1)-(4), wherein the test sample of the biliary atresia diagnosis or auxiliary diagnosis kit includes whole blood, serum, plasma, cerebrospinal fluid from the subject One or more of , tissue/tissue lysate, cell culture supernatant, semen, saliva sample, amniotic fluid, villi; preferably whole blood, serum or plasma.

(6) According to the use described in any one of the above (1)-(5), the biliary atresia diagnosis or auxiliary diagnosis product is suitable for performing one or more of the following detection methods: biological mass spectrometry, electrophoresis, chromatography method, immunofluorescence, immunochemiluminescence, immunoturbidimetry, western blot, and dot blot.

(7) According to the use according to any one of the above (1)-(6), the subject of the product is a child or an adult, preferably a child.

(8) Diagnostic or auxiliary diagnostic markers for biliary atresia, the diagnostic or auxiliary diagnostic markers include r-GT, ALT, AST, TBA, DBIL and IBIL.

(9) According to the marker described in (8) above, when the increase of r-GT, ALT, AST, TBA, DBIL relative to the reference level is combined with the decrease of IBIL relative to the reference level, the diagnosis or auxiliary diagnosis is biliary atresia, Wherein, the reference level represents the level of the corresponding index from the subjects of the same age group representing non-biliary atresia; preferably, the non-biliary atresia subjects of the same age group are healthy, choledochal cyst or physiological jaundice. subjects of the same age group.

(10) The marker according to (8) or (9) above, the diagnostic or auxiliary diagnostic marker further includes one or more of GGT, IgG4, TBIL, and IFN-β; preferably, when GGT, IgG4 The increase of one or more of TBIL, IFN-β relative to the reference level, then further diagnosis or auxiliary diagnosis is biliary atresia, wherein, the reference level represents the same age group from the representative non-biliary atresia. The level of the index; preferably, the non-biliary atresia subjects of the same age group are healthy, choledochal cyst or physiological jaundice subjects of the same age group.

(11) The marker according to any one of the above (8)-(10), wherein the marker is detected from children or adults, preferably from children.

(12) Use of a reagent for detecting any of the markers described in (8)-(11) above in preparing a product for biliary atresia diagnosis or auxiliary diagnosis, the product including a diagnostic reagent, a detection chip, and a kit.

(13) The use according to the above (12), wherein the test sample or test sample of the product includes whole blood, serum, plasma, cerebrospinal fluid, tissue or tissue lysate from the subject to be tested One or more of , semen, saliva sample, amniotic fluid, villi; preferably whole blood, serum or plasma.

(14) The use according to any one of the above (12)-(13), wherein the product is suitable for performing one or more of the following detection methods: biological mass spectrometry, electrophoresis, chromatography, Immunofluorescence, immunochemiluminescence, immunoturbidimetry, immunoblotting, and dot blot.

5. The use of IFNAR blockers in the prevention and treatment of biliary atresia and its complications

(1) Use of an IFNAR blocker in any one of (b1) to (b8);

(b1) Preparation of medicines for the treatment of diseases

(b2) preparing a product that reduces the jaundice rate;

(b3) preparing a product that reduces the level of pro-inflammatory factors;

(b4) preparing a product that increases the level of IL-17a;

(b5) preparing a product that reduces the level of inflammatory monocytes;

(b7) preparing a product that increases the proportion of Kupffer cells;

(b8) Preparation of a product that enhances the phagocytic ability of Kupffer cells

(2) The use according to (1) above, wherein the IFNAR blocking agent is an IFNAR blocking antibody.

(3) According to the use according to the above (2), the blocking antibody of IFNAR is a neutralizing antibody or a functional antibody.

(4) According to the use described in (3) above, the blocking antibody for IFNAR is a monoclonal antibody, preferably a humanized monoclonal antibody against IFNAR.

(5) The use according to the above (2), wherein the antibody is a chimeric antibody, a modified antibody, a humanized antibody, a fully human antibody, a bispecific antibody, a multispecific antibody or an antibody fragment.

(6) The use according to the above (5), wherein the antibody fragment includes Fab, F(ab')2, Fd, Fv, scFv, scFv-Fc, diabody and antibody minimum recognition unit.

(7) The use according to (5) above, wherein the humanized antibody is selected from one or more of Anifrolumab, Faralimomab, MEDI-545, Rontalizumab, Sifalimumab, AGS-009, IFNαKinoid, and NI-0101.

(8) The use according to any one of (1) to (6) above, wherein the medicament comprises a pharmaceutically acceptable carrier.

(9) The use according to any one of the above (1)-(7), wherein the medicine is a freeze-dried powder, an injection or an oral liquid injection capsule.

(10) According to the use described in (1)-(9) above, the medicament is prepared in a dosage form suitable for children or adults, preferably a dosage form for children.

(11) The use according to any one of the above (1) to (10), wherein the biliary atresia is biliary atresia, liver fibrosis caused by biliary atresia, or liver cirrhosis caused by biliary atresia.

(12) The use according to any one of (1) to (11) above, wherein the pro-inflammatory factor is IFN-γ.

(13) The use according to any one of (1) to (12) above, wherein the inflammatory monocytes are Ly6G ⁺ Ly6C ⁺ CD11b ⁺ cells.

6. Use of BAFF and its blockers in the diagnosis and treatment of the occurrence and development of biliary atresia

(1) Use of a reagent for detecting BAFF in the preparation of a disease diagnosis product, which is a biliary atresia disease diagnosis product or a biliary atresia disease diagnosis product for the degree of liver fibrosis; preferably, the product includes detection reagents, detection Test paper or strip, detection chip or kit; preferably, the reagent for detecting BAFF is a reagent for detecting the protein level or mRNA level of BAFF.

(2) According to the use described in (1) above, when BAFF increases relative to a reference level, biliary atresia is diagnosed, and the reference level represents the level of the corresponding index from subjects of the same age group representing non-biliary atresia; preferably Specifically, the subjects of the same age group without biliary atresia are healthy, physiological jaundice, choledochal cyst or liver cancer subjects of the same age group; BAFF level is positively correlated with liver fibrosis, when the subject is diagnosed with After biliary atresia, the higher the level of BAFF, the more severe the degree of liver fibrosis diagnosed as biliary atresia.

(3) According to the purposes described in any one of the above (1)-(2), the product also includes one or more combinations of the following detection reagents: a reagent for detecting GGT, a reagent for detecting IgG4, a reagent for detecting ALT Reagents for the detection of AST, reagents for the detection of TBIL, reagents for the detection of DBIL, reagents for the detection of IBIL, reagents for the detection of TBA, reagents for the detection of IFN-β.

(4) According to the use described in any one of the above (1)-(3), the test sample of the product includes whole blood, serum, plasma, cerebrospinal fluid, tissue or tissue lysate from the subject to be tested, At least one of semen, saliva sample, amniotic fluid, and villi.

(5) According to the use according to the above (4), the test sample of the product is selected from at least one of tissue or tissue lysate, whole blood, plasma, and serum; preferably, the tissue or tissue lysis The solution is liver tissue or liver tissue lysate.

(6) According to the use according to any one of the above (1)-(5), the product further includes a sample treatment reagent, and the sample treatment reagent includes at least one of a sample lysis reagent, a sample purification reagent and a protease inhibitor kind.

(7) According to the use described in any one of the above (1)-(6), the product is suitable for performing one or more of the following detection methods: indirect immunofluorescence method, particle agglutination method, antibody confirmation experimental detection method , rapid diagnostic reagent method, enzyme-linked immunosorbent assay, radioimmunoassay, immunodiffusion method, colloidal gold method, biological mass spectrometry, electrophoresis, chromatography, immunofluorescence, immunochemiluminescence, immunoturbidimetry, immunoassay Blotting, immunodot blot, flow cytometry, and immunohistochemical analysis.

(8) According to the use according to any one of the above (1)-(7), the subject of the product is a child or an adult, preferably a child.

(9) According to the use according to any one of the above (1)-(8), the product further comprises a detection reagent for detecting MMP7; preferably, the reagent for detecting MMP7 is its protein or mRNA level; preferably, when MMP7 is relatively An increase in the reference level was further diagnosed as biliary atresia.

(10) A diagnostic kit for biliary atresia or biliary atresia fibrosis, the kit includes: a detection reagent for BAFF, and a detection reagent for MMP7; preferably, the detection reagent detects protein or mRNA level; preferably , BAFF and/or MMP7 increase relative to a reference level, then biliary atresia is diagnosed, and the reference level represents the level from the corresponding index of the same age group representing non-biliary atresia; preferably, the non-biliary atresia Age group subjects were healthy, physiological jaundice, choledochal cyst or liver cancer subjects of the same age group; BAFF level was positively correlated with liver fibrosis, when the subject was diagnosed with biliary atresia, the higher the BAFF level , the more severe the degree of hepatic fibrosis was diagnosed as biliary atresia disease.

(11) The diagnostic kit according to (10), wherein the sample source for which the kit is applicable is whole blood, serum, plasma, cerebrospinal fluid, tissue or tissue lysate, semen, saliva sample, amniotic fluid, villus at least one; preferably, whole blood, serum or plasma.

(12) The diagnostic kit according to (10), comprising an anti-BAFF antibody and an anti-human MMP-7 antibody; preferably, the antibody is a monoclonal antibody.

(13) The diagnostic kit according to (10), which is suitable for performing one or more of the following detection methods: indirect immunofluorescence method, particle agglutination method, antibody confirmation experimental detection method, rapid diagnostic reagent method, enzyme-linked immunosorbent assay, radioimmunoassay, double immunodiffusion method, colloidal gold method, biological mass spectrometry, electrophoresis, chromatography, immunofluorescence, immunochemiluminescence, immunoturbidimetry, western blotting, immunoassay Dot blot, flow cytometry, and immunohistochemical analysis.

(14) The diagnostic kit according to any one of (10)-(13), the kit further comprises sample processing reagents, sample dilution reagents, positive or negative control solutions, washing solutions, stop solutions, and preservatives. one or more.

(15) The diagnostic kit according to any one of (10)-(14), wherein the subject to which the kit is applicable is a child or an adult, preferably a child.

(16) Use of a specific blocking agent for BAFF in any one of (c1) to (c7);

(c1) Preparation of medicines for preventing and treating diseases

(c2) preparing a product that reduces the rate of jaundice;

(c3) preparing a product that reduces IgG4 levels;

(c4) preparing a product that reduces liver function indexes;

(c5) preparing a product that reduces the level of pro-inflammatory factors;

(c6) preparing a product that increases the proportion of Kupffer cells;

(c7) preparing a product that improves the phagocytic ability of Kupffer cells;

(17) The use according to (16) above, wherein the specific blocking agent for BAFF blocks the binding of BAFF to at least one BAFF receptor selected from the group consisting of BCMA, TACI and BAFF-R; or, the BAFF The specific blocking agent of BAFF weakens or disables the function of BAFF.

(18) The use according to any one of (16) to (17) above, wherein the specific blocking agent for BAFF is a blocking antibody for BAFF.

(19) The use according to any one of the above (18), wherein the antibody is a neutralizing antibody or a functional antibody.

(20) The use according to (19) above, wherein the neutralizing antibody neutralizes membrane-bound B cell activating factor (mbBAFF), soluble trimeric B cell activating factor (sBAFF), and soluble 60-mer B cell activating factor At least one form of human B cell activating factor.

(21) The use according to (18) above, wherein the antibody is an anti-BAFF monoclonal antibody, preferably an anti-BAFF humanized monoclonal antibody.

(22) The use according to any one of the above (18), wherein the antibody is a chimeric antibody, a modified antibody, a humanized antibody, a fully human antibody, a bispecific antibody, a multispecific antibody or an antibody fragment.

(23) The use according to (22) above, wherein the antibody fragment comprises Fab, F(ab')2, Fd, Fv, scFv, scFv-Fc, bispecific antibody and antibody minimum recognition unit.

(24) The use according to (18) above, wherein the blocking antibody is selected from Tabalumab, Lymphostat B, anti BAFF scFv, and anti BAFF scFv Fc.

(25) According to the use according to any one of the above (16)-(24), the medicine further comprises other active ingredients; preferably, the active ingredients are selected from MHC-I blocking antibodies, MHC-II blocking antibodies At least one of a blocking antibody and an IFNAR blocking antibody.

(26) The use according to any one of the above (16)-(25), wherein the medicament further comprises a pharmaceutically acceptable carrier.

(27) The use according to any one of the above (16)-(26), wherein the medicament is a freeze-dried powder, an injection or an oral liquid injection capsule.

(28) According to the use according to any one of the above (16)-(27), the applicable object of the medicament is children or adults, preferably children.

(29) A pharmaceutical composition for preventing and/or treating biliary atresia, liver fibrosis caused by biliary atresia or liver cirrhosis caused by biliary atresia, the pharmaceutical composition comprising: the pharmaceutical composition At least one of a), b) and c) is included: further, at least two of a), b) and c) are included; further, a), b) and c) are included;

a) Agents that inhibit MHC-I and/or II signaling pathways;

b) specific blockers of IFNAR;

c) Specific blockers of BAFF.

(30) The pharmaceutical composition according to (29) above, wherein the specific blocking agent for BAFF is a blocking antibody for AFF; preferably, the agent for inhibiting MHC-I and/or II signaling pathway is MHC-I The blocking antibody and/or the blocking antibody of MHC-II; preferably, the specific blocking agent of IFNAR is the blocking antibody of IFNAR; preferably, the blocking antibody is a humanized antibody or a fully human antibody .

(31) The pharmaceutical composition according to (30) above, wherein the blocking antibody is a monoclonal antibody; or the blocking antibody is a bispecific antibody, a trispecific antibody or a multispecific antibody.

(32) The pharmaceutical composition according to (30) above, wherein the blocking antibody of BAFF is selected from Tabalumab, Lymphostat B, anti BAFF scFv, anti BAFF scFv Fc; The blocking antibody of MHC-I or the blocking antibody of MHC-II is selected from IMMU-114, Hu1D10 and Lym-1; the blocking antibody of IFNAR is selected from Anifrolumab, Faralimomab, MEDI-545, Rontalizumab, Sifalimumab, AGS -009, IFNαKinoid, NI-0101.

(33) The pharmaceutical composition according to any one of the above (29)-(32), wherein the dosage form of the pharmaceutical composition is lyophilized powder, injection or oral liquid injection capsule.

(34) The pharmaceutical composition according to any one of the above (29)-(33), wherein the applicable object of the medicine is children or adults, preferably children.

Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Unless otherwise stated, immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, etc. employed in the present invention are routine skills in the art.

In the following examples, the experimental methods without specific conditions are usually in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer. The materials, reagents, etc. used in this example, unless otherwise specified, are reagents and materials obtained from commercial sources.

The materials and methods involved in the following examples are as follows:

1. Liver Biopsy Sample Processing

Liver biopsies are obtained during laparoscopy or Kasai surgery. Samples were collected in sterile phosphate buffered saline (PBS) supplemented with 0.5% BSA and 1 mM EDTA (pH=8.0) (FACS buffer). Each sample was evaluated for histopathology, and spare tissue was used to isolate intrahepatic lymphocytes: liver tissue (0.05-0.2 g) was washed 3 times with FACS buffer to eliminate blood contamination and remove intravascular lymphocytes. Transfer the sample to a 70 μm cell strainer pre-wetted with 2 mL of FACS buffer, place it in a 50 mm petri dish, cut into < ¹ mm pieces with ophthalmic scissors, and mince gently with the plunger of a 1 mL insulin syringe. . An additional 2 mL of FACS buffer was added to wash the cell strainer. A total of 4 mL of cell suspension was filtered through a cell strainer (Falcon, round bottom tube with cell strainer cap). To remove most of the hepatocytes, cells were centrifuged at 50 x g for 2 min. The collected supernatant was further centrifuged at 1800 rpm for 5 minutes. The supernatant (liver homogenate) was stored in a freezer at -80°C. The cell pellet was resuspended in 1-2 mL of erythrocyte lysis buffer for 5 minutes at room temperature (RT). Before centrifugation, 10 mL of FACS buffer was added to stop the reaction. The cell pellet was resuspended in 10 mL of 35% Percoll and centrifuged at 2000 rpm for 30 min at 4°C. The resulting cell pellets (lymphocytes) were washed once in PBS and then resuspended in FACS buffer for phenotypic flow cytometry analysis.

2. Multiplex cytometric bead assay for the determination of immunoglobulin

Immunoglobulin concentrations (IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD and IgE) in plasma, liver homogenates and cell culture supernatants were performed by LEGENDplex ^™ CBA immunoassay according to the manufacturer's instructions (Biolegend) Determination. The liver homogenate was centrifuged at high speed (13,000 rpm, 5 min, 4° C.), and the supernatant was collected and tested for dilution. Plasma and cell culture supernatants were diluted 5-fold for immunoglobulin assays. Samples were incubated with the bead mix and secondary antibody for 2 hours. Detection antibody was added and incubated for an additional 30 minutes. Beads were washed and resuspended in wash buffer and analyzed using a FACSAria flow cytometer (BD Science). The concentrations of immunoglobulins in the liver are expressed in milligrams of protein, which were determined by the Bradford method from the corresponding liver homogenate samples using the BCA protein assay kit (Beyotime Biotechnology) according to the manufacturer's instructions.

3. Use Bio-plex Pro ^TM human cytokine standard 27-plex and 37-plex kits to determine Ctrl-other (children in other control groups, liver cancer or liver tumors, physiological jaundice), Ctrl-CC (CC control group) , concentrations of 64 cytokines, chemokines and growth factors in plasma and liver homogenate samples from children with choledochal cyst (control group) and BA group (children with BA), according to the manufacturer (Bio-Rad Laboratories) The pre-optimized protocol, 37-layer panel. The liver homogenate was centrifuged at high speed (13,000 rpm, 5 min, 4°C), and the supernatant was collected for testing. Data were collected and analyzed using a Bio-Rad Bio-Plex 200 instrument equipped with Bio-Plex Manager software version 6.0 (Bio-Rad laboratory). Protein concentrations were determined using the BCA protein assay kit (Beyotime Biotechnology) for concentration normalization.

4. ELISA detection and quantification of autoantibodies

96-well clear polystyrene microplate

Coat with 100 μL of recombinant antigen (10 μg/mL) in PBS overnight at 4°C. After washing with PBS-T (PBS+0.05% Tween20), the microplates were blocked with 10% FBS dissolved in PBS-T (200 μL) for 2 hours and washed thoroughly with PBS-T. 100 μL of serum dilution (1:100 in PBS) and liver homogenate were added to the plate for 1 hour at room temperature with agitation. After a similar wash, 100 μL of alkaline phosphatase-conjugated rabbit anti-human IgG or IgM diluted 1:1000 in PBS-T was added and incubated for 1 hour at room temperature with agitation. After 3 washes, 100 μL of p-nitrophenyl phosphate (Sigma) was developed at a concentration of 1 mg/mL in 1 mL of diethanolamine buffer (pH 9.8) for 1 hour to generate alkaline phosphatase activity. The mean optical density (OD) of each sample was measured at 450 nm on an ELISA reader (Bio-rad). Serum results are expressed in ELISA activity units, while liver homogenate results are expressed in ELISA activity units (per mg protein).

5. Immunofluorescence Staining (IF)

Paraffin-embedded sections from liver biopsies have been fixed and embedded according to the following procedure. Briefly, sections were dewaxed in xylene and placed in an oven at 70 °C for 1 h, followed by rehydration in 100%, 90%, 70% ethanol sequentially. Antigens were recovered with acid buffer (pH 6.0) in a water bath at 95°C for 20 minutes. Endogenous peroxidase was inactivated by blocking nonspecific binding sites by incubating in ₃ % H2O2 for 15 min followed by 30 min preincubation with ₁₀ % normal goat serum. Sections were incubated with primary antibodies overnight at 4°C in the dark in a humidified chamber. Sections were then washed twice with PBS for 5 min each, incubated with secondary antibodies for 1 hr at room temperature in the dark, and mounted with DAPI-bearing antifade Mounting Medium (VECTASHIELD Antifade Mounting Medium - H-1200, Vector Laboratories, CA, USA) for nuclear staining. Immunofluorescence images were acquired using a Leica TCS SP8 inverted fluorescence microscope (Leica Microsystems) using a 20×0.75 dry objective. Primary antibodies used to stain sections include anti-CD10, RAG1 and RAG2 antibodies. The fluorescein-conjugated secondary antibodies used in this study were goat anti-rabbit IgG H&L-Cy3 (1:3000), goat anti-mouse IgG1-Alexa Fluor 488 (1:500), donkey anti-mouse IgG-Alexa Fluor 488 ( 1:500). Positively stained cells were counted using ImageJ software.

6. Rhesus rotavirus (RRV)-induced biliary atresia (BA) mouse model

BALB/C mice were purchased from Guangdong Provincial Laboratory Animal Center. RRV-free adult male and female BALB/C mice were housed together. Using the Whitten effect (Dalal et al., 2001; Van Der Lee and Boot, 1955), the maximum number of pregnancies was obtained in the same week. Once the females are pregnant, they are placed in separate cages. Newborn pups were randomly injected with 50 μL of PBS or intraperitoneally infected with 50 μL of PBS containing 1.5×10 ⁶ PFU (plaque forming units) RRV. In the therapeutic experiment, 3 hours before RRV infection, mice were given B cell depleting antibody (5 μg, Clone: 18B12, mouse IgG2a, Absolute Antibody), MHC-I blocking antibody (6 μg, Clone: 28-14- 8, mouse IgG2a) and MHC-II (IA/IE) blocking antibody (6 μg, Clone: M5/114, rat IgG2b), anti-IFNAR (0.1 mg, Clone: MAR1-5A3, mouse IgG1). The same dose of each antibody was repeated every 48 hours. All protocols have been approved by the Animal Ethics Committee of Guangzhou Medical University, China. Mice were monitored daily and euthanized on day 11.

Mononuclear cells were prepared from perfused livers by lightly mincing and passing through a 40 μm cell strainer, centrifuged and resuspended in 35% Percoll, followed by centrifugation at 2000 r.p.m for 30 min at 4°C and erythrocyte lysis . Phenotypic analysis was performed as described above. Different B cell subtypes are identified by the following antibodies: CD45, CD3, B220, CD20, IgD, IgM, CD21, CD23, CD43, CD5. Stained cells were analyzed with FACSCalibur. PI dye was added to exclude dead cells prior to flow cytometry acquisition. Analyze data with FlowJo.

To detect intracellular cytokines, hepatic single cell suspensions were plated in a 96-well U-bottom plate in RPMI1640 medium containing 10% heat-inactivated FBS, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. cultured in PMA (100 ng/mL), iodomycin (1 ng/mL) and monensin (2 μM) for 4-6 hours. Cells were surface stained with anti-CD45, CD4, CD3, CD8, then fixed, permeabilized with permeabilization/fixation buffer, and intracellularly stained with anti-IFN-γ and IL-17A antibodies. Expression of IFN-γ and IL-17 was analyzed in CD4T and CD8T cells. Beads were added to count cells prior to surface staining. Zombie dye was added to exclude dead cells. Analyze data with FlowJo.

7. Histological Staining

Fresh liver tissue was fixed with 4% paraformaldehyde and embedded in paraffin, then sliced (2 μm thick) and stained with hematoxylin and eosin (H&E) for morphological evaluation and Masson’s trichrome stain for morphological evaluation. Fibrosis is detected.

8. Phagocytosis of fluorescently labeled E. coli by Kupffer cells

Single cell suspensions of liver samples were prepared and seeded into 96-well plates (2×10 ⁶ /well), then incubated with 5 μL/well of E. coli slurry for 2 hours in a cell culture incubator. After washing the wells three times with ice-cold phagocytosis assay buffer, the cell surface of human samples was stained with CD68 and VSIG4, and the surface of mouse cells was stained with CD45, CD3, CD19, CD11b, F4/80, Iy6G, Ly6C . Stained cells were immediately analyzed with FACSCalibur. PI was added to exclude dead cells prior to flow cytometry acquisition. Phagocytosis activity was expressed as the ratio of FITC+Kupffer cells.

9. Single-cell RNA sequencing and analysis

Single-cell RNA sequencing: The dead cells in the single-cell suspension were removed by PI staining and running. Single-cell 5'-end RNA sequencing library construction was performed using 10x GENOMICS' Chromium Single Cell 5'Library Kit: After mixing the single-cell suspension with RT-PCR master mix, it was mixed with isolation oil and nano-sized gel beads to obtain single-cell 5'-end RNA sequencing. The cells are transferred together with the oil droplet emulsion to the single-cell 5' chip. Within a single cell, RNA transcripts are uniquely barcoded and reverse transcribed into barcoded cDNAs. After purification, amplification, end repair, and sequencing adapters, a single-cell RNA sequencing library was constructed. Part of the single-cell RNA-sequencing library described above was isolated, and a TCR- and BCR-enriched library was generated using the Chromium Single Cell V(D)J Enrichment kit. All libraries were sequenced on the Illumina Novaseq 6000 platform.

Single-cell sequencing analysis: After splitting the 5'-single-cell sequencing data from the computer, use Cell Ranger v2.1.1 (10x GENOMICS) to compare it with the human transcriptome (GRCh38). Convert UMI matrix to Seurat object using Seurat v 3.1.2. Cells with acceptable quality (200-5000 gene counts, UMI counts below 50,000, and mitochondrial gene proportions below 12.5%) were filtered before downstream analysis. Normalization In(UMI-per-10000+1) was performed on the original gene expression of each cell. Dimensionality reduction of single-cell data, definition of cell types, single-cell pseudo-temporal developmental trajectory analysis, BCR/TCR analysis, differential gene analysis of cell subsets, etc. Reference (Cell 2019;179(5):1160-76 e24.) .

10. Flow Cytometry

(1) Obtain intraoperative liver tissue specimens.

(2) Obtaining liver mononuclear lymphocytes: First, cut the liver with tissue scissors on ice, fully cut and grind it, and then filter it into a single suspension cell under a 70um filter screen to remove the residual excess connective in the liver grinding solution. organize. Then, the liver tissue was centrifuged at 50g at 4°C for 2 minutes, and then the supernatant was collected and passed through a 40um blue filter flow tube. Then continue to 1800rpm, centrifuge at 4°C for 5 minutes, save the supernatant for subsequent experiments (detect cytokines), and use 2mL of red blood cell lysate for precipitation to fully lyse for 5min at room temperature (you can shake several times during the period). Then, the reaction was terminated by diluting to 10 mL of PBS solution, and centrifuged at 1800 rpm for 5 minutes at 4 °C, then resuspended the pelleted cells with 35% percol buffer, and centrifuged at 4 °C and 2000 rpm for 30 minutes to remove the supernatant and the centrifuged tissue. Resuspend with 1 mL of FACS (2% FBS+PBS) solution and count.

(3) Cell staining: The obtained single cell suspension was plated on a 96-well plate according to the flow cytometry panel method, with approximately 1*10 ⁶ cells per well. It was then centrifuged at 4°C, 1800 rpm. Remove the supernatant, and then stain according to the (T phenotype, B developmental phenotype, Myeliod) phenotype: add 30 μL of antibody solution (antibody dilution 1:200) to each well, and mix well with a 100 μL spray gun. Try to avoid bubble formation. Incubate for 30 minutes in a 4°C refrigerator protected from light. Then add 200 μL of FACS to each well to stop the dilution, centrifuge at 1800 rpm at 4° C. for 5 minutes, remove the supernatant, and repeat once. Finally, use 100 μL of FACS containing PI to resuspend (1:100) and wait for the machine.

Intracellular staining: First, perform cell surface membrane receptor staining, add 30 μL of antibody solution (antibody dilution 1:200) to each well, and mix thoroughly with a 100 μL spray gun to avoid the formation of air bubbles to affect staining and exclude marginal effects. Incubate for 30 minutes in a 4°C refrigerator protected from light. Then add 200 μL of FACS to each well to stop the dilution, centrifuge at 1800 rpm at 4° C. for 5 minutes, aspirate the supernatant, and repeat once. Then use the fixative solution, incubate in a refrigerator at 4 °C for 30 minutes in the dark, then centrifuge the supernatant at 1800 rpm at 4 °C, resuspend the cells with 1X permeabilization solution, and incubate at 4 °C for 15 minutes in the dark at 1800 rpm at 4 °C Centrifuge to remove the supernatant, prepare internal staining antibody solution with 1X membrane breaking solution 1:100, then add 30 μL of internal staining antibody solution to each well, protect from light, incubate at 4°C for about 1 hour, and then add 150 μL of 1X disrupting solution to each sample well. Cells were resuspended in membrane fluid, centrifuged at 1800 rpm at 4°C (repeated once), and finally cells were resuspended in 100 μL of FACS and waited for the machine to be loaded.

11. RNA extraction and RT-qPCR

RNA was extracted from cells and tissues using the phenol-chloroform method. Reverse transcription into cDNA was performed using a reverse transcription kit (Vazyme). On the ABI Prism 7700 real-time fluorescence quantitative PCR instrument, using SYBR Green Master Mix (Vazyme) and the gene-related detection primers involved in the examples of human fibrosis genes (COL1A1 and COL1A2), viral replication genes (VP6 and NSP3), etc., at 384 RT-qPCR (Usage Biosystems) was performed in triplicate in well plates.

Example 1. Defects in B cell production and autotolerance lead to expansion of autoreactive B cells in children with BA

Existing studies have reported that the hematopoiesis of the liver occurs in the human fetal liver and stops after birth (Holt and Jones, 2000). There is still a complete B cell development process.

method:

Single-cell RNA sequencing and analysis were performed on BA patients (n=6) and corresponding control liver tissues (6 patients with choledochal cyst and 6 patients with other diseases) to study the development and autoreactivity of B cells in the liver of children with BA. The phenomenon of B cell expansion. Among them, the t-SNE map was used to analyze the distribution of cell subsets in different stages of B cell development, the violin map was used to display the gene expression of each B cell subset, and the heat map was used to analyze the different development of B cells. Stage marker gene expression, and a bar graph showing the distribution of B cell subsets across samples within different groups. The abundance of precursor B cells in the liver of BA patients (n=6), CC patients (n=6), and other control patients (n=6) was obtained by single-cell analysis, and correlated with the age of the individual ( days, 0-3000 days) for correlation analysis. Using single-cell sequencing combined with BCR and other bioinformatics to analyze the number of mutated nucleotides in the Fv regions of the heavy and light chains of intrahepatic B cell subsets in BA children, CC patients, and other control patients, and to detect CSig in them , IgM, IgD expression ratio.

B cells were detected by flow cytometry and immunofluorescence experiments. Among them, a variety of B cells were isolated from liver biopsy, bone marrow and PBMC, respectively, and RNA sequence analysis was used to detect the expression of their marker genes, and the heat map and two-dimensional principal component analysis (PC) map were used to show the B cells on different samples. Condition. Further, the inventors used immunofluorescence staining to display the expression of RAG1 and RAG2 in precursor B cells in liver samples, and compared Ctrl-CC (choledochal cyst, n= 4) The number of RAG1/2+ cells per square millimeter field in the liver of BA and BA (n=16) subjects.

The concentration of TNFSF8/CD30L and its expression in T cells and NK cells were detected using the Bio-plex Pro Human Cytokine Standard 37-plex Kit.

result:

Figure 1 shows the results of B cell development and autoreactive B cell expansion in the liver of children with BA.

Among them, A in Figure 1 shows the distribution of cell subsets in different stages of B cell development on t-SNE; Figure 1 B shows hematopoietic stem cells (HSC), pre-pro-B, pro-B, pre-B and immature B cell subsets of the violin diagram of the expression of marker genes; Figure 1 C is a heat map showing the expression of marker genes at different stages of B cell development, the cells are arranged according to pseudo-chronological developmental trajectories; Figure 1 D is the display of different groups within Bar graph of the distribution of B cell subsets across samples, where blocks represent different samples and are color-coded by their derivation groups. The above single-cell transcriptome studies have found that a variety of B cells related to the entire B cell developmental stage, including hematopoietic stem cells ( HSCs) (CD34+SPINK2+SOX4+IL1B+), pre-pro-B (CD19-CD34+CD10+RAG1+RAG2+), progenitor B cells (pro-B, progenitor) (CD19+CD34loCD10+RAG1+RAG2+), pre- Somatic B cells (precursor, pre-B) (CD19+CD10+CD34-RAG1+RAG2+), immature B cells (immature, CD19+CD10loRAG1/2lo/-), transitional B cells (transitional, CD19+CD24intCD38int),

(CD19+CD27-IGHD+), CD27+classic memory (CD27+memory), CD27-non-classic memory (CD27-memory), plasmablasts (PB) (CD27+CD38+MKI67+) and plasma cells (PC) ( CD27+CD38+TNFRSF17+) (see Figure 1, panels A to D), therefore, surprisingly, the inventors found that hepatic B cell lymphogenesis remains intact after birth.

In Figure 1, E is the result of flow cytometry detection of B cell precursors in the liver and bone marrow. The inventors performed a large number of RNA sequence analysis on HSCs, and screened out the developing cells from liver biopsy, bone marrow and PBMC, respectively. B cells. Figure 1, F, is a heatmap showing marker gene expression in B cells at different stages isolated from liver biopsies, bone marrow, and peripheral blood, with data from batch RNA-seq analysis. In Figure 1, G is a two-dimensional principal component analysis (PC) diagram showing the development of B cells in different samples. It can be seen from the figure that B cells at multiple stages are expressed in bone marrow, liver, and peripheral blood. , it can also be seen that in liver and bone marrow, the transcriptional profiles of corresponding B cells at their respective developmental stages are almost identical (FIG. 1F and FIG. 1G). The above results demonstrate that B cells are generated in the liver, bone marrow and PBMC without stopping after birth.

In Figure 1, H is the immunofluorescence result of the expression of RAG1/2 and CD10 in precursor B cells in liver samples: precursor B cells [RAG1/RAG2+ (green) and CD10+ (red) double positive) exist in liver samples ], the figure shows that in the liver biopsy of BA patients, RAG1 and RAG2 are expressed in pre-B cells (shown as CD10+ B cells); in Figure 1, I is a graph showing the abundance of precursor B cells in the liver of BA patients Dot plot, BA patients were significantly higher than controls; significance was obtained by Student's t-test, ** indicates P<0.01, the results show that BA compared with Ctrl-CC (choledochal cyst, n=4) subjects (n=16) The number of RAG1/2+ cells per square millimeter field in the subject's liver was significantly increased (p<0.01).

It can be seen that both flow cytometric sorting experiments (Pro-, Pre-, immature, transitional B) and immunofluorescence experiments indicated the presence of B cell precursors (CD10 ⁺ RAG1/2 ⁺ ) in the infant liver (Fig. 1). F to I).

J in Figure 1 is a scatter plot of the abundance of precursor B cells in the liver of different children, showing that the single-cell analysis method also found that the abundance of precursor B cells in the liver is related to the age (days, days) of the individual. Inversely proportional, in the figure r represents Pearson's correlation coefficient. As can be seen, the abundance of B cell precursors gradually decreased with age, and abnormal B cell proliferation was seen in BA patients relative to other control diseases (Figure 1, J).

In Figure 1, K is the result of the number of mutated nucleotides in the Fv regions of the heavy and light chains of the intrahepatic B cell subsets of different children: heavy and light chain B cell subsets of the children with BA The number of mutated nucleotides in the Fv region of the chain was significantly less than that of the control group; in Figure 1, L is the histogram of the Ig class composition of memory B cell (CSR) subsets in different children: memory B cells in children with BA ( The CSR) subpopulation had significantly less class-converted BCR proportions, where CSIg represents class-converted Ig. From the results of the BCR study in the figure, it can be seen that the memory B cells, plasma cells and plasmablasts in the liver of BA patients have somatic hypermutation (SHM) in the variable regions of the heavy chain (IGH) and light chain (IGK) of the light chain (IGK). ) and antibody class switch recombination (CSR) were significantly lower than those in the control group (K-L in Figure 1), indicating that B cells in the liver of children with BA have the characteristics of autoantibodies.

In Figure 1, M is a graph showing the content of TNFSF8 in the liver of different children with BA: children with BA have a significantly higher concentration of TNFSF8; Mann–Whitney U test, *P<0.05, ***P<0.001 ; N in Figure 1 is the t-SNE map of TNFSF8 expression in T cells and NK cells; the color intensity of each cell is proportional to the normalized UMI count [In(UMI-per-10000+1)] (from 1 to 7). The results of N in Figure 1 suggest that the decreased CSR in the liver of BA patients may be related to the increased expression of TNFSF8 (CD30L) by T and NK cells, as TNFSF8/CD30L can inhibit CD40-mediated TNFSF8/CD30L in the absence of inflammatory cytokines.

CSR of B cells.

in conclusion:

The inventors' team found that B cell production in the liver persists after birth, at least for the first few months of life, and further found that these B cells form a functional hub, and their dysregulation leads to autoantibodies in children with BA accumulation and liver failure. The above evidence also suggests that B cell modification therapy may be an effective method to restore liver immune function in children with BA.

On the premise that Example 1 demonstrates that defects in B cell generation and self-tolerance lead to the expansion of autoreactive B cells in children with BA, in order to further study the pathogenesis of BA caused by defects in B cell development and tolerance and its impact on BA disease To study the mechanism of process effect, and to study the correlation between BA and other immune indicators that may be related to the development of B cells, the following experiments in Examples 2-7 were carried out.

Example 2. Biliary atresia with high IgM/IgG4 ratio has a good prognosis

method:

By measuring the concentrations of immunoglobulin subtypes (including IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD and IgE) in serum of children with BA (26 cases) after Kasai surgery, and then detecting all types of IgM and IgE in serum IgG4, and the ratio of IgM/IgG4 was compared to evaluate the prognosis of BA patients after Kasai surgery.

result:

Figure 2 is a graph showing the survival curve of BA children with high IgM/IgG4 ratio and low IgM/IgG4 ratio in serum after Kasai surgery; the high and low levels were classified by bioinformatics cluster analysis on 26 children.

As can be seen from Figure 2, the BA children with high IgM/IgG4 ratio (13 cases) had an jaundice-free survival probability of about 62.5%, and the BA children with low IgM/IgG4 ratio (13 cases) had an jaundice-free survival probability of about 12.5%. , there was a significant difference (P<0.05), in which the IgM/IgG4 ratio can be compared with the reference level obtained from the comparison of the poor prognosis of the BA Kasai surgery. In this experiment, the prognosis of the BA Kasai surgery The IgM/IgG4 of the poor population was mostly <0.01, while the IgM/IgG4 of the population with good prognosis after BA Kasai surgery were all >0.01, and the IgM/IgG4 ranged from 0.012 to 0.076). According to the results in the figure, it can be reasonably concluded that children with BA with a high IgM/IgG4 ratio have a higher jaundice-free survival rate after Kasai surgery, that is, a better prognosis.

in conclusion:

Subgroups of BA patients with high IgM/IgG4 ratios in serum (or similarly select plasma, whole blood) have significantly longer jaundice-free survival times after Kasai surgery, so reagents for the detection of IgM and IgG4 ratios (IgM/IgG4) can be used to prepare In products for prognostic analysis of biliary atresia (e.g. test reagents, test strips or strips, test chips or kits), when the expression level of IgM/IgG4 is increased relative to a reference population (e.g. healthy controls, people with poor BA prognosis) , the diagnosis or auxiliary diagnosis of biliary atresia has a good prognosis (eg, longer jaundice-free survival, and correspondingly, also reduces BA or BA hepatobiliary injury brings other problems, which can increase weight, reduce liver fibrosis or liver cirrhosis degree, etc.).

Example 3. Increased Ro/SSA-specific IgG is an indication of biliary atresia

method:

ELISA was used to detect and quantify IgM (DsDNA-specific IgM, Nucleosome-specific IgM, RNP-specific IgM, Ro/SSA-specific IgM), Ro/SSA-specific IgM in serum and liver of BA children, CC control group, and other control groups. /SSA-specific autoreactive IgG concentration and comparative analysis.

Result description:

Figure 3 is a graph showing the concentrations of IgM and IgG in serum and liver of children with BA, CC control group and other control groups. As can be seen from Figure 3, the concentrations of various IgMs (including DsDNA-specific IgM, Nucleosome-specific IgM, RNP-specific IgM, and Ro/SSA-specific IgM) in children with BA were significantly lower in serum and liver. There was a significant difference between the CC control group and other control groups (P<0.05 or 0.01 or 0.001); at the same time, the concentrations of Ro/SSA-specific autoreactive IgG in serum and liver in children with BA were significantly higher than The CC control group and other control groups showed significant difference (P<0.05 or 0.01 or 0.001). It can be seen that the IgM antibodies of the children with BA are significantly decreased, and the Ro/SSA-specific autoreactive IgG antibodies are significantly increased. At the same time, the Ro/SSA-specific IgG is the recipient of the predictive performance of distinguishing BA from non-BA (CC) subjects. The operating characteristic (ROC) plot is shown in Figure 4: Ro/SSA-specific autoreactive IgG was able to differentiate BA subjects from CC cases very effectively (AUC = 0.767), using Ro/SSA-specific autoreactivity IgG is one of the diagnostic markers for BA.

in conclusion:

The autoantibody assay found that BA patients had significantly lower levels of natural IgM antibodies and significantly higher levels of Ro/SSA-specific IgG autoantibodies. Therefore, Ro/SSA-specific IgG autoantibodies can be used as a marker for BA disease diagnosis or auxiliary diagnosis. One of the substances, and the reagents for detecting Ro/SSA-specific IgG autoantibodies (or further adding reagents for detecting IgM on this basis, especially the reagents for natural IgM listed in the examples) can be used to prepare biliary atresia diagnosis or auxiliary diagnosis In products, such as detection chips, test strips or strips, test kits, etc.

Example 4. MHC-I and/or II blocking antibodies significantly alleviate the disease phenotype of RRV-induced BA model mice

method:

Examples 2 and 3 have proved that a variety of antibodies can be used as effective BA diagnostic markers, so whether there are some specific antibodies that can be used for the prevention and treatment of BA diseases, the inventor team used RRV to induce BALB/c suckling mice to construct BA The disease model was tested using MHC-I/II blocking antibody (anti-MHC I/II). The group settings and the number of experimental animals were: control group mice (n=34), RRV group mice ( n=29), RRV-anti-MHC I mice (n=6), and RRV-anti-MHC II mice (n=22), and the dosing regimen was prophylactic and therapeutic (3 hours before RRV infection and Repeat dosing every 48 hours).

Flow cytometry was used to detect the abundance of IFN-γ+CD4+ T cells and IFN-γ+CD8+ T cells in the liver of mice in each animal model group to further study the effect of MHC-I/II blocking antibody on BA. preventive and therapeutic effects.

Flow cytometry was used to detect the presence of B1-a, B cell precursors and mature B cells in the liver of mice in the control group and RRV group at 11 days; Ratio of intrahepatic B1-a, B cell precursors, mature B cells in I mice and RRV-anti-MHC II mice.

RT-qPCR was used to detect the relative expression levels of RRV virus genes in control mice, RRV mice, RRV-anti-MHC I mice and RRV-anti-MHC II mice.

The concentrations of Cx3cr1 and C1q in the liver of the mice in the control group, the mice in the RRV group, the RRV-anti-MHC I mice and the RRV-anti-MHC II mice were detected by ELISA.

Flow cytometry was used to detect the phagocytosis of FITC fluorescently labeled Escherichia coli (E. The ratio of Kupffer cells in different groups of mice and their respective phagocytic abilities were compared. The method is as follows: Liver immune cells (2*10 ⁶ /well) were mixed with FITC fluorescently labeled E. coli (abcam, ) in The round-bottomed 96-well plate was co-incubated for 2 hours in a 37°5% _CO2 incubator, washed twice with the wash buffer in the kit, and stained with APC-VSIG4, BV785-CD68 flow antibody (1:100) on ice After 40 minutes, Kupffer (CD68+VSIG4+) cells were finally distinguished by flow cytometry, and the phagocytosis of E. coli by Kupffer cells was detected.

Staining analysis: HE staining was used to analyze the liver sections of mice in the control group, RRV group and anti-MHC-I/II group; Liver sections were analyzed by Masson's trichrome staining.

result:

A in Figure 5 is a graph of the weight gain of the control group mice, the RRV group mice, the RRV-anti-MHC I mice and the RRV-anti-MHC II mice. As can be seen from A in Figure 5, on the 11th day, the body weight of the mice in the RRV group increased by about 230%, and continued to decrease, which was significantly different from that of the control mice, RRV-anti-MHC I mice, and RRV-anti-MHC mice. II mice showed significant differences (P<0.001, P<0.05, P<0.001); the weight gain of RRV-anti-MHC I mice and RRV-anti-MHC II mice was about 400%, and it has been increasing There was no significant difference with the control group mice (P>0.05). It can be seen from A in Figure 5 that anti-MHC I/II can effectively block the antigen presentation mediated by MHC I/II, which can significantly increase the body weight of RRV mice and achieve the effect of preventing and treating BA.

B in Figure 5 is a graph of the jaundice-free rate of mice in the control group, mice in the RRV group, RRV-anti-MHC I mice and RRV-anti-MHC II mice. It can be seen from B in Figure 5 that in the RRV mouse group, all the mice developed jaundice on the 8th day; on the 11th day, the jaundice rate of the RRV-anti-MHC II mice was about 85%, while that of the control mice, RRV-anti-MHC II mice was about 85%. The anti-MHC I mice were all without jaundice, and the three groups were significantly different from the RRV group mice (P<0.001). That is, after anti-MHC I/II administration, the anicterus rate of RRV mice was significantly improved, and anti-MHC I/II showed significant BA preventive and therapeutic effects.

C in Figure 5 is the IFN-γ ⁺ CD4 ⁺ T cells, IFN-γ ⁺ CD8 ⁺ T cells in the liver of the control mice, the RRV group mice, the RRV-anti-MHC I mice and the RRV-anti-MHC II mice. Cell abundance map: The concentration of IFN-γ ⁺ CD4 ⁺ T cells in RRV group mice was significantly higher than that in control mice, indicating that BA mice had increased IFN-γ ⁺ CD4 ⁺ T cell concentrations; RRV-anti-MHC II The proportion of IFN-γ ⁺ CD4 ⁺ T cells in the mice and the blank control was the same, which was significantly different from that in the RRV group (P<0.001); there was no significant difference between the RRV-anti-MHC I mice and the RRV mice , the concentration is higher than the other two groups, indicating that anti-MHC II treatment can prevent and treat BA by significantly reducing the concentration of IFN-γ ⁺ CD4 ⁺ T cells in BA mice; IFN-γ in the liver of mice in the RRV group ⁺ CD8 ⁺ T cells were significantly higher than that in control mice (P<0.01), indicating that the concentration of IFN-γ ⁺ CD8 ⁺ T cells in BA mice was increased; RRV-anti-MHC I/II mice and control mice The intrahepatic concentration of IFN-γ ⁺ CD8 ⁺ T cells was comparable (P>0.05), and significantly lower than that in RRV mice (P<0.05, P<0.001), indicating that anti-MHC I/II can significantly reduce BA mice by reducing the The concentration of IFN-γ ⁺ CD8 ⁺ T cells in BA was achieved to prevent and treat BA.

D in Figure 5 is the result of flow cytometry detection of B1-a, B cell precursors and mature B cells in the liver of mice in the control group and the RRV group at 11 days, which is similar to the results of human infants and young children observed in Example 1. results are consistent.

In Figure 5, E is the ratio of B1-a, B cell precursors, and mature B cells in the liver of control mice, RRV mice, RRV-anti-MHC I mice and RRV-anti-MHC II mice. It can be seen from E in Figure 5: there is no significant difference in the concentration of B1-a cells between the RRV group and the control group (P>0.05), RRV-anti-MHC I mice, RRV-anti-MHC II mice Compared with the mice in the RRV group, the concentration of B1-a cells was significantly decreased (P<0.05), indicating that anti-MHCI/II can reduce the concentration of B1-a cells; the B cell precursors of the mice in the RRV group were compared with those in the control group. The concentration was significantly increased (P<0.001), and the concentration of B cell precursor cells in RRV-anti-MHC I mice and RRV-anti-MHC II mice was significantly lower than that in RRV group mice (P<0.001), and the There was no significant difference in the control group (P>0.05), indicating that anti-MHC I/II can reduce the increase in the concentration of B cell precursors caused by BA disease; the concentration of mature B cells in the RRV group was significantly lower than that in the control group ( P<0.001), there was no significant difference between RRV-anti-MHC I mice and RRV group mice (P>0.05), and the concentration of mature B cells in RRV-anti-MHC II mice was significantly lower than that in RRV group mice (P>0.05). P<0.001), there was no significant difference with the control mice, indicating that anti-MHC II can reduce the increase in the concentration of mature B cells caused by BA disease.

A in Figure 6 is a graph of the relative expression levels of RRV virus genes in the control group mice, the RRV group mice, the RRV-anti-MHC I mice and the RRV-anti-MHC II mice. NSP3 and VP6 are non-structural proteins and structural proteins of RRV virus. It can be seen from the figure that the mice in the control group do not express NSP3 and VP6, while the mice in the RRV group highly express NSP3 and VP6. It can significantly reduce the expression of NSP3 and VP6, that is, anti-MHC I/II administration not only does not destroy the virus clearance function of mice after RRV infection, but can significantly inhibit the proliferation of RRV virus.

B in Figure 6 is a graph of the intrahepatic Cx3cr1 and C1q concentrations of the control mice, the RRV group mice, the RRV-anti-MHC I mice and the RRV-anti-MHC II mice. Cx3cl1 and C1q can inhibit the activation of autoreactive CD8 ⁺ T cells. It can be seen from the figure that the concentrations of Cx3cl1 and C1q in RRV mice were significantly lower than those in control mice (P<0.05, P<0.01). -MHC I/II mice had significantly higher concentrations of Cx3cl1 and C1q compared to RRV mice (P<0.01, P<0.001). It shows that the concentration of Cx3cl1 and C1q in BA mice is reduced, and the activation of autoreactive CD8 ⁺ T cells is increased, while anti-MHC I/II can increase the concentration of Cx3cl1 and C1q, inhibit the activation of autoreactive CD8 ⁺ T cells, so as to achieve prevention and the purpose of treating BA.

In Figure 6, C and D are the results of Kupffer cells in the liver of control mice, RRV group mice, RRV-anti-MHC I mice and RRV-anti-MHC II mice. Among them, C in Figure 6 is different The results of phagocytosis of fluorescently labeled Escherichia coli by Kupffer cells in the liver of mice in each group. It can be seen that the ability of Kupffer cells in the RRV group to phagocytose Escherichia coli pathogens was significantly lower than that of the control group, while the anti-MHC I/II group had a significantly lower ability to phagocytose Escherichia coli pathogens. The ability of murine Kupffer cells to phagocytose Escherichia coli pathogens was close to or even slightly higher than that of the control group, so the ability of Kupffer cells to phagocytose Escherichia coli pathogens was significantly recovered after anti-MHC I/II administration; D in Figure 6 is the liver of different groups of mice The phagocytic ability and proportion of Kupffer cells. It can be seen from the figure that the proportion and phagocytic ability of Kupffer cells in the RRV group were significantly lower than those in the control group, while the phagocytic ability and proportion of Kupffer cells in the anti-MHC I/II group It was close to the control group (no significant difference, p>0.05), so the proportion and phagocytic ability of Kupffer cells were significantly restored after anti-MHC I/II administration.

In Figure 6, E is the HE staining image of the liver sections of mice in the control group, the RRV group and the anti-MHC-I/II group. As can be seen from the figure, compared with the RRV mice, the anti-MHC-I/II group mice had less lymphocyte infiltration around the hepatic hilum area, and the lobular structure was more complete, indicating that anti-MHC-I/II can reduce the liver Inflammation, has significant preventive and therapeutic effects on BA.

In Figure 6, F is the Masson's trichrome staining of liver sections of mice in the control group, the RRV group and the anti-MHC-I/II group. It can be seen from the figure that the degree of liver fibrosis in the anti-MHC-I/II group mice was lower than that in the RRV mice, indicating that anti-MHC-I/II treatment can reduce the degree of liver fibrosis and has a significant effect on BA. Preventive and therapeutic effects.

in conclusion:

The above experimental results show that anti-MHC-I/II can significantly block MHC class I or MHC class II-mediated antigen presentation, significantly improve body weight loss caused by BA disease, increase the rate of no jaundice, and reduce the inflammatory factor IFN- The level of γ effectively inhibits the proliferation of RRV virus and does not destroy the virus clearance function of mice after RRV infection, inhibits the activation of autoreactive CD8 ⁺ T cells, increases the number of Kupffer cells and phagocytic function, and reduces lymphocyte infiltration. Decreased liver fibrosis. In conclusion, the liver injury of RRV-induced BA mouse model in neonatal BALB/c mice can be ameliorated by anti-MHC-I/II, and anti-MHC therapy can significantly prevent and treat biliary atresia diseases. Therefore, agents for inhibiting MHC-I and/or II signaling pathways (eg, MHC-I/II blocking antibodies, their bispecific antibodies, monoclonal antibodies, etc.) can be used to prepare prophylactic and/or therapeutic biliary tract in blocked drugs.

Example 5. r-GT (r-glutamyl transferase), ALT (alanine aminotransferase), AST (aspartate aminotransferase), TBA (total bile acids), DBIL (direct bilirubin) An increase in bilirubin) combined with a decrease in IBIL (indirect bilirubin) is an indication of biliary atresia

It is known that biliary atresia is difficult to distinguish from other diseases such as physiological jaundice and choledochal cyst. This example continues to explore whether there are biochemical indicators to distinguish them.

method:

Blood and liver biopsies from healthy controls (n=2471), subjects with BA (n=927), choledochal cyst (CC) (n=248) or physiologic jaundice (n=9297) were collected via electronic medical records Cohort characteristics of immune cells, especially clinical biochemical indicators of liver function and relative values, application of LASSO regression models to identify specific clinical indicators between BA patients and non-BA control subjects: only 12,943 electronic medical record reports recorded more than 20 % of the index is included in the machine learning process, from which 10% of the subjects subset is randomly selected as the dataset for the training process, and the remaining 90% of the subjects are used to adopt the ROC curve (receiver operating characteristic, receiver operating characteristic). ), to verify the predictive performance of BA-specific clinical indicators according to the AUC (area under the curve) to explore whether the selected indicators can truly distinguish children with BA from CC, physiological jaundice, and non-disease control subjects.

result:

Through regression models, 58 significant coefficients (clinical indicators) were identified as group parameters, using heat maps to show healthy controls (n=2471) or those with BA (n=927), choledochal cyst (CC) (n=248 ) or physiological jaundice (n=9297) in the relative values of clinical indicators in these 58 coefficients, the results are shown in Figure 7, it can be clearly seen from the figure that the r-glutamyl Transferase (r-GT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), persistently elevated total bile acid (TBA), and direct bilirubin (DBIL) levels were significantly elevated In contrast, patients with CC or physiologic jaundice showed comparable long-term outcomes and long-term outcomes to healthy control subjects. In addition, it can be seen from Fig. 7 that the change trend of the index IBIL (indirect bilirubin) is inconsistent with the aforementioned index. Therefore, further, we need to find out whether it can be used as a joint detection index. Therefore, Fig. 8 shows the TBA, DBIL, The scatter plot of IBIL concentration showed that compared with non-BA children, in BA children, especially in the early stage, the concentrations of TBA and DBIL were significantly increased (consistent with the results in Figure 7), while the concentration of IBIL was significantly decreased (p < 0.05). The above results predict that the combined value of decreased IBIL with increased r-GT, ALT, AST, TBA, and DBIL may provide an additional biomarker combination for the detection of BA patients.

The above prediction results were further verified by the ROC curve, and the results were displayed by the ROC curve. As shown in Figure 9, the combined values of decreased IBIL and increased r-GT, ALT, AST, TBA, and DBIL were very effective in distinguishing BA subjects from healthy controls (AUC=0.978), physiologic jaundice (AUC=0.967) ) or CC cases (AUC=0.806). Therefore, an increase in r-GT, ALT, AST, TBA, and DBIL combined with a decrease in IBIL is an effective indication for biliary atresia.

in conclusion:

This example found that DBIL, TBA, liver enzymes (r-GT, ALT and AST) continued to rise in BA patients, while IBIL showed a continuous downward trend from the earliest onset. ROC curves validated the combined values of decreased IBIL and increased r-GT, ALT, AST, TBA, and DBIL, which were very effective in distinguishing BA subjects from healthy controls, physiologic jaundice, or CC cases. Therefore, the increase of r-GT, ALT, AST, TBA, and DBIL combined with the decrease of IBIL is an effective indication for biliary atresia, and can be used as a combined biomarker in the diagnosis of biliary atresia, especially in the early diagnosis. The detection of children with biliary atresia greatly reduces the prognostic risk of biliary atresia disease.

Example 6. IFNAR blocker can significantly alleviate the disease phenotype of RRV-induced BA model mice

IFNAR is a receptor that binds type I interferons (interferon-alpha/beta). This example further studies the correlation of type I interferon, its receptors and receptor blockers in BA disease, so as to investigate whether it can play a role in the diagnosis, prevention and treatment of BA.

method:

RRV induced BALB/c suckling mice to build a BA disease model (in the experiment, the control group, the RRV-BA mouse group, and the Anti-IFNAR group were set), the level of IFN-β in the liver tissue of the mice was detected, and the IFNAR blocking antibody ( Anti-IFNAR) test, the body weight, jaundice rate changes, IFN-γ (in liver and/or spleen) and IL-17a abundance changes in CD4T and CD8T cells before and after activation, inflammatory The number of monocytes, Kupffer cells and phagocytosis.

IFN-β in human plasma was detected by multiple cytokines to further verify the correctness of the mouse experiment. Among them, HC group, BA disease control group (control, choledochal cyst or physiological jaundice) and BA group were set respectively.

result:

Figures 10 to 15 are schematic diagrams showing that Anti-IFNAR can effectively alleviate the disease phenotype of RRV-BA model mice in this example, wherein,

Figure 10 shows that, compared with the control group (n=10), in the liver tissue of RRV virus-induced BA model mice (n=10), the level of IFN-β was significantly increased, indicating the activation of type I interferon pathway.

As can be seen in Figure 11, the control mice (n=3) had no jaundice and had a normal body size; the RRV-BA mice (n=3) had obvious jaundice and were smaller in size by comparison; while Anti-IFNAR treated RRV- After BA mice (n=6), most of the mice were similar to the control group, and no jaundice occurred, and the incidence of jaundice in the mice was also reduced to 33.3%. Therefore, Anti-IFNAR can significantly increase the body weight and reduce the incidence of jaundice in BA mice, and the survival of the mice is significantly improved.

Further analysis of the mechanism of inhibiting the activation of type I IFN to improve BA found that compared with the RRV-induced BA model mouse group, after blocking IFNAR with anti-IFNAR, the CD4 ⁺ T cells and CD8 ⁺ T cells in the liver and spleen were significantly reduced. (p<0.05), at the same time, the proportion of activated CD4 ⁺ T cells and CD8 ⁺ T cells in the spleen was also significantly decreased (p<0.05), the results are shown in Figure 12 . It was shown that the activation of type I IFN was involved in the activation of T cells in BA mice, and Anti-IFNAR treatment inhibited the activation of CD4 ⁺ T cells and CD8 ⁺ T cells in the liver and/or spleen.

Figure 13 shows that, compared with RRV-induced BA model mice, after blocking IFNAR by means of anti-IFNAR, it was also found that IFN-γ was significantly reduced in anicteric mice (p<0.05). Anti-IFNAR treated RRV In -BA mice, the abundance of IFN-γ ⁺ CD4T and IFN-γ ⁺ CD8 T cells was significantly decreased, whereas the abundance of IL-17a ⁺ CD4 T cells was significantly increased in the liver of anicteric mice (p<0.05). The above shows that IFNAR blocker can reduce the expression of inflammatory cytokines expressed by T cells in BA disease and also the expression of IFN-γ, an activating factor of BA liver fibrosis, and make the expression of IL-17a close to that of the control group, which plays a certain role. The role of prevention and treatment of BA disease.

Fig. 14 shows that in RRV mice without jaundice that use anti-IFNAR to block IFNAR, inflammatory monocytes decreased, Kupffer cells with pathogen clearance function increased, and the phagocytosis of Kupffer itself was also enhanced. These results suggest that the increase of inflammatory monocytes, the decrease of Kupffer cells with pathogen scavenging function, and the weakened phagocytic function are the pathogenesis of type I IFN activation and participation in RRV-induced BA disease, and anti-IFNAR can effectively prevent the above mechanism. and treatment BA.

Figure 15 shows that the level of IFN-β in the plasma of BA patients was significantly higher than that of the healthy peers and the BA disease control group (p<0.0001); this result is consistent with the aforementioned mouse experiments, and also shows that there is type I type in BA disease Abnormal activation of the interferon pathway.

in conclusion:

There is activation of type I interferon pathway in RRV virus-induced mice, which is manifested as decreased body weight, 100% jaundice rate, significantly increased levels of IFN-β and IFN-γ in liver tissue, and IL-17a levels. Significantly decreased, increased CD4 ⁺ and CD8 ⁺ T cells before and after activation of liver and spleen, increased inflammatory monocytes, decreased Kupffer cells and decreased phagocytic capacity, and was also seen in human BA patients in serum/plasma Elevated levels of IFN-β. Using an antibody that blocks IFNAR, mice were found to have a significantly lower incidence of jaundice, a significant increase in body weight, a significant reduction in IFN-β and IFN-γ levels, a significant increase in IL-17a levels, and CD4 and CD8 T cells before and after liver activation. Decreased proportions, decreased inflammatory monocytes, increased pathogen-clearing Kupffer cells, and enhanced phagocytosis of Kupffer itself. Therefore, BA disease is accompanied by activation of type I IFN, IFNAR blockers (such as blocking antibodies) , neutralizing antibodies) are effective BA preventive and therapeutic drugs.

Example 7. The elevation of BAFF in liver/blood is positively correlated with BA and its liver fibrosis process. BAFF blockers can significantly alleviate BA and its liver fibrosis process in in vitro and in vivo experiments

BAFF (B cell activating factor) is one of many B cell activators, and is a ligand of receptors TNFRSF13B/TACI, TNFRSF17/BCMA and TNFRSF13C/BAFFR. This example further studies the role of BAFF in BA disease and its liver fibrosis process. In order to study whether it can play a role in the diagnosis, prevention and treatment of BA (including delaying the progress of liver fibrosis and cirrhosis of BA).

method:

1. Human specimen experiment:

Collect and prepare liver and blood (including whole blood, serum, and plasma) specimens from BA patients and control patients. The steps for collecting clinical specimens and data include:

The medical histories of children with biliary atresia and other control groups, especially laboratory test indicators related to liver injury, were obtained from the hospital's electronic medical record system. Ethics Statement: This project has been approved by the Medical Ethics Committee of Guangzhou Women and Children's Medical Center. Experiments were performed in accordance with the "International Ethical Guidelines for Research Involving Humans" as stated in the Declaration of Helsinki. Informed consent for project research will be obtained from the patient or the patient's legal guardian. Inclusion criteria for subjects: Subjects for this study were recruited from children undergoing hepatobiliary surgery in the Department of Hepatobiliary Surgery, Guangzhou Women's and Children's Medical Center.

Control children and BA children were judged according to the following criteria:

(1) Control: Children aged 0-4 months with hepatobiliary disease (choledochal cyst, liver cancer or liver tumor, physiological jaundice) requiring surgical treatment.

(2) Biliary atresia: Children with obstructive jaundice aged 0-4 months should be diagnosed with BA by intraoperative cholangiography and/or liver biopsy.

(3) Exclusion criteria: (1) Children with systemic inflammatory response syndrome or multisystem malformations; (2) Children with unclear primary disease diagnosis; (3) Children whose parents refuse to participate in the study or who cannot obtain parental authorization Son.

For the collected liver and blood samples, ELISA method was used to detect the concentration of BAFF in the liver and blood samples. Use enzyme-linked immunosorbent assay kit (Bio-rad company) to detect the concentration of BAFF, the specific operation is carried out according to the instructions; detect the concentration of γ-glutamyltransferase (GGT) (tested by the hospital laboratory); CBA produced by Biolegend company (cytometric bead array) immunoglobulin kit to detect the concentration of immunoglobulin (IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgE, IgD); analyze the correlation between BAFF concentration and the above indicators; RT-qPCR method detection Gene expression levels of BAFF receptors in B cells.

The obtained livers were stained with HE and masson, and the liver lymphocyte infiltration, inflammatory cells and fibrosis were detected and scored according to the Ishak score. The Ishak scoring standard of liver pathological sections is shown in Table 1.

Table 1. Liver Ishak scoring criteria

Clustering and dimensionality reduction analysis was performed on the collected multidimensional data using the nonlinear dimensionality reduction algorithm and graphing method of t-SNE (t-distributed stochastic domain embedding); linear correlation analysis and other analysis methods were used to study the intrahepatic hepatic function of children with BA. Concentration levels of BAFF and their relationship with Ro/SSA-IgG autoantibody levels.

Gene expression, cell number and distribution of BAFF sources, CD14 ⁺ cells/CD68 ⁺ cells/CD20 ⁺ B cells/IgG4 ⁺ cells and SMA ⁺ fibroblasts were detected by immunofluorescence. Among them, the steps of immunofluorescence to detect the expression of IgG4 in the liver of BA and the location of IgG4 ⁺ cells in the liver include: taking the paraffin sections of the liver tissues of the control and BA patients, placing them in xylene to deparaffinize, adding water, and heat-recovering the exposed antigens, PBS Wash for 3 minutes, 3 times, add 1% normal goat serum to block for 1 hour at room temperature, suck off the blocking solution, directly add primary antibody dropwise, keep moist at 4°C overnight. Wash twice with pre-cooled PBS and incubate with labeled secondary antibody for 1 hour at room temperature, protected from light. Washed twice with PBS. Take pictures under a fluorescence microscope or a laser confocal microscope.

2. In vitro cell experiments:

The naive B cells were activated with BAFF in vitro, and the expression of IgG4 in the culture supernatant was detected by ELISA. Among them, the experimental steps of activating B cells in vitro include: separating peripheral blood mononuclear cells (PBMC) from peripheral blood, sorting CD45+CD19+IgD+CD27- by flow sorting machine, and using 100,000 cells per well Seed in a 96-well plate, CpG ⁺ IL-4 ⁺ CD40L activated B cells, with or without BAFF, cultured for at least 7 days, and the supernatant was collected to detect the expression of IgG4.

Experimental study on the increased expression of fibrosis-promoting genes with human recombinant immunoglobulin IgG4 in vitro: In vitro stimulation of human stellate cell line LX-2 with human recombinant IgG4, detection of fibrosis-related gene expression levels, and analysis of IgG4-related fibrosis The relationship between genes, the specific process includes: in vitro culture of hepatic stellate cell line HSC, 37°, 5% CO ₂ , induction of differentiation (+TGF-β), and stimulation with IgG4 for three days at the same time, collecting cells, extracting RNA, and RT-qPCR was performed to detect the expression of fibrosis-related genes SMA, COL1A1, COL1A2, COL3A1, and ACTA1;

Since abnormal autoimmunity induced by perinatal pathogen (eg, viral) infection is a possible pathogenesis of BA, pathogens including bacterial and viral infection cause activation of the body's type I interferon pathway, and LPS levels are elevated. In vitro experiments to study whether virus infection promotes BAFF-mediated liver injury: LPS from pathogens and inflammatory factor IFN-β were used to stimulate the cell line THP1 that had differentiated into macrophages, and the expression of BAFF was observed.

Further, B cells and monocytes in peripheral blood were sorted, a B cell-monocyte co-culture system was established, and IFN-β stimulation or simultaneous administration of BAFF neutralizing antibody (Anti-BAFF) was given to block the effect of BAFF. , respectively, collect the supernatant and detect the expression of IgG4, observe the relationship between IFN-β and IgG4 expression, and the effect of adding BAFF blocking agent.

3. In vivo animal experiments:

Establishing an animal model of biliary atresia: (1) Animals: adult BALB/c pregnant mice, specific pathogen-free grade (SPF grade), purchased from Guangdong Provincial Medical Laboratory Animal Center. The animals were reared in the SPF environment of the Laboratory Animal Center of Guangzhou Medical University. After the pregnant mice gave birth to newborn mice (an average of 8 newborn mice per pregnant mouse) with an average weight of 1.5 g, the newborn mice were randomly selected according to the experimental group for the experiment. This experimental animal disposal method conforms to animal ethics standards. (2) Modeling method: Neonatal BALB/c mice were intraperitoneally injected with monkey MMU18006 rotavirus 20 μL (titer 1.0×10 ⁶ PFU) within 24 hours of birth to establish BA mouse model. (3) Observe the survival status of mice: including mouse survival rate, growth weight, skin jaundice and changes in liver function.

Effect experiment of Anti-BAFF: On the established mouse model of acute biliary atresia, paired experiments were performed according to the experimental requirements, and the BA model was divided into different experimental groups: 1) control mouse group (intraperitoneal administration of PBS); 2) BA mouse model group induced by RRV; 3) BA mouse + anti-BAFF group (wherein, the anti-mouse BAFF antibody is rabbit IgG, mAb (Sandy-2) (preservative free) Prod.No.AG-20B-0063PF- C100, AdipoGen LIFE SCIENCES), dose of 2μg/g, intraperitoneal injection, administered on the 1st, 3rd, 6th, and 9th days after virus infection, and the experiment was terminated on the 12th day; 4) BA mice + IgG (rabbit source, clone 18B12, purchased from abcam) isotype control group (administration method is the same as BA mice + anti-BAFF group).

In the above experiments, synchronously before and after the experiment and during the process, the hepatobiliary appearance, jaundice characteristics, body weight and survival rate of the mice in the above groups were observed; necrotic cells and lymphocyte infiltration in the liver were also observed (by HE staining), etc. Changes in immune cell function; flow cytometry to detect immune cell subtypes in the liver and spleen; ELISA to detect the expression level of BAFF; CBA to detect the expression of TNF-α, IFN-β, IFN-γ and other cytokines; hospital laboratory testing Liver function indicators ALT, AST, DBIL, TBA levels; use immunofluorescence and flow cytometry to detect the expression of CD4 ⁺ T cells, CD8 ⁺ T cells, B220 ⁺ cells, CD138 ⁺ cells in liver and/or spleen tissue , Detect the number and function of Kupffer cells, and the expression of IgG1 (the function of mouse IgG1 is similar to that of human IgG4).

result:

1. Experimental results of human specimens:

Figure 16 shows the correlation between the levels of BAFF, the concentration of BAFF in the liver and serum, the correlation between the concentration of BAFF in serum and the concentration of γ-glutamyltransferase (GGT), the level of BAFF in the liver and the serum and the concentration of BAFF in the liver and serum of different children. Result plot of the correlation between lymphocyte infiltration and the degree of fibrosis. Among them, 50 cases of BA children and 25 cases of disease control group (choledochal cyst, CC) were selected for the test. The results are shown in A in Figure 16. Detected by ELISA, the level of BAFF in the serum and liver of BA was both significantly higher than the CC control group (p<0.0001), and the concentration of BAFF in the liver was positively correlated with the concentration of BAFF in the serum (r=0.4082, p=0.0003) (Fig. 16, B); in addition, in other non-BA liver disease controls (including liver cancer, cholestasis, physiologic jaundice, n=18), the level of BAFF in the liver was also close to that of the CC control group (n=35) and was significantly different (p<0.0001) from the BA patient group (n=53) ) (C in Figure 16). Meanwhile, the Receiver Operating Characteristic (ROC) plot of the predictive performance of BAFF in distinguishing BA from non-BA (CC) subjects is shown in Figure 16, Panel D: BAFF is able to distinguish BA subjects from CC cases very effectively ( AUC=0.923). Therefore, BAFF can be one of the indicators or markers for the early diagnosis of BA disease.

Further, after correlation analysis with various liver function indexes of clinical detection, it was found that BAFF in serum was positively correlated with the concentration of γ-glutamyltransferase (GGT) in serum, r=0.4485, p=0.0023 , see E in Figure 16. GGT is the most sensitive liver function indicator to detect whether there is liver disease caused by bile duct damage, but the specificity is not high. The level of GGT is still elevated in other causes such as tumors or viral hepatitis. The combination of GGT improves the specificity of GGT and can become one of the indicators or markers for the early diagnosis of BA disease.

In addition, when the obtained liver specimens were stained and the liver lymphocyte infiltration and fibrosis degree were scored according to the Ishak score, it was found that the level of BAFF in the liver was positively correlated with the lymphocyte infiltration and the degree of fibrosis in the liver, as shown in Figure 16. F. Therefore, BAFF can not only be used as an index or marker for the diagnosis of biliary atresia, but also provide a diagnostic basis for the diagnosis of hepatic fibrosis in biliary atresia. That is, when the level of BAFF is higher, it can predict liver fibrosis in biliary atresia The more serious the degree of transformation.

At the same time, the study found that BAFF gene is mainly expressed in myeloid cell subsets, as shown in Figure 17 A and B, where A in Figure 17 is a t-SNE map, showing that BAFF gene is mainly expressed in myeloid cell subsets; FIG. 17B is a violin plot showing the expression levels of genes encoding BAFF receptors in B cell subsets and the expression levels of genes encoding receptors that bind IgG constant regions in myeloid cells.

On the basis of the above, by DAPI immunofluorescence staining, it was also found that CD14 ⁺ and CD68 ⁺ co-stained with BAFF in the liver, and the number of cells co-stained with CD14 ⁺ in the BA liver was significantly increased, as shown in Figure 17 C, D, again It provides a basis for the increased expression of BAFF in BA. Therefore, combined with the results of Figure 16, it can be determined that BAFF is an effective detection index or biomarker for diagnosing BA disease.

Further, through human specimen experiments, it was also proved that BAFF promotes the increase of IgG4 secretion and mediates the process of liver fibrosis, and the results are shown in E to G in Figure 17 . Among them, E and F in Figure 17 show that there was a significant increase in the level of immunoglobulin IgG4 in the plasma and liver of BA patients (p<0.05), and IgG4 was positively correlated with the level of BAFF; G in Figure 17 shows , IgG4 ⁺ cells were significantly increased in BA livers and distributed around SMA ⁺ fibroblasts by DAPI immunofluorescence staining. Therefore, IgG4 can also assist BAFF as an index or marker for BA diagnosis or BA liver fibrosis diagnosis.

In addition, in Example 3, it was found that the Ro/SSA-IgG autoantibodies in the liver and serum of children with BA were significantly increased, and it was further found here that the concentration of BAFF in the liver of children with BA was related to the relationship between Ro/SSA-IgG autoantibodies. The levels are positively correlated, see Figure 17, H. Therefore, in addition to using BAFF or further adding GGT and IgG4 as the diagnostic markers for BA as before, it can also be considered to add Ro/SSA-IgG autoantibodies, and similarly, the r-GT described in Example 5 above can also be added. , ALT, AST, TBA, DBIL and IBIL detection reagents are used as indicators or biomarkers for BA diagnosis to further improve the accuracy of diagnosis or auxiliary diagnosis of BA. In addition, the reported MMP7 can also be added as a marker to achieve the purpose of increasing the sensitivity and specificity of synergistic diagnosis and reducing false positives and false negatives.

2. Results of in vitro cell experiments:

As mentioned above, E to G in Fig. 17 have been verified through human specimen experiments that BAFF promotes the increase of IgG4 secretion and mediates liver fibrosis. Further, in the experiment of activating native B cells in vitro and detecting the expression of IgG4 in the culture supernatant, it was also found that the stimulation of BAFF was consistent with the aforementioned human specimen experiments, that is, BAFF significantly promoted the secretion of IgG4 (A in Figure 18). . The above results indicate that BAFF promotes the expression of IgG4.

In the following in vitro experiments, human recombinant IgG4 was used to stimulate human stellate cell line LX-2, and it was found that human recombinant IgG4 significantly promoted the expression of fibroblast-related genes COL1A2 and ACTA1, p<0.05 (Figure 18). B), proves that BAFF participates in the process of liver fibrosis in BA disease by promoting the secretion of IgG4, further proves that IgG4 and BAFF can be used as indicators or biomarkers for the diagnosis of liver fibrosis in BA.

Figure 18C shows pathogen infection promotes BAFF-mediated liver injury. It was found that when the cell line THP1 differentiated into macrophages was stimulated with the pathogen toxin component LPS and the inflammatory factor IFN-β produced by the stimulated body, the expression of BAFF could be significantly promoted (p<0.0001).

Further experiments in the B cell-monocyte co-culture system also found that IFN-β can promote the expression of IgG4, and when the effect of BAFF was blocked by adding anti-BAFF in the co-culture system, the level of IgG4 decreased. was significantly reduced (D in Figure 18), indicating that the type I interferon pathway activated by viral infection can promote the secretion of IgG4 through BAFF, which is involved in the production of BA disease and the process of liver fibrosis in BA disease, and the specific blocker of BAFF It can effectively inhibit the secretion of IgG4, thereby preventing the occurrence and development of BA diseases.

3. In vivo animal experiment results:

The BA model established by infection with rhesus rotavirus (RRV) within 24 hours of birth found that at the 6th day, the BAFF between the RRV model group and the control group had begun to show a significant difference (p<0.05), and by the 12th day At the end of the modeling day, the BAFF of the RRV modeling group and the control group showed a very significant difference (p=0.002), so it can be seen that with time, the level of BAFF in the plasma of RRV-infected mice gradually increased (Figure 19 middle A).

One day after RRV infection, BAFF neutralizing antibody (anti-BAFF) was used to observe the changes in mouse phenotype and immune cell function to test whether anti-BAFF has preventive and therapeutic effects. The results showed that after blocking BAFF with anti-BAFF, when the jaundice rate in the RRV group reached 100%, the jaundice rate in the anti-BAFF administration group was only 30%, and the jaundice rate in the mice also decreased significantly at the end of the experiment. Difference, p=0.001 (B in Figure 19). Meanwhile, compared with the RRV model group, the weight loss of the anti-BAFF group was also significantly improved, p<0.05 (C in Figure 19).

Further, the results of HE staining showed that necrosis and lymphocyte infiltration in the liver of BA mice increased, and after BAFF was specifically blocked by anti-BAFF, the necrosis and lymphocyte infiltration in the liver were significantly reduced, which was close to the control group (Fig. 19 D).

In addition, the foregoing Example 5 describes that the detection reagents of r-GT, ALT, AST, TBA, DBIL and IBIL can be combined as markers for the diagnosis of BA, and this example further proves that the anti-BAFF administration group is better than the control group. The levels of liver function indexes ALT, AST, DBIL, and TBA in the group were significantly decreased, p<0.05 (E in Figure 19). The expression levels of IFN-γ in both CD4 ⁺ T cells and CD8 ⁺ T cells were also significantly decreased in liver and spleen, p<0.01 (Fig. 20, panel A). Therefore, the above experiments demonstrate that blocking BAFF can inhibit the secretion of the toxic cytokine IFN-γ from T cells, and the liver function is also significantly improved, thereby significantly affecting BA and the process of liver damage (such as liver fibrosis/cirrhosis). inhibitory and protective effects.

In other respects, compared with the RRV model group, in the liver of the anti-BAFF administration group, the number of Kupffer cells with pathogen clearance function was significantly increased (p<0.001), and the phagocytic function of Kupffer cells was significantly enhanced (B in Figure 20). . The ELISA results showed that the BAFF neutralizing antibody could significantly reduce the BAFF concentration in the plasma of BA mice (p<0.0001) (C in Figure 20). In the detection results of cytokines, it was also found that compared with the RRV-induced BA model mice group, the levels of cytokines TNF-α, IFN-β and IFN-γ in the plasma of the mice in the anti-BAFF administration group were also significantly higher. (p<0.05), and was close to the blank control group (D in Figure 20). Furthermore, it was also found in the experiment that compared with the RRV-induced BA model mouse group, the anti-BAFF administration group could significantly reduce the The expression of IgG1 in plasma cells in mouse spleen, p<0.05, and the IgG1 level in the anti-BAFF administration group was close to the blank control group (E in Figure 20), while in mice, the function of IgG1 was similar to that of IgG4 in humans. The functions are similar, so it is also consistent with the results of E to G in Figure 17 of the human specimen experiment and A in Figure 18 of the in vitro cell experiment, that BAFF promotes the increase of IgG4 secretion and mediates liver fibrosis. As a drug, anti-BAFF can prevent and treat BA and its progression to liver fibrosis and even cirrhosis.

in conclusion:

The results of this example demonstrate that BAFF and its regulated signaling pathways play an important role in the diagnosis of biliary atresia disease and its fibrosis process. Among them, in the process of occurrence and development of BA caused by viruses, for example, the levels of BAFF in serum/plasma and liver are positively correlated, and the level of BAFF in the liver is also related to the level of jaundice, weight loss, and liver lymph nodes of BA. Cell infiltration, liver fibrosis score, IFN-β and other phenotypes or indicators were positively correlated; further, it was found that BAFF was positively correlated with GGT, Ro/SSA-IgG autoantibodies, and it was also found that IgG4 in BA liver and plasma was positively correlated. The level of IgG4 increased, and the increase of IgG4 in serum/plasma was proportional to the increase of BAFF in serum/plasma. BAFF could significantly stimulate the expression of IgG4, while IgG4 promoted the expression of fibroblast-related genes and accelerated the expression of BA. fibrosis process. Thus, this example clarifies the mechanism of the occurrence/development of BA caused by elevated BAFF levels, and innovatively proposes the combination of BAFF with IgG4, GGT, Ro/SSA-IgG autoantibodies, etc. as a diagnostic marker for BA and its fibrosis progression. . Correspondingly, in terms of prevention and treatment, by using specific blockers of BAFF, such as neutralizing antibody anti-BAFF, it can inhibit the secretion of TNF-α/IFN-β/IFN-γ, enhance the phagocytic function of Kupffer cells, Inhibit the secretion of pro-fibrotic immunoglobulin IgG1 (corresponding to human IgG4), reduce liver cell necrosis and lymphocyte infiltration and other liver damage, reduce liver damage indicators (ALT, AST, DBIL, TBA), delay liver fibrosis, and reduce jaundice rate , Inhibit weight loss, achieve significant prevention and treatment of biliary atresia and its liver fibrosis/cirrhosis process.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

Claims

Use of an agent that inhibits MHC-I and/or MHC-II signaling pathway in any one of (a1) to (a8);

(a1) preparing a medicine for preventing and treating biliary atresia;

(a2) preparing a product that reduces the rate of jaundice;

(a3) preparing a product that reduces the level of pro-inflammatory factors;

(a4) preparing a product that inhibits RRV virus gene expression;

(a5) preparing a product that increases the levels of Cx3cl1 and C1q;

(a6) preparing a product that inhibits the activation of autoreactive CD8 + T cells;

(a7) preparing a product that increases the proportion of Kupffer cells;

(a8) Preparation of a product that increases the phagocytic ability of Kupffer cells.
The use according to claim 1, wherein the reagent is a blocking antibody of MHC-I and/or MHC-II.
The use according to claim 2, wherein the reagent is a bispecific blocking antibody of MHC-I and MHC-II.
The use according to claim 1 or 2, the blocking antibody is a monoclonal antibody.
The use according to claim 4, wherein the blocking antibody is an HLA-DR blocking antibody.
The use according to claim 5, wherein the HLA-DR blocking antibody is selected from at least one of IMMU-114, Hu1D10 and Lym-1.
The use according to claim 2, wherein the reagent is a chimeric antibody, modified antibody, humanized antibody or fully human antibody of MHC-I and/or MHC-II.
The use according to claim 1, wherein the reagent is an antibody fragment that blocks MHC-I and/or MHC-II.
According to the use of claim 8, the antibody fragment comprises Fab, F(ab')2, Fd, Fv, scFv, scFv-Fc, diabody and antibody minimal recognition unit.
The use according to any one of claims 1-9, the medicament comprising at least one of a pharmaceutically acceptable excipient, carrier, buffer and stabilizer;

Preferably, the medicine is lyophilized powder or injection;

Preferably, the medicament is prepared in a dosage form suitable for adults or children;

Preferably, the medicament is prepared in a dosage form suitable for use in children.