Treatment of primary biliary cholangitis (PBC) with tolerizing nanoparticles

Abstract

How to treat primary biliary cholangitis (PBC). [Solution] The present disclosure relates to methods of treating primary biliary cholangitis (PBC) using tolerizing, immunomodulating nanoparticles that encapsulate PBC-associated antigens.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 270,447, filed October 21, 2021, and U.S. Provisional Patent Application No. 63 / 369,574, filed July 27, 2022, which are incorporated by reference in their entireties herein.

[0002] The present disclosure relates to methods of treating primary biliary cholangitis (PBC) using tolerizing, immunomodulating nanoparticles that encapsulate PBC-associated antigens. [Background technology]

[0003] Primary biliary cholangitis (PBC), also known as primary biliary cirrhosis, is a rare autoimmune disease of the liver. PBC affects women more frequently, with an estimated prevalence of approximately 580 cases per million women in the United States. 1 PBC is characterized by immune-mediated destruction of intrahepatic bile ducts via autoreactive T and B cells, leading to cholestasis (reduced or cessation of bile flow) and a progressive course that ultimately results in end-stage liver disease. 2、3 .

[0004] Autoimmune activity in PBC primarily targets intracellular proteins (e.g., mitochondrial and nuclear proteins), with approximately 95% of patients exhibiting circulating antimitochondrial antibodies. 4、5 Symptoms of PBC include a buildup of toxic bile acids in the liver, abnormal liver enzyme levels, liver dysfunction, liver fibrosis, cirrhosis, bone pain, fatigue, itching, and dry eyes and mouth.

[0005] There is no cure for PBC. The current standard of care for PBC relies on regulators of bile acid synthesis, such as ursodeoxycholic acid (UDCA). 6、7Current standard treatments are associated with significant side effects that fail to address the underlying immune imbalances that lead to disease pathology with low response rates. These therapeutic approaches can only delay disease progression and time to liver transplantation. Summary of the Invention

[0006] Tolerizing immune-modifying particles (TIMPs) containing one or more antigens have been previously described for the treatment of immune-mediated disorders (e.g., autoimmune diseases and allergies) through the induction of antigen-specific immune tolerance (WO2013 / 192532 and WO2015 / 023796, which are incorporated herein by reference). In several preclinical models of autoimmune diseases and allergies, TIMPs have demonstrated efficacy in inducing T cell tolerance. Induction of antigen-specific tolerance to PBC autoantigens using TIMPs encapsulating PBC-specific antigens (TIMP-PBC) may improve or potentially cure PBC.

[0007] Provided herein are methods of treating PBC in a subject, comprising administering TIMP-PBC to the subject, wherein the TIMP-PBC is administered at a dose determined based on the subject's body weight. In various embodiments, the TIMP-PBC is administered at a dose of 0.01-12 mg / kg. In various embodiments, the TIMP-PBC is administered at a fixed dose of 1 mg-800 mg. In various embodiments, the TIMP-PBC is administered at a dose of about 0.01-12 mg / kg, about 0.05-10 mg / kg, about 0.01-5 mg / kg, about 0.1-10 mg / kg, about 1-8 mg / kg, about 1.5-10 mg / kg, about 2-12 mg / kg, about 2-10 mg / kg, about 3-10 mg / kg, about 4-10 mg / kg, about 4-12 mg / kg, or about 5-12 mg / kg. In various embodiments, TIMP-PBC is administered at a dose of about 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 3.0 mg / kg, 4.0 mg / kg, 5 mg / kg, 6 mg / kg, 8.0 mg / kg, 10 mg / kg, or 12 mg / kg. In various embodiments, TIMP-PBC is administered in a fixed dose of about 1 mg, 2 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, or 800 mg.

[0008] In various embodiments, the TIMP-PBC encapsulates one or more PBC antigens or antigenic epitopes. In various embodiments, the PBC antigen is a mitochondrial protein. In various embodiments, the PBC antigen is a nuclear protein. In various embodiments, the PBC antigen is selected from the group consisting of pyruvate dehydrogenase complex E2 subunit (PDC-E2), gp210, nucleoporin 62, Sp100, PML, CENP A, CENP B, CENP C, dsDNA, histones, branched-chain 2-oxoacid dehydrogenase complex (BCOADC), smooth muscle proteins, soluble liver antigens, liver / kidney microsomes, centromere proteins, tubulin, actin, vimentin, desmin, cytokeratin, F-actin, UDP-glucuronosyltransferase family 1 member A complex (UGTA1), formiminotransferase cyclodeaminase, asialoglycoprotein receptor (ASGPR), cardiolipin, h-Lamp-2, proteinase 3, CYP 2C9, CYP 2A6, and CYP 450 2D6.

[0009] In various embodiments, the TIMP-PBC encapsulates a PDC-E2 peptide that includes an antigenic epitope. In various embodiments, the TIMP-PBC encapsulates PDC-E2 amino acids 155-185 (PDC-E2) having the amino acid sequence KVGEKLSEGDLLAEIETDKATIGFEVQEEGY (SEQ ID NO: 1). 155~185 ) is enclosed.

[0010] In various embodiments, TIMP-PBC is administered in a single dose or multiple doses. In various embodiments, TIMP-PBC is administered in two doses, one week apart. In various embodiments, TIMP-PBC is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every six months, or once a year.

[0011] In various embodiments, the TIMP-PBC consists of poly(lactic-co-glycolic acid) (PLGA) particles encapsulating one or more PBC antigens and a suitable buffer or excipient. In various embodiments, the TIMP-PBC particles are surface functionalized. In various embodiments, the TIMP-PBC particles are surface functionalized by carboxylation. In various embodiments, the TIMP-PBC particles have a negative zeta potential. In various embodiments, the negative zeta potential of the TIMP-PBC particles is from about -100 mV to about 0 mV. In various embodiments, the zeta potential of the particles is about -100 mV to about -25 mV, about -100 to about -30 mV, about -80 mV to about -30 mV, about -75 mV to about -30 mV, about -70 mV to about -30 mV, about -75 to about -35 mV, about -70 to about -25 mV, about -60 mV to about -30 mV, about -60 mV to about -35 mV, or about -50 mV to about -30 mV. In various embodiments, the zeta potential is about -25mV, -30mV, -35mV, -40mV, -45mV, -50mV, -55mV, -60mV, -65mV, -70mV, -75mV, -80mV, -85mV, -90mV, -95mV or -100mV.

[0012] In various embodiments, the size or diameter of the TIMP-PBC particles is from 0.05 μm to about 10 μm. In various embodiments, the diameter of the TIMP-PBC particles is from 0.1 μm to about 10 μm. In various embodiments, the diameter of the TIMP-PBC particles is from 0.1 μm to about 5 μm. In various embodiments, the diameter of the TIMP-PBC particles is from 0.1 μm to about 3 μm. In various embodiments, the diameter of the TIMP-PBC particles is from 0.3 μm to about 5 μm. In various embodiments, the diameter of the TIMP-PBC particles is from about 0.3 μm to about 3 μm. In various embodiments, the diameter of the TIMP-PBC particles is from about 0.3 μm to about 1 μm. In various embodiments, the diameter of the TIMP-PBC particles is from about 0.4 μm to about 1 μm. In various embodiments, the TIMP-PBC particles have a diameter of about 100-10,000 nm, about 100-5,000 nm, about 100-3,000 nm, about 100-2,000 nm, about 300-5,000 nm, about 300-3,000 nm, about 300-1,000 nm, about 300-800 nm, about 400-800 nm, or about 200-700 nm. In various embodiments, the TIMP-PBC particles have a diameter of about 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm. In various embodiments, the negatively charged particles have a diameter of between 400 nm and 800 nm.

[0013] In various embodiments, the TIMP-PBC is administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, or orally.

[0014] In various embodiments, TIMP-PBC is administered at a concentration of about 0.005 mg / mL to about 50 mg / mL. In various embodiments, TIMP-PBC is administered at a concentration of about 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 3.25 mg / mL, 3.5 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12.5 mg / mL, 15 mg / mL, 17.5 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, or 50 mg / mL. In various embodiments, TIMP-PBC is administered via intravenous infusion lasting about 1, 2, 3, 4, 5, 6, 7, or 8 hours.

[0015] In various embodiments, administering TIMP-PBC to a subject in need thereof alleviates one or more symptoms of PBC. In various embodiments, the symptoms of PBC are selected from the group consisting of hepatitis, cirrhosis, cholestasis, liver dysfunction, liver failure, liver fibrosis, increased liver immune infiltration, elevated bile acid levels, elevated liver enzyme levels, (ALT, ALP, AST, gamma-glutamyl transpeptidase), elevated bilirubin levels, circulating antimitochondrial antibodies (AMA), circulating antinuclear antibodies (ANA), fatigue, itchy skin, pruritus, dry eyes and mouth, abdominal pain, splenomegaly, musculoskeletal pain, edema, fluid accumulation, skin xanthomas, jaundice, hyperpigmentation, osteoporosis, high cholesterol, diarrhea, steatorrhea, hypothyroidism, and weight loss.

[0016] In various embodiments, administering TIMP-PBC to a subject in need thereof reduces the duration and severity of an inflammatory immune response to one or more PBC antigens. In various embodiments, the inflammatory immune response is a T cell response, a B cell response, a Th1 response, a myeloid cell response, and / or an antibody response. In various embodiments, the efficacy of TIMP-PBC in alleviating one or more symptoms of PBC and / or reducing the duration and severity of an inflammatory immune response to one or more PBC antigens is determined from an assay of one or more biological samples from the subject. In various embodiments, the biological sample is selected from the group consisting of whole blood, peripheral blood, peripheral blood mononuclear cells (PBMCs), serum, plasma, urine, cerebrospinal fluid (CSF), stool, tissue biopsy, and / or bone marrow biopsy.

[0017] In various embodiments, administering TIMP-PBC to a subject reduces the level of activated antigen-specific T cells. In various embodiments, administering TIMP-PBC to a subject reduces the level of activated antigen-specific CD4+ and / or CD8+ T cells.

[0018] In various embodiments, administering TIMP-PBC to a subject reduces the level of T cell infiltration in the liver, hi various embodiments, treatment with TIMP-PBC reduces liver immune infiltration by about 5% to 100%, or about 2- to 100-fold, inclusive.

[0019] In various embodiments, administering TIMP-PBC to a subject reduces the level of antimitochondrial antibodies in the blood, hi various embodiments, treatment with TIMP-PBC reduces the level of antimitochondrial antibodies by about 5% to 100%, or about 2-100 fold, inclusive, as compared to the subject's baseline measurement and / or as compared to healthy subjects.

[0020] In various embodiments, administering TIMP-PBC to a subject reduces the level of antinuclear antibodies in the blood, hi various embodiments, treatment with TIMP-PBC reduces the level of antinuclear antibodies by about 5% to 100%, or about 2-100 fold, inclusive, relative to the subject's baseline measurement and / or relative to healthy subjects.

[0021] In various embodiments, administering TIMP-PBC to a subject reduces the level of IgM in the blood, hi various embodiments, treatment with TIMP-PBC reduces the level of IgM by about 5%-100%, or about 2-100 fold, relative to the subject's baseline measurement and / or relative to healthy subjects.

[0022] In various embodiments, administering TIMP-PBC to a subject reduces the levels of anti-Gp210 antibodies, anti-Sp100 antibodies, kynurenine and / or soluble CD14 in the blood. In various embodiments, treatment with TIMP-PBC reduces the levels of anti-Gp210 antibodies, anti-Sp100 antibodies, kynurenine and / or CD14 by about 5%-100%, or about 2-100 fold, relative to the subject's baseline measurements and / or relative to healthy subjects.

[0023] In various embodiments, the disclosure provides a method of treating PBC or a PBC-related condition in a subject in need thereof, comprising administering to the subject a composition comprising TIMP-PBC, alone or in combination with a therapeutic agent useful for treating PBC. In various embodiments, the therapeutic agent useful for treating PBC is a steroid, a corticosteroid, a nonsteroidal immunosuppressant, an immunomodulatory agent, a monoclonal antibody, a cytokine and chemokine targeted therapy, a JAK inhibitor, a chimeric antigen receptor (CAR) T cell therapy, a regulatory T cell (Treg) therapy, a B cell targeted therapy, an antiviral agent, a synthetic bile acid, or a surgical treatment. In various embodiments, the therapeutic agent is ursodeoxycholic acid (UDCA), obeticholic acid (OCA), fibrates, peroxisome proliferator-activated receptor delta (pparδ) agonists, ileal bile acid transporter (IBAT) inhibitors, ustekinumab, tauroursodeoxycholic acid, 6α-ethylchenodeoxycholic acid, setanaxib, moexipril, abatacept, fibroblast growth factors, statins, colchicone, CD20 targeted therapy, CD19 targeted therapy, CD40 / CD40L targeted therapy, CD80 / CD86 targeted therapy, B cell targeting factor (BAFF) targeted therapy, B cell maturation antigen (BCMA) targeted therapy, anti-IL6 therapy, anti-IFN therapy, amifampridine, cyclosporine, cyclophosphamide, batoclimab, inebilizumab, nipocalimab, pozelimab, rituximab, satralizumab, seladelpar, pentoxifylline, tocilizumab, tofacitinib, trebrutinib, abolstatin, fenofibrate, bezafibrate, linelixibat, methotrexate, butyrate, palmitate, or liver transplant. In various embodiments, the steroid or corticosteroid is selected from the group including beclomethasone, cyclosonide, fluticasone furoate, mometasone, budenoside, fluticasone, triamcinolone, loteprednol, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, betamethasone, or hydrocortisone.

[0024] In various embodiments, the therapeutic agent is administered prior to, concomitantly with, or subsequent to administration of TIMP-PBC. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of TIMP-PBC. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months prior to administration of TIMP-PBC. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years prior to administration of TIMP-PBC. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years prior to administration of TIMP-PBC. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, or 7 days after administration of TIMP-PBC. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, or 7 weeks after administration of TIMP-PBC. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration of TIMP-PBC.In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years after administration of TIMP-PBC.

[0025] Also contemplated is a composition comprising the TIMP-PBC described herein for use in treating primary biliary cholangitis. In various embodiments, the present disclosure provides for the use of a composition comprising the TIMP-PBC described herein in the preparation of a medicament for treating primary biliary cholangitis.

[0026] It is understood that each feature or embodiment, or combination described herein is a non-limiting illustrative example of any of the aspects of the invention, and is therefore meant to be combinable with any other feature or embodiment, or combination described herein. For example, when a feature is described in language such as "one embodiment," "some embodiments," "certain embodiments," "further embodiments," "particular exemplary embodiments," and / or "another embodiment," each of these types of embodiments is a non-limiting example of the feature that is intended to be combined with any other feature or combination of features described herein, without listing all possible combinations. Such features or combinations of features apply to any of the aspects of the invention. When examples of values ​​that fall within a range are disclosed, any of these examples are contemplated as possible endpoints of the range, and any and all numerical values ​​between such endpoints are contemplated, and any and all combinations of upper and lower limits are envisioned. [Brief description of the drawings]

[0027] [Figure 1] CNP-104 inhibits antigen-specific T cell proliferation in a PBC mouse model. PDC-E2-specific T cell proliferation in mice treated with CNP-104 lot 2107GM07. PDC-E2-specific T cell proliferation in the CNP-104 treatment group was compared to the control unloaded CNP group in a PBC mouse model. **** represents p<0.0001. [Diagram 2] CNP-104 inhibits anti-mitochondrial IgG antibodies in a PBC mouse model. PDC-E2-specific IgG anti-mitochondrial antibody (AMA) titers were determined from mice primed with PDC-E2 155-185 and treated with CNP-104 or control unloaded nanoparticles (CNPs) by ELISA. [Figure 3A]CNP-104 inhibits antigen-specific T cell responses in the DTH model. C57BL / 6 mice were primed with PDC-E2-peptide emulsified in CFA on day 0. On days 0 and 7 after priming, mice were treated with CNP104 at a dose of 2.5 mg dose / mouse (10 mg / kg HED). On day 14 after priming, mice were challenged intradermally with PDC-E2-peptide (single lot, FIG. 3A) or ovalbumin (negative control). The thickness of the pinna of each ear was measured immediately after and 24 hours after challenge with PDC-E2-peptide and OVA. The change in pinna thickness (ΔT) of both ears of each mouse was calculated to evaluate the DTH response. [Figure 3B] CNP-104 inhibits antigen-specific T cell responses in the DTH model. C57BL / 6 mice were primed with PDC-E2-peptide emulsified in CFA on day 0. On days 0 and 7 after priming, mice were treated with CNP104 at a dose of 2.5 mg dose / mouse (10 mg / kg HED). On day 14 after priming, mice were challenged intradermally with PDC-E2-peptide (multiple lots, FIG. 3B) or ovalbumin (negative control). The thickness of the pinna of each ear was measured immediately after and 24 hours after challenge with PDC-E2-peptide and OVA. The change in pinna thickness (ΔT) of both ears of each mouse was calculated to evaluate the DTH response. [Figure 4] CNP-104 inhibits leukocyte infiltration in the liver in a PBC mouse model. The level of leukocyte (CD45+) infiltration in the liver was determined by flow cytometry from mice primed with PDC-E2155-185. [Diagram 5]CNP-104 increases regulatory T cells in the liver. On the day of the experiment (day 0), mice were primed with 2-octinoic acid conjugated to bovine serum albumin (2OA-BSA) / CFA by intraperitoneal injection (ip). 2OA-BSA ip priming was repeated with IFA on day 14. On days 0 and 2, mice were ip injected with 100 ng of pertussis toxin. On days 0 and 14, mice were subcutaneously injected with 5 mg / kg poly(I:C). On day 7, mice were primed with PDC-E2-peptide / CFA emulsion. Mice were treated intravenously with CNP104 on days 7 and 14. Livers were analyzed for Tregs on day 55. CNP104 treatment increased the frequency of Tregs in the liver compared to untreated untreated controls. [Figure 6A] 1 is a timeline of events for the clinical trial evaluating the safety and efficacy of CNP-104. [Figure 6B] 1 is a timeline of events for the clinical trial evaluating the safety and efficacy of CNP-104. [Figure 7] HLA haplotypes of PBC patients, the presence of antimitochondrial antibodies (AMA), and alkaline phosphatase levels in the blood of 70% of PBC patients show HLA haplotype restriction for PDC-E2. 70% and 76% of PBC patients are AMA positive and have elevated ALP levels, respectively. [Figure 8] 1 shows CD4+ T cell, CD8+ T cell, and biliary epithelial cell counts assessed from liver biopsies using a multiplex immunofluorescence panel. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] There is a need for therapies to address immune imbalances in PBC, leading to amelioration of disease symptoms and improved outcomes without the risk of toxic side effects. The present disclosure provides methodologies for monitoring the induction and maintenance of immune tolerance in subjects with PBC after receiving immunotherapy.

[0029] The headings herein are for the convenience of the reader and are not intended to be limiting. Further aspects, embodiments, and variations of the invention will be apparent from the detailed description and / or drawings and / or claims.

[0030] definition Unless otherwise stated, the following terms used in this Application, including the specification and claims, have the definitions given below.

[0031] As used in this specification and the appended claims, the indefinite articles "a" and "an" and the definite article "the" include plural and singular referents unless the context clearly dictates otherwise.

[0032] The term "about" or "approximately" refers to an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. Whenever the term "about" or "approximately" appears before a first number in a series of two or more numerical values, it is understood that the term "about" or "approximately" applies to each of the numerical values ​​in the series.

[0033] As used herein, "particle" refers to any non-tissue derived composition of matter, which may be a sphere or sphere-like entity, a bead, or a liposome. The terms "particle", "immunomodulating particle", "carrier particle", and "bead" may be used interchangeably depending on the context. In addition, the term "particle" may be used to encompass beads and spheres.

[0034] As used herein, "negatively charged particles" refer to particles that have been modified to have a net surface charge that is less than zero.

[0035] "Carboxylated particles" or "carboxylated beads" or "carboxylated spheres" include any particle that has been modified to contain carboxyl groups on its surface. In some embodiments, the addition of carboxyl groups improves phagocyte / monocyte uptake of the particle from the circulation, for example, through interaction with scavenger receptors such as MARCO. Carboxylation of particles can be accomplished using any compound that adds carboxyl groups.

[0036] As used herein, the term "Th cells" or "helper T cells" refers to CD4 + Refers to cells. CD4 + T cells assist other leukocytes in immunological processes, including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. T cells are activated when peptide antigens are presented to them by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs).

[0037] As used herein, the term "Th1 cells" refers to a subset of Th cells that produce proinflammatory mediators. Th1 cells secrete cytokines to promote immune responses and play a role in host defense against pathogens, in part by mediating the recruitment of neutrophils and macrophages to infected tissues. Th1 cells secrete cytokines including IFN-gamma, IL2, IL-10, and TNF alpha / beta to orchestrate defense against intracellular pathogens such as viruses and some bacteria.

[0038] "Polypeptide" and "protein" refer to polymers composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds or peptide bond isosteres. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The terms "polypeptide" and "protein" are not limited to a minimum length of the product. The term "protein" typically refers to large polypeptides. The term "peptide" typically refers to short polypeptides. Thus, peptides, oligopeptides, dimers, multimers, etc. are included in the definition. Both full-length proteins and fragments thereof are encompassed in the definition. The terms "polypeptide" and "protein" also include post-expression modifications of a polypeptide or protein, such as glycosylation, acetylation, phosphorylation, etc. Furthermore, for purposes of this disclosure, a "polypeptide" can include "modifications" to the native sequence, such as deletions, additions, substitutions (which may be conservative in nature or may include substitutions with any of the 20 amino acids commonly found in human proteins, or with any other natural or non-natural or atypical amino acids), and chemical modifications (e.g., addition of or substitution with a peptidomimetic). These modifications may be deliberate, by site-directed mutagenesis or by chemical modification of amino acids to remove or attach chemical moieties, or may be accidental, by mutations that occur via the host cell producing the protein or by errors due to PCR amplification prior to host cell transfection.

[0039] As used herein, "antigenic moiety" or "antigen" refers to any moiety, e.g., peptide, that is recognized by the host's immune system. Examples of antigenic moieties include, but are not limited to, autoantigens, allergens, enzymes, and / or bacterial or viral proteins, peptides, drugs or components.

[0040] "Pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, buffers, etc., such as phosphate buffered saline, 5% aqueous solution of dextrose, and emulsions (e.g., oil / water or water / oil emulsions). Non-limiting examples of excipients include adjuvants, binders, fillers, diluents, disintegrants, emulsifiers, wetting agents, lubricants, glidants, sweeteners, flavoring agents, and coloring agents. Suitable pharmaceutical carriers, excipients, and diluents are described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995). Preferred pharmaceutical carriers depend on the intended mode of administration of the active agent. Typical modes of administration include enteral (e.g., oral) or parenteral (e.g., subcutaneous, intramuscular, intravenous, or intraperitoneal injection; or topical, transdermal, or transmucosal administration).

[0041] "Pharmaceutically acceptable" or "pharmacologically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained, or with any components present outside or inside the individual's body.

[0042] As used herein, the term "subject" encompasses mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class, humans, non-human primates such as chimpanzees, and other ape and monkey species, domestic animals such as cows, horses, sheep, goats, pigs, domestic animals such as rabbits, dogs, and cats, and laboratory animals including rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds, fish, and the like. The term does not denote a particular age or sex.

[0043] The term "epitope" refers to a portion of any molecule that can be recognized and bound by a selective binding agent at one or more of the antigen-binding regions. Epitopes usually consist of chemically active surface groupings of molecules, such as amino acids or carbohydrate side chains, and have specific three-dimensional structural characteristics, as well as specific charge characteristics. As used herein, epitopes can be contiguous or non-contiguous. Furthermore, epitopes can be mimics (mimotopes) in that they contain the same or similar three-dimensional structure and / or amino acid sequence as the epitope used to generate antibodies, but do not contain any, or only some, of the amino acid residues found in the target used to stimulate the antibody immune response. As used herein, a mimotope is not considered a different antigen than the epitope bound by the selective binding agent, which recognizes the same three-dimensional structure and / or amino acid sequence of the epitope and mimotope.

[0044] The term "symptom" is used herein to mean any physical or observable manifestation of a disorder, regardless of whether the disorder is a general feature of that disorder. The term "symptom" may mean all such manifestations or any subset thereof.

[0045] The term "therapeutically effective amount" is used herein to indicate an amount of an antigen-specific composition of the present disclosure effective to ameliorate or alleviate the symptoms or signs of the disease being treated.

[0046] The terms "treat," "treated," and "treatment" as used with respect to the methods herein refer to the elimination, reduction, inhibition, or amelioration, either temporarily or permanently, partially or completely, of one or more clinical symptoms, symptoms, or progression of an event, disease, or condition. Such treatment need not be absolute to be useful. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, a stabilized (i.e., not worsening) state of disease, a delay or slowing of disease progression, an improvement or palliative of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not treated. Those in need of treatment include those already with the condition or disorder, as well as those prone to having the condition or disorder, or those in whom the condition or disorder is to be prevented.

[0047] particle The size and charge of the particles are important for tolerance induction. Particles vary in size and charge based on the antigen encapsulated within the particle, but generally, the particles described herein are effective in inducing tolerance if they are between about 100 nanometers and about 1500 nanometers and have a negative charge of 0 to about -100 mV. In various embodiments, the particles are 400 to 800 nanometers in diameter and have a charge of about -25 mV to -70 mV. The average particle size and charge of the particles can be slightly altered in the lyophilization process, so both the post-synthesis average and the post-lyophilization average are described. As used herein, the terms "post-synthesis size" and "post-synthesis charge" refer to the size and charge of the particles before lyophilization. The terms "post-lyophilization size" and "post-lyophilization charge" refer to the size and charge of the particles after lyophilization.

[0048] In some embodiments, the particles are non-metallic. In these embodiments, the particles may be formed from polymers. In a preferred embodiment, the particles are biodegradable in the individual. In this embodiment, the particles can be provided to the individual over multiple doses without the particles accumulating in the individual. Examples of suitable particles include polystyrene particles, PGA particles, PLA particles, PLGA particles, PLURIONICS stabilized polypropylene sulfide particles, and diamond particles.

[0049] Preferably, the particle surface is made of a material that minimizes non-specific or undesirable biological interactions. Interactions between the particle surface and the stroma may be a factor that plays a role in lymphatic uptake. The particle surface may be coated with a material to prevent or reduce non-specific interactions. Steric stabilization by coating the particle with a hydrophilic layer such as poly(ethylene glycol) (PEG) and its copolymers, such as PLURONICS® (including copolymers of poly(ethylene glycol)-bl-poly(propylene glycol)-bl-poly(ethylene glycol)), may reduce non-specific interactions with interstitial proteins, as demonstrated by improved lymphatic uptake after subcutaneous injection. All these facts suggest the relevance of the particle's physical properties in terms of lymphatic uptake. Biodegradable polymers may be used to make all or part of the polymer and / or particle and / or layer. Biodegradable polymers may degrade, for example, as a result of functional groups reacting with water in solution. The term "degradation" as used herein refers to becoming soluble, either by a decrease in molecular weight or by the conversion of hydrophobic groups to hydrophilic groups. Polymers containing ester groups, such as polylactide and polyglycolide, generally undergo spontaneous hydrolysis.

[0050] The particles disclosed herein may also contain additional components. For example, carriers may have imaging agents incorporated or conjugated to them. An example of carrier nanospheres with imaging agents that are currently commercially available is Kodak X-Site Nanospheres. Inorganic quantum confined luminescent nanocrystals, known as quantum dots (QDs), have emerged as ideal donors in FRET applications: their high quantum yield and tunable size-dependent Stokes shift allow them to emit different sizes from blue to infrared when excited with a single ultraviolet wavelength. (Bruchez, et al., Science, 1998, 281, 2013; Niemeyer, CM Angew.Chem.Int.Ed.2003, 42, 5796; Waggoner, A.Methods Enzymol.1995, 246, 362; Brus, LEJ Chem.Phys.1993, 79, 5566). Quantum dots, such as hybrid organic / inorganic quantum dots based on a class of polymers known as dendrimers, can be used in biological labeling, imaging, and optical biosensing systems. (Lemon, et al., J. Am. Chem. Soc. 2000, 122, 12886). Unlike traditional synthesis of inorganic quantum dots, the synthesis of these hybrid quantum dot nanoparticles does not require high temperatures or highly toxic and unstable reagents. (Etienne, et al., Appl. Phys. Lett. 87, 181913, 2005).

[0051] The particles can be formed from a wide range of materials. The particles are preferably composed of materials suitable for biological use. For example, the particles can be composed of glass, silica, polyesters of hydroxycarboxylic acids, polyanhydrides of dicarboxylic acids, or copolymers of hydroxycarboxylic acids and dicarboxylic acids. More generally, the carrier particles can be composed of polyesters of linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked, alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxy hydroxy acids, or polyanhydrides of linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked, alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, alkenyl, heteroaryl, or alkoxy dicarboxylic acids. Furthermore, the carrier particles can be quantum dots or composed of quantum dots, such as quantum dot polystyrene particles (Joumaa et al. (2006) Langmuir 22:1810-6). Carrier particles containing a mixture of ester and anhydride bonds (e.g., copolymers of glycolic acid and sebacic acid) may be used. For example, the carrier particles may include materials including polyglycolic acid polymers (PGA), polylactic acid polymers (PLA), polysebacic acid polymers (PSA), poly(lactic-co-glycolic) acid copolymers (PLGA or PLG, the terms are interchangeable), poly(lactic-co-sebacic) acid copolymers (PLSA), poly(glycolic-co-sebacic) acid copolymers (PGSA), polypropylene sulfide polymers, poly(caprolactone), chitosan, and the like. Other biocompatible, biodegradable polymers useful in the present invention include polymers or copolymers of caprolactone, carbonates, amides, amino acids, orthoesters, acetals, cyanoacrylates, and degradable urethanes, as well as their copolymers with linear or branched, substituted or unsubstituted, alkanyl, haloalkyl, thioalkyl, aminoalkyl, alkenyl, or aromatic hydroxy or dicarboxylic acids.In addition, biologically important amino acids with reactive side groups, such as lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine, or their enantiomers, can be included in copolymers with any of the aforementioned materials to provide reactive groups for conjugation to antigenic peptides and proteins or conjugation sites. Biodegradable materials suitable for the present invention include diamond, PLA, PGA, polypropylene sulfide, and PLGA polymers. Biocompatible but non-biodegradable materials can also be used in the carrier particles of the present invention. For example, non-biodegradable polymers of acrylate, ethylene-vinyl acetate, acyl-substituted cellulose acetate, non-degradable urethane, styrene, vinyl chloride, vinyl fluoride, vinyl imidazole, chlorosulfonated olefins, ethylene oxide, vinyl alcohol, TEFLON® (DuPont, Wilmington, Del.), and nylon can be used.

[0052] In certain embodiments, the particles are copolymers having a molar ratio of about 80:20 to about 100:0, or about 20:80 to 100:0. Suitable copolymer ratios for the current tolerizing immunomodulating particles can be 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0. In certain embodiments, the particles are PLURONICS stabilized polypropylene sulfide particles, polyglycolic acid particles (PGA), polylactic acid particles (PLA), or poly(lactic-co-glycolic acid) particles, or carboxylated polyglycolic acid particles (PGA), carboxylated polylactic acid particles (PLA), or carboxylated poly(lactic-co-glycolic acid) particles. In certain embodiments, the particles have a copolymer ratio of polylactic acid / polyglycolic acid 80:20, polylactic acid / polyglycolic acid 90:10, or polylactic acid / polyglycolic acid 50:50. In various embodiments, the particles are poly(lactic-co-glycolic acid) particles and have a copolymer ratio of polylactic acid:polyglycolic acid of about 50:50.

[0053] It is contemplated that the particles may further comprise a surfactant. The surfactant may be anionic, cationic, or nonionic. Poloxamer and poloxamine family surfactants are commonly used in particle synthesis. Surfactants that may be used include, but are not limited to, PEG, Tween-80, gelatin, dextran, Pluronic L-63, polyvinyl alcohol (PVA), polyacrylic acid (PAA), methylcellulose, lecithin, dimethylaminobenzaldehyde (DMAB), and poly(ethyl methacrylate) (PEMA). In addition, biodegradable and biocompatible surfactants include, but are not limited to, vitamin E TPGS (Da-tocopheryl polyethylene glycol 1000 succinate), polyamino acids (e.g., polymers of lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine, or their enantiomers), and sulfate polymers. In certain embodiments, two surfactants are used. For example, when the particles are produced by double emulsion method, the two surfactants can include a hydrophobic surfactant for the first emulsion and a hydrophobic surfactant for the second emulsion. In certain embodiments, the polypeptide antigen is encapsulated in the particles by the single emulsion process. In further embodiments, the polypeptide antigen is more hydrophobic. Sometimes, the double emulsion process leads to the formation of large particles that can lead to leakage of hydrophilic active ingredients and low entrapment efficiency. Binding and Ostwald ripening are two mechanisms that can destabilize double emulsion droplets, while diffusion of hydrophilic active ingredients through the organic phase is the main mechanism for low levels of entrapped active ingredients. In some embodiments, it can be beneficial to reduce the nanoparticle size. One strategy to achieve this is to apply a second strong shear rate. The leakage effect can be reduced by using polymer concentration and polymer molecular weight, which is accompanied by an increase in the viscosity of the inner aqueous phase and an increase in the surfactant molecular weight.In certain embodiments, particles encapsulating antigens are produced by nanoprecipitation, co-precipitation, inert gas condensation, sputtering, microemulsion, sol-gel techniques, layer-by-layer techniques, or ionic gelation. Several methods for producing nanoparticles have been described in the literature and are incorporated herein by reference.

[0054] antigen An antigen refers to a discreet portion of a molecule, such as a polypeptide or peptide sequence, a three-dimensional structural formation of a polypeptide or peptide, a polysaccharide or a polynucleotide, that can be recognized by a host immune cell. Antigen specificity refers to the ability of a subject's host cells to recognize and generate an immune response to the antigen alone, or to a molecule closely similar to the antigen, as well as to an epitope or mimotope.

[0055] "Anergy", "tolerance", or "antigen-specific tolerance" refers to the insensitivity or reprogramming of T cells to T cell receptor-mediated stimulation. Such reprogramming is generally antigen-specific and persists after exposure to antigenic peptides has ceased. This reprogramming results in the induction of regulatory T cells, Tr1 cells, and T cell anergy. For example, insensitivity in T cells is characterized by lack of effector cytokine production, lack of proliferation, or lack of activation. Reprogramming occurs when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD3-mediated signal) in the absence of a second signal (costimulatory signal) or in the presence of an inhibitory signal (negative costimulatory or regulatory cytokine). Under these conditions, reexposure of the cells to the same antigen (even if reexposure occurs in the presence of costimulatory molecules) results in failure to produce cytokines and subsequently failure to proliferate. Thus, failure to produce cytokines prevents proliferation. However, anergic T cells can proliferate when cultured with (e.g., IL-2).

[0056] It is contemplated that the tolerization therapy described herein is antigen-specific. For example, the TIMP administered as a tolerization therapy encapsulates one or more antigens associated with the tolerization therapy and the associated disease or condition being treated. It is contemplated that the TIMP used in the tolerization therapy comprises one or more PBC antigens. The one or more PBC antigens may be whole proteins, polypeptides, or peptides that contain a PBC antigen epitope.

[0057] In certain embodiments, one, two, three or more antigens or antigen peptides are used in TIMPs. In certain embodiments, one or more PBC antigens are encapsulated in TIMPs by covalent attachment to the inner surface of the particle (see, for example, US Patent Publication No. 20190282707, incorporated herein by reference). In certain embodiments, it is contemplated that the sequences of two or more PBC antigens are linked in a fusion protein and encapsulated in TIMPs as described herein. Certain PBC epitopes are described in Shimoda et al. (Gastroenterology 124:1915-1925, 2003). Methods for producing TIMPs with linked epitopes are described in US Patent Publication No. 20190365656, incorporated herein by reference.

[0058] How to use Primary biliary cholangitis (PBC, formerly known as primary biliary cirrhosis) is a prototypic autoimmune liver disease characterized by destructive lymphocytic cholangitis primarily targeting the E2 component of the mitochondrial pyruvate dehydrogenase complex (PDC-E2) and specific antimitochondrial autoantibodies (AMA). 1 The prevalence of PBC in the United States is estimated to be approximately 580 cases per million women. 2 There is a significant unmet medical need in this rare disease and an opportunity for TIMP-PBC to make a positive impact on subjects affected by this disease.

[0059] Liver failure is the most common cause of death in patients with PBC2 The current standard of care for PBC (ursodeoxycholic acid) has reduced the likelihood of needing a liver transplant and extended patient lifespan. However, up to 40% of patients with PBC have a suboptimal biochemical response to treatment as determined by either binary response criteria and / or prognostic models. 1 .

[0060] Currently, treatments in clinical trials for PBC fall into multiple categories: synthetic bile acids, bile acid inhibitors, peroxisome proliferator-activated receptor (PPAR) gamma agonists, and immunosuppressants such as rituximab and abetacept. However, these classes of therapies do not address the underlying cause of liver pathology, which is driven by an antigen-specific autoimmune attack against the liver directly by CD8 T cells and indirectly by antibodies.

[0061] Provided herein is a method of treating PBC in a subject, comprising administering TIMP-PBC to the subject, wherein the TIMP-PBC is administered at a dose level determined based on the subject's body weight. It is also contemplated that the TIMP-PBC may be administered at a fixed dose, regardless of the subject's body weight. In various embodiments, a method of treating PBC in a subject, comprising administering TIMP-PBC to the subject, wherein the TIMP-PBC is administered at a dose of 0.01-12 mg / kg based on the subject's body weight, or at a fixed dose of 1 mg-800 mg, is contemplated. Also provided herein is a method of reducing an inflammatory immune response to a PBC antigen in a subject suffering from PBC, comprising administering TIMP-PBC to the subject, wherein the TIMP-PBC is administered at a dose of 0.01-12 mg / kg based on the subject's body weight, or at a fixed dose of 1 mg-800 mg.

[0062] Also contemplated is TIMP-PBC being administered at a dose of about 0.01-12 mg / kg, about 0.05-10 mg / kg, about 0.01 to about 5 mg / kg, about 0.1 to about 10 mg / kg, about 1-8 mg / kg, about 1.5-10 mg / kg, about 2-12 mg / kg, about 2-10 mg / kg, about 3-10 mg / kg, about 4-10 mg / kg, about 4-12 mg / kg, or about 5-12 mg / kg. Optionally, TIMP-PBC is administered at a dose of about 0.01 mg / kg, 0.02 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 6 mg / kg, 8.0 mg / kg, 10 mg / kg, or 12 mg / kg. Alternatively, the TIMP-PBC is administered in a dose of about 1 mg, 2 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, or 800 mg. In another embodiment, TIMP-PBC is administered at a concentration of about 0.005 mg / mL to about 50 mg / mL, optionally about 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 3.25 mg / mL, 3.5 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12.5 mg / mL, 15 mg / mL, 17.5 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, or 50 mg / mL.

[0063] It is contemplated that TIMP-PBC is administered in a single dose or multiple doses.In various embodiments, TIMP-PBC is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every six months, once a year, once every two years, once every three years, once every four years, once every five years, once every six years, once every seven years, once every eight years, once every nine years, or once every ten years.In certain embodiments, TIMP-PBC is administered in two doses at weekly intervals.

[0064] In various embodiments, the TIMP-PBC is administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, or orally. It is contemplated that when the TIMP-PBC is injected intravenously, it may be via an intravenous infusion lasting about 1, 2, 3, 4, 5, 6, 7, or 8 hours.

[0065] TIMP-PBC therapy is intended to alleviate, reduce or ameliorate one or more symptoms of PBC, including, but not limited to, hepatitis, cirrhosis, cholestasis, liver dysfunction, liver failure, liver fibrosis, increased liver immune infiltration, elevated bile acid levels, elevated liver enzyme levels, (ALT, ALP, AST, gamma-glutamyl transpeptidase), elevated bilirubin levels, circulating antimitochondrial antibodies (AMA), circulating antinuclear antibodies (ANA), fatigue, itchy skin, pruritus, dry eyes and mouth, abdominal pain, splenomegaly, musculoskeletal pain, edema, fluid accumulation, skin xanthomas, jaundice, hyperpigmentation, osteoporosis, high cholesterol, diarrhea, steatorrhea, hypothyroidism, and weight loss.

[0066] TIMP-PBC therapy is also contemplated to reduce, shorten or ameliorate the duration and severity of an inflammatory immune response to one or more PBC antigens in a subject. Inflammatory immune responses include T cell responses, B cell responses, Th1 responses, myeloid cell responses, and / or antibody responses. In various embodiments, the efficacy of TIMP-PBC in alleviating one or more symptoms of PBC and / or reducing the duration and severity of an inflammatory immune response to one or more PBC antigens is determined from assays of one or more biological samples from a subject as described herein.

[0067] Screening Methods It is contemplated that the induction and maintenance of immune tolerance is monitored in a subject suffering from PBC, and the subject is being treated, or about to be treated, with an antigen-specific tolerization therapy comprising a TIMP encapsulating a PBC antigen as described herein.

[0068] Methods for screening cell types, cytokines, or other tolerance measures from subjects undergoing the tolerization therapy described herein are known in the art. Methods for assessing tolerance are performed using techniques such as flow cytometry, mass cytometry (CyTOF), ELISA, ELISPOT, in vitro or ex vivo cell stimulation assays (including but not limited to cell proliferation assays, macrophage stimulation assays), measurement of autoantibodies, or measurement of immunoglobulin (Ig) serotypes, for example by ImmunoCap assay.

[0069] In various embodiments, the subject's immune tolerance state is determined from assaying one or more biological samples from the subject, including whole blood, peripheral blood, peripheral blood mononuclear cells (PBMCs), serum, plasma, urine, cerebrospinal fluid (CSF), stool, tissue biopsy, and / or bone marrow biopsy. In various embodiments, assaying the biological sample includes analyzing the level and / or presence or absence of cell surface proteins, extracellular proteins, intracellular proteins, nucleic acids, metabolites, enzymes, and / or combinations thereof associated with the disease or disorder.

[0070] The cells assayed from the biological sample include immune cells, non-immune cells, and / or combinations thereof. The immune cells include innate immune cells, adaptive immune cells, and / or combinations thereof. The innate immune cells assayed from the biological sample are antigen-presenting cells (APCs). Exemplary innate immune cells assayed from the biological sample include monocytes, macrophages, neutrophils, granulocytes, dendritic cells, mast cells, eosinophils, basophils, and / or combinations thereof. The adaptive immune cells assayed from the biological sample include effector immune cells such as CD4+ T cells, CD8+ T cells, B cells, NK cells, NK-T cells, and / or combinations thereof. In various embodiments, the T cells are Th1 cells, Th2a cells, Treg cells, and Tr1 cells.

[0071] In certain embodiments, the cells assayed from the biological sample are epithelial cells, stromal cells, endothelial cells, fibroblasts, pericytes, adipocytes, mesenchymal stem cells, hematopoietic stem cells, hematopoietic progenitor cells, hepatocytes, hepatic sinusoidal endothelial cells (LSECs), and / or Kupffer cells.

[0072] One aspect of a subject's immune tolerance state and immune signature is determined by analyzing one or more proteins from one or more biological samples from the subject. In various embodiments, the proteins are cytokines and / or chemokines. In various embodiments, the proteins are cell signaling proteins. In various embodiments, the cytokines and chemokines are IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-12p70, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL- 26, IL-27, IL-27b, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6 , CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23 , CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2(MCP-1), CXCL3(MIP-1α, CXCL4(MIP-1β, CXCL5(RANTES), CX CL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, GM-CSF, IFN-α, IF In various embodiments, the protein is selected from the group consisting of TNF-β, IFN-γ, TNF-α, TGF-β1, TGF-β2, TGF-β3, soluble CD14, and / or combinations thereof. In various embodiments, the protein is a protease. In various embodiments, the protease is an aspartic acid protease, a cysteine ​​protease, a metalloprotease, a serine protease, and / or a threonine protease.In various embodiments, the protease is selected from the group consisting of ADAM1, ADAM2, ADAM7, ADAM8, ADAM9, ADAM10, ADAM11, ADAM12, ADAM15, ADAM17, ADAM18, ADAM19, ADAAM20, ADAM21, ADAM22, ADAM23, ADAM28, ADAM29, ADAM30, ADAM33, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28. In various embodiments, the protein associated with apoptosis is selected from the group consisting of P53, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, BCL-2, BCL-XL, MCL-1, CED-9, A1, BFL1, BAX, BAK, DIVA, BCL-XS, BIK, BIM, BAD, BID, and EGL-1. Several methods for assaying proteins from biological samples have been described in the literature, including enzyme-linked immunosorbent assay (ELISA), Western blot, and mass spectrometry. In various embodiments, the protein is one or more immunoglobulins (Ig). In various embodiments, the Ig is selected from the group consisting of IgA, IgD, IgE, IgM, and / or variants thereof. In various embodiments, the immunoglobulins are antigen-specific.Several methods for detecting immunoglobulins from biological samples have been described in the literature, including ELISA and ImmunoCap.

[0073] One aspect of a subject's immune tolerance status and immune signature is determined by analyzing one or more cell surface proteins from a biological sample. In various embodiments, the cell surface proteins include CD1c, CD2, CD3, CD4, CD5, CD8, CD9, CD10, CD11b, CD11c, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, TACI, CD25, CD27, CD28, CD30, CD30L, CD31, CD32, CD32b, CD34, CD33, CD38, CD39, CD40, CD40-L, CD41b, CD42a, CD42b, CD43, CD44, CD45, CD46, CD47, CD48, CD49, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD61, CD62, CD63, CD64, CD65, CD66, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD77, CD78, CD79, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107, CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD118, CD D45RA, CD47, CD45RA, CD45RO, CD48, CD52, CD55, CD56, CD58, CD61, CD66b, CD69, CD70, CD72, CD79, CD68, CD84, CD86, CD93, CD94, CD95, CRACC, BLAME, BCMA, CD103, CD107, CD112, CD120a, CD120b, CD123, CD125, CD127, CD134, CD135, CD140a, CD141, CD154, CD155, CD160, CD161, CD163, CD 172a, XCR1, CD203c, CD204, CD206, CD207CD226, CD244, CD267, CD268, CD269, CD355, CD358, CRTH2, NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F , NKG2H, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, KIR3DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2D S5, DAP12, KIR3DS, NKp44, NKp46, TCR, BCR, integrin, FcβεRI, MHC-I, MHC-II, IL-1R, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-3Rα, CSF2RB, IL-4R, IL-5Rα, CSF2RB, IL-6Rα, gp130, IL-7Rα, IL-9R, IL-10R, IL-12Rβ1, IL-12Rβ2, IL-13Rα1, IL-13Rα2, IL-15Rα, IL-21R, IL23R, IL-27Rα, IL-31Rα, OSMR,CSF-1R, cell surface IL-15, IL-10Rα, IL-10Rβ, IL-20Rα, IL-20Rβ, IL-22Rα1, IL-22Rα2, IL-22Rβ, IL-28RA, PD -1, PD-1H, BTLA, CTLA-4, PD-L1, PD-L2, 2B4, B7-1, B7-2, B7-H1, B7-H4, B7-DC, DR3, LIGHT, LAIR, LTα1β 2, LTβR, TIM-1, TIM-3, TIM-4, TIGIT, LAG-3, ICOS, ICOS-L, SLAM, SLAMF2, OX-40, OX-40L, GITR, GITRL, TL1A, HVEM, 41-BB, 41BB-L, TL-1A, TRAF1, TRAF2, TRAF3, TRAF5, BAFF, BAFF-R, APRIL, TRAIL, RANK, AIT R, TRAMP, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CLECL9a, DC-SIGN, IGSF4A, SIGLEC, EGFR, PDGFR, VEGFR, FAP, α-SMA, FAS, FAS-L, FC, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, PECAM-1, MICA, MICB, UL16, ULBP1, ULBP2, ILBP3, ULBP4, ULBP5, ULBP6, MULT1, RAE1 α, β, γ, δ, and ε, H60a, H60b, H60c, GPR15, ST2, and / or combinations thereof. Integrins include α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, β8, and / or combinations thereof. TCRs include α, β, γ, δ, ε, ζ chains, and / or combinations thereof. Several methods for assaying cell surface protein expression have been described in the literature, including flow cytometry and mass cytometry (CyTOF).

[0074] In various embodiments, the subject's immune tolerance status and immune signature are determined by analyzing the subject's liver function. In various embodiments, the subject's liver function is determined by assaying total cholesterol, triglycerides, LDL cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyltransferase (GGT), alkaline phosphatase (ALP), albumin, total protein, total bilirubin, globulin, creatine kinase (CK), and lactate dehydrogenase (LDH).

[0075] One aspect of the subject's immune tolerance state and immune signature is determined by analyzing one or more metabolites from a biological sample. In various embodiments, the metabolite is a proinflammatory metabolite. In various embodiments, the metabolite is an anti-inflammatory metabolite. In various embodiments, examples of proinflammatory metabolites include acids, lipids, sugars, amino acids, lactate, trimethylamine N-oxide, O-acetylcreatine, L-carnitine, choline, succinate, glutamine, fatty acids, cholesterol, 3-hydroxybutyrate, 3'-sialyllactose, arachidonic acid, prostaglandins (G2 and H2), PGD2, PGE2, PGF2a, PGI2, TXA2, leukotrienes (A4, B4, C4, D4, E4), kynurenine, 3-hydroxykynurenine, lipoxin A4, and lipoxin B4. In various embodiments, examples of anti-inflammatory metabolites include 2-amino-3-carboxymucone 6-semialdehyde, picolinic acid, anthranilic acid, 3-hydroxylanthranilic acid, glutaryl co-A, NAD+, quinolinic acid, arginine, butyrate, and adenosine.

[0076] Lists of human metabolites that can be assayed from biological samples can be found in the literature, including (Psychogios et al., 2011), (Wishart et al., HMDB: the Human Metabolome Database. Nucleic Acids Res. 2007 Jan;35(Database issue):D521-6, 2007), and the Human Metabalome Database (HMDB), which are incorporated herein by reference.

[0077] In certain embodiments, the tolerance state of the subject is determined by analyzing nucleic acids from a biological sample. In various embodiments, the nucleic acids are DNA and / or RNA, including but not limited to single-stranded DNA, double-stranded DNA, mRNA, rRNA, tRNA, siRNA, miRNA, long non-coding RNA (long ncRNA, lncRNA), non-coding RNA (ncRNA), and mitochondrial RNA. In various embodiments, the tolerance state of the subject is determined by assaying gene expression from a biological sample. In various embodiments, the tolerance state is determined by assaying gene expression associated with immune function, antibodies, foreign body response, metabolism, apoptosis, cell death, necrosis, ferroptosis, autophagy, cell migration, endocytosis, phagocytosis, pinocytosis, tight junction regulation, cell adhesion, differentiation, and / or combinations thereof. In various embodiments, the tolerance state is determined by assaying gene expression associated with immune suppression. In various embodiments, the tolerance state is determined by assaying gene expression associated with immune activation. In various embodiments, the immune tolerance state is determined by assaying gene expression associated with immune regulatory function. In various embodiments, nucleic acid analysis is used to generate immune tolerance signatures. Several methodologies for high-throughput gene expression analysis have been described in the literature, including RNA sequencing (RNA-seq), single-cell RNA sequencing (scRNA-seq), exome sequencing, and microarray-based analysis.

[0078] The biological sample is optionally assayed after in vivo and / or ex vivo stimulation with one or more stimuli, such as an antigen and one or more activating agents. It is contemplated that the T cells, B cells, and immunoglobulins used in the assay are antigen-specific. Exemplary T cells include effector memory T cells, antigen-specific T cells, activated antigen-specific T cells, Th1 cells, Th17 cells, T follicular helper (TFH) cells, TH0 cells, or other antigen-specific T cells. B cells include effector B cells, memory B cells, plasma cells, and Breg cells. In certain embodiments, T cells are identified based on the expression of proteins listed in Table 1. [Table 1]

[0079] In various embodiments, the subject's immune tolerance state is determined by obtaining one or more samples, such as whole blood, from the subject from before the first dose on the day of administration of TIMP-PBC (day 1), 14 days after administration of the second dose, and then 90 days after the second dose (e.g., 90 days, 180 days, 270 days, and 360 days after the second dose). The whole blood can then be processed to isolate peripheral blood mononuclear cells (PBMCs), basophils, neutrophils, plasma, and serum for downstream analysis. The cells isolated from the one or more samples taken from the subject are assayed and analyzed using methods such as those described below.

[0080] In various embodiments, the subject's immune tolerance state determined prior to administration of TIMP-PBC serves as a baseline. In various embodiments, the subject's baseline is determined from assay of one or more biological samples 1, 2, 3, 4, 5, 6, or 7 days prior to administration of TIMP-PBC. In various embodiments, the subject's baseline is determined from assay of one or more biological samples 1, 2, 3, or 4 weeks prior to administration of TIMP-PBC. In various embodiments, the subject's baseline is determined from assay of one or more biological samples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months prior to administration of TIMP-PBC.

[0081] In various embodiments, the subject's immune tolerance status is determined after administration of TIMP-PBC. In various embodiments, the subject's immune tolerance status is determined from assay of one or more biological samples 1, 2, 3, 4, 5, 6, or 7 days after administration of TIMP-PBC. In various embodiments, the subject's baseline is determined from assay of one or more biological samples 1, 2, 3, or 4 weeks after administration of TIMP-PBC. In various embodiments, the subject's baseline is determined from assay of one or more biological samples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration of TIMP-PBC. In various embodiments, the subject's immune tolerance status determined after administration of TIMP-PBC is compared to a baseline. In various embodiments, the subject's immune tolerance status determined after administration of TIMP-PBC is compared to a healthy subject.

[0082] In various embodiments, the subject's immune tolerance status is determined based on the assessment of liver fibrosis. In various embodiments, liver fibrosis is assessed by imaging. In various embodiments, liver fibrosis is assessed by FibroScan, ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), magnetic resonance elastography (MRA), ultrasound elastography, transient elastography, point shear wave elastography, or two-dimensional shear wave elastography. In various embodiments, liver fibrosis is assessed based on one or more biochemical markers. In various embodiments, the biochemical markers are from whole blood, peripheral blood, peripheral blood mononuclear cells (PBMCs), serum, plasma, urine, cerebrospinal fluid (CSF), stool, tissue biopsy, and / or bone marrow biopsy. In various embodiments, the biochemical markers are identified from the assay of cells, proteins, peptides, enzymes, metabolites, cells, and / or one or more biochemical substances. In various embodiments, the protein is a cytokine, a chemokine, a protease, a cell surface protein, a soluble protein, and / or an antibody. In various embodiments, the cell is an immune cell or a non-immune cell. In various embodiments, the immune cell is a T cell, a B cell, a NK cell, a NKT cell, a monocyte, a macrophage, a dendritic cell, an eosinophil, or a basophil. In various embodiments, the cell is an epithelial cell, a stromal cell, an endothelial cell, a fibroblast, a pericyte, an adipocyte, a mesenchymal stem cell, a hematopoietic stem cell, a hematopoietic progenitor cell, a hepatocyte, a hepatic sinusoidal endothelial cell (LSEC), an erythrocyte, a platelet, and / or a Kupffer cell. In various embodiments, the biochemical markers are total cholesterol, triglycerides, LDL cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyl transferase (GGT), alkaline phosphatase (ALP), albumin, total protein, total bilirubin, globulin, creatine kinase (CK), and lactate dehydrogenase (LDH), hyaluronic acid, PIINP, TIMP-1, type 4 collagen 7S, ProC3, or M2BPGi.In various embodiments, the metabolite is an acid, a lipid, a sugar, an amino acid, lactate, trimethylamine N-oxide, O-acetylcreatine, L-carnitine, choline, succinate, glutamine, fatty acid, cholesterol, 3-hydroxybutyrate, 3'-sialyllactose, arachidonic acid, prostaglandins (G2 and H2), PGD2, PGE2, PGF2a, PGI2, TXA2, leukotrienes (A4, B4, C4, D4, E4), kynurenine, 3-hydroxykynurenine, lipoxin A4, lipoxin B4, 2-amino-3-carboxymucon 6-semialdehyde, picolinic acid, anthranilic acid, 3-hydroxyanthranilic acid, glutaryl co-A, NAD+, quinolinic acid, arginine, butyrate, and adenosine.

[0083] A subject's immune tolerance signature is generated using one or more of the following parameters assayed from one or more biological samples obtained from the subject and stimulated in vivo and / or ex vivo: A. The proportion of effector T cells in the total T cell population. B. The percentage of Treg cells in the total T cell population. C. The proportion of effector B cells in the total B cell population; D. Specific IgG and / or IgM levels; E. Levels of inflammatory cytokines and chemokines; F. Anti-inflammatory cytokine and chemokine levels G. Liver enzyme levels H. Levels of inflammatory metabolites, and I. Levels of anti-inflammatory metabolites.

[0084] The immune tolerance signature is indicative of the maintenance of immune tolerance if 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the parameters listed in (a)-(i) above are indicative of the maintenance of immune tolerance. In various embodiments, the immune tolerance signature is indicative of the maintenance of immune tolerance if at least 2 / 9 of the parameters listed in (a)-(i) above are indicative of the maintenance of immune tolerance. In various embodiments, the subject is determined to not require treatment with a TIMP if 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the parameters listed in (a)-(i) above are indicative of the maintenance of immune tolerance. In various embodiments, the subject is determined to not require treatment with a TIMP if at least 3 / 9 of the parameters listed in (a)-(i) above are indicative of the maintenance of immune tolerance.

[0085] A subject's immune tolerance signature generated using one or more of the parameters described herein is indicative of weakened and / or absent immune tolerance before or after treatment with TIMP-PBC if: a. the percentage of effector T cells in the total T cell population is between 0.01% and 100% (e.g., about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (including all values ​​and ranges therebetween) relative to the subject's baseline measurement and / or relative to a healthy subject; and / or b. the percentage of T cells in the total T cell population is 0.01-100% (e.g., about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (including all values ​​and ranges therebetween) relative to the subject's baseline measurement and / or relative to a healthy subject; and / or c. the percentage of effector B cells in the total B cell population is between 0.01% and 100% (e.g., about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (including all values ​​and ranges therebetween) relative to the subject's baseline measurement and / or relative to a healthy subject; and / or d. The levels of IgG and / or IgM are about 0.01% to 100% (e.g., about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (including all values ​​and ranges between those values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% or about 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges between those values)) and / or e. The level of inflammatory cytokines / chemokines is increased or decreased by about 0.01% to 100% (e.g., about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%) relative to the subject's baseline measurement and / or relative to a healthy subject. , about 90%, about 95%, or about 100% (including all values ​​and ranges between those values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% or about 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges between those values)); and / or f. The levels of anti-inflammatory cytokines and chemokines are increased by about 0.01% to 100% (e.g., about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95 ... %, about 90%, about 95%, or about 100% (including all values ​​and ranges between those values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% or about 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges between those values)); and / or g. The level of liver enzymes is increased or decreased by about 0.01% to 100% (e.g., about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 100%, about 15 ... an increase of about 95%, or about 100% (including all values ​​and ranges between those values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% or about 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges between those values)); and / or h. The level of an inflammatory metabolite is increased or decreased by about 0.01% to 100% (e.g., about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 100%, about 15 ... %, about 95%, or about 100% (including all values ​​and ranges between those values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% or about 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges between those values), and / or i. the level of an anti-inflammatory metabolite is increased or decreased by about 0.01% to 100% (e.g., about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, A decrease of about 90%, about 95%, or about 100% (including all values ​​and ranges between those values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% or about 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges between those values).

[0086] The efficacy of TIMP-PBC in alleviating one or more symptoms of PBC and / or reducing the duration and severity of the inflammatory immune response to PBC antigens is determined from assaying one or more biological samples from a subject. Biological samples include whole blood, peripheral blood, peripheral blood mononuclear cells (PBMC), serum, plasma, urine, cerebrospinal fluid (CSF), stool, tissue biopsy, and / or bone marrow biopsy. In various embodiments, assaying the biological sample includes analyzing the levels and / or presence or absence of cell surface proteins, extracellular proteins, intracellular proteins, nucleic acids, metabolites, enzymes, and / or combinations thereof.

[0087] In various embodiments, the efficacy of TIMP-PBC in alleviating one or more symptoms of PBC and / or reducing the duration and severity of an inflammatory immune response to a PBC antigen is determined based on assaying cells from one or more biological samples from a subject before and after treatment with TIMP-PBC. In various embodiments, the cells are immune cells, non-immune cells, and / or combinations thereof. In various embodiments, the immune cells include innate immune cells, adaptive immune cells, and / or combinations thereof. The innate immune cells assayed from the biological sample are antigen-presenting cells (APCs). Exemplary innate immune cells assayed from the biological sample include monocytes, macrophages, neutrophils, granulocytes, dendritic cells, mast cells, eosinophils, basophils, and / or combinations thereof. The adaptive immune cells assayed from the biological sample include effector immune cells such as CD4+ T cells, CD8+ T cells, B cells, NK cells, NK-T cells, and / or combinations thereof. In various embodiments, the T cells are Th1 cells, Th2a cells, Treg cells, and Tr1 cells.

[0088] In certain embodiments, the cells assayed from the biological sample are epithelial cells, stromal cells, endothelial cells, fibroblasts, pericytes, adipocytes, mesenchymal stem cells, hematopoietic stem cells, hematopoietic progenitor cells, hepatocytes, hepatic sinusoidal endothelial cells (LSECs), and / or Kupffer cells.

[0089] In various embodiments, the efficacy of TIMP-PBC in alleviating one or more symptoms of PBC and / or reducing the duration and severity of an inflammatory immune response to one or more PBC antigens is determined based on assays of cell surface proteins from one or more biological samples from a subject before and after treatment with TIMP-PBC. In various embodiments, the cell surface proteins are CD1c, CD2, CD3, CD4, CD5, CD8, CD9, CD10, CD11b, CD11c, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, TACI, CD25, CD27, CD28, CD30, CD30L, CD31, CD32, CD32b, CD34, CD33, CD38, CD39, CD40, CD40-L, CD41b, CD42a, CD42b, CD42c, CD42d, CD42e, CD42f, CD42g, CD42h, CD42h, CD42i, CD42m ... 2b, CD43, CD44, CD45, CD45RA, CD47, CD45RA, CD45RO, CD48, CD52, CD55, CD56, CD58, CD61, CD66b, CD69, CD70, CD72, CD79, CD68, C D84, CD86, CD93, CD94, CD95, CRACC, BLAME, BCMA, CD103, CD107, CD112, CD120a, CD120b, CD123, CD125, CD127, CD134, CD135, CD14 0a, CD141, CD154, CD155, CD160, CD161, CD163, CD172a, XCR1, CD203c, CD204, CD206, CD207CD226, CD244, CD267, CD268, CD269, C D355, CD358, CRTH2, NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL 1, KIR3DL2, KIR3DL3, KIR3DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, DAP12, KIR3DS, NKp44, NKp46, TCR, BCR, integrin, FcβεRI, MHC-I, MHC-II, IL-1R, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-3Rα, CSF2RB, IL-4R, IL-5Rα, CSF2RB, IL-6Rα, gp130, IL-7Rα, IL-9R,IL-10R, IL-12Rβ1, IL-12Rβ2, IL-13Rα1, IL-13Rα2, IL-15Rα, IL-21R, IL23R, IL-27Rα, IL-31Rα, OSMR, CSF-1R, cell surface IL-15, IL-10Rα, IL-10Rβ, IL-20Rα, IL-20Rβ, IL-22Rα1, IL-22Rα2, IL-22Rβ, IL-28RA, PD-1, PD-1H, BTLA, CTLA-4, P D-L1, PD-L2, 2B4, B7-1, B7-2, B7-H1, B7-H4, B7-DC, DR3, LIGHT, LAIR, LTα1β2, LTβR, TIM-1, TIM-3, TIM-4, TIGIT, LA G-3, ICOS, ICOS-L, SLAM, SLAMF2, OX-40, OX-40L, GITR, GITRL, TL1A, HVEM, 41-BB, 41BB-L, TL-1A, TRAF1, TRAF2, TRAF 3, TRAF5, BAFF, BAFF-R, APRIL, TRAIL, RANK, AITR, TRAMP, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR 10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CLECL9a, DC-SIGN, IGSF4A, SIGLEC, EGFR, PDGFR, VEGFR , FAP, α-SMA, FAS, FAS-L, FC, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, PECAM-1, MICA, MICB, UL16, ULBP1, ULBP2, ILBP3, ULBP4, ULBP5, ULBP6, MULT1, RAE1α, β, γ, δ, and ε, H60a, H60b, H60c, GPR15, ST2, and / or combinations thereof. Integrins include α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, β8, and / or combinations thereof. TCRs include α, β, γ, δ, ε, ζ chains and / or combinations thereof. Several methods for assaying cell surface protein expression have been described in the literature, including flow cytometry and mass cytometry (CyTOF).

[0090] In various embodiments, treatment with TIMP-PBC reduces expression of inflammatory cell surface proteins by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (including all values ​​and ranges therebetween) relative to the subject's baseline measurement and / or relative to a healthy subject). In various embodiments, the cell surface protein is reduced by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges therebetween). In various embodiments, the cell surface protein is CD14.

[0091] In various embodiments, treatment with TIMP-PBC reduces expression of anti-inflammatory cell surface proteins by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%) relative to a subject's baseline measurement and / or relative to a healthy subject. or 50% or about 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges between these values).

[0092] In various embodiments, the efficacy of TIMP-PBC in alleviating one or more symptoms of PBC and / or reducing the duration and severity of an inflammatory immune response to a PBC antigen is determined based on assays of proteins from one or more biological samples from a subject before and after treatment with TIMP-PBC. In various embodiments, the proteins are cytokines and / or chemokines. In various embodiments, the proteins are cell signaling proteins. In various embodiments, the cytokines and chemokines are IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-12p70, IL-13, IL-14, IL-15, IL-16, IL-17, IL-17p70, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL-57, IL-58, IL-59, IL-60, IL-61, IL-62, IL-63, IL-64, IL- L-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-27b, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL 12, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2(MCP-1), CXCL3(MIP-1α, CXCL4(MIP-1β, CXCL5(RANTES), CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, GM-CSF, IFN-α, IFN-β, IFN-γ, TNF-α, TGF-β1, TGF-β2, TGF-β3, and / or combinations thereof.

[0093] In various embodiments, the protein is a protease. In various embodiments, the protease is an aspartic acid protease, a cysteine ​​protease, a metalloprotease, a serine protease, and / or a threonine protease. In various embodiments, the protease is selected from the group consisting of ADAM1, ADAM2, ADAM7, ADAM8, ADAM9, ADAM10, ADAM11, ADAM12, ADAM15, ADAM17, ADAM18, ADAM19, ADAAM20, ADAM21, ADAM22, ADAM23, ADAM28, ADAM29, ADAM30, ADAM33, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28. In various embodiments, the protein associated with apoptosis is selected from the group consisting of P53, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, BCL-2, BCL-XL, MCL-1, CED-9, A1, BFL1, BAX, BAK, DIVA, BCL-XS, BIK, BIM, BAD, BID, and EGL-1. Several methods for assaying proteins from biological samples have been described in the literature, including enzyme-linked immunosorbent assay (ELISA), Western blot, and mass spectrometry. In various embodiments, the protein is one or more immunoglobulins (Ig). In various embodiments, the Ig is selected from the group consisting of IgA, IgD, IgE, IgM, and / or variants thereof. In various embodiments, the immunoglobulins are antigen-specific.Several methods for detecting immunoglobulins from biological samples have been described in the literature, including ELISA and ImmunoCap.

[0094] In various embodiments, treatment with TIMP-PBC reduces levels of inflammatory proteins by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%) relative to a subject's baseline measurement and / or relative to a healthy subject. or about 2 to 1 / 100 (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values). Treatment with TIMP-PBC reduces levels of anti-inflammatory proteins by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (e.g., by about these values) relative to the subject's baseline measurement and / or relative to a healthy subject). 55%, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges between those values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% or about 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges between those values).

[0095] In various embodiments, treatment with TIMP-PBC increases the levels of antinuclear antibodies (ANA), anti-Gp210 antibodies, anti-Sp100 antibodies, IgM, or antimitochondrial antibodies by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%) relative to the baseline measurement in a subject and / or relative to a healthy subject. , about 95%, or about 100% (including all values ​​and ranges between these values)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% or about 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges between these values)).

[0096] In various embodiments, the efficacy of TIMP-PBC in alleviating one or more symptoms of PBC and / or reducing the duration and severity of an inflammatory immune response to a PBC antigen is determined based on assaying metabolites from one or more biological samples from a subject before and after treatment with TIMP-PBC. In various embodiments, the metabolite is a pro-inflammatory metabolite. In various embodiments, the metabolite is an anti-inflammatory metabolite. In various embodiments, examples of proinflammatory metabolites include acids, lipids, sugars, amino acids, lactate, trimethylamine N-oxide, O-acetylcreatine, L-carnitine, choline, succinate, glutamine, fatty acids, cholesterol, 3-hydroxybutyrate, 3'-sialyllactose, arachidonic acid, prostaglandins (G2 and H2), PGD2, PGE2, PGF2a, PGI2, TXA2, leukotrienes (A4, B4, C4, D4, E4), kynurenine, 3-hydroxykynurenine, lipoxin A4, and lipoxin B4. In various embodiments, examples of anti-inflammatory metabolites include 2-amino-3-carboxymucon 6-semialdehyde, picolinic acid, anthranilic acid, 3-hydroxylanthranilic acid, glutaryl co-A, NAD+, quinolinic acid, arginine, butyrate, and adenosine. Treatment with TIMP-PBC may reduce levels of inflammatory metabolites by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (all values ​​in between) relative to the subject's baseline measurement and / or relative to a healthy subject. or 50% or about 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges therebetween).In various embodiments, treatment with TIMP-PBC increases the level of anti-inflammatory metabolites by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about ...30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%) relative to the subject's baseline measurement and / or relative to a healthy subject. 55%, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges between those values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% or about 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges between those values).

[0097] In various embodiments, the efficacy of TIMP-PBC in alleviating one or more symptoms of PBC and / or reducing the duration and severity of the inflammatory immune response to PBC antigens is determined based on an assay of liver fibrosis from one or more liver biopsies. In various embodiments, the efficacy of TIMP-PBC in alleviating one or more symptoms of PBC and / or reducing the duration and severity of the inflammatory immune response to PBC antigens is determined based on an assessment of liver fibrosis by imaging. In various embodiments, liver fibrosis is assessed by FibroScan, ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), magnetic resonance elastography (MRA), ultrasound elastography, transient elastography, point shear wave elastography, or two-dimensional shear wave elastography.

[0098] In various embodiments, the efficacy of TIMP-PBC in alleviating one or more symptoms of PBC and / or reducing the duration and severity of the inflammatory immune response to PBC antigens is determined based on assaying the level of hepatic immune infiltration from one or more liver biopsies.

[0099] In various embodiments, the liver biopsy is a core liver biopsy. In various embodiments, treatment with TIMP-PBC reduces liver fibrosis by about 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about ...30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 6 or 50% or about 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges between those values).

[0100] In various embodiments, treatment with TIMP-PBC increases liver immune infiltration by about 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (e.g., about these values) relative to a subject's baseline measurement and / or relative to a healthy subject. 55%, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges between those values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% or about 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges between those values).

[0101] In various embodiments, the efficacy of TIMP-PBC in alleviating one or more symptoms of PBC and / or reducing the duration and severity of the inflammatory immune response to PBC antigens is determined based on the subject's score on a clinical disease scale. In various embodiments, the clinical disease scale is the PBC-40 scale, modified PBC-40 scale, Improved Liver Fibrosis (ELS) score, PBC-27 scale, GLOBE scale, UK-PBC scale, Average Worst Daily Itch Scale, 5-D Itch Scale, Visual Analog Scale (VAS), Scheuer scale, Nakanuma staging, fibrosis score, bile duct loss score, FIB-4 index, or APRI. Several clinical disease scoring systems have been described in the literature and references can be found in: 8~10 and is incorporated herein by reference.

[0102] In various embodiments, the efficacy of TIMP-PBC in alleviating one or more symptoms of PBC and / or reducing the duration and severity of the inflammatory immune response to PBC antigens is determined based on assessment of the following before and after administration of TIMP-PBC: A. Levels of infiltrating T cells from core liver biopsies. B. Levels of antigen-specific IFN-γ production in the blood. C. Serum ALP levels. D. Serum bilirubin levels. E. Serum ALT levels. F. Serum AST levels. G. Serum GGT levels. H. Levels of anti-Gp210 antibodies in the blood. I. Levels of anti-Sp100 antibodies in the blood. J. Serum soluble CD14 levels. K. Serum kynurenine levels. L. Serum IgM levels. M. Improved Liver Fibrosis (ELF) score. N. Levels of hepatitis and fibrosis in liver core biopsies. O. Average daily worst itch score. P.PBC-40 scale score.

[0103] The efficacy of TIMP-PBC in ameliorating one or more symptoms of PBC is determined from one or more of the following parameters assayed from one or more biological samples obtained from a subject: A. The proportion of effector T cells in the total T cell population. B. The percentage of Treg cells in the total T cell population. C. The proportion of effector B cells in the total B cell population; D. Specific IgG and / or IgM levels; E. Levels of inflammatory cytokines and chemokines; F. Anti-inflammatory cytokine and chemokine levels G. Liver enzyme levels H. Levels of inflammatory metabolites, and I. Levels of anti-inflammatory metabolites.

[0104] In various embodiments, the efficacy of TIMP-PBC for ameliorating one or more symptoms of PBC is determined from the results of assays of 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the parameters listed in (a)-(i) above.

[0105] In various embodiments, the efficacy of TIMP-PBC to ameliorate one or more symptoms of PBC is identified based on HLA haplotype, the presence / level of anti-mitochondrial antibodies, and the level of alkaline phosphatase in the blood.

[0106] In various embodiments, the efficacy of TIMP-PBC in improving one or more symptoms of PBC and / or reducing the duration and severity of the inflammatory response to PBC antigens is determined based on the use of an alternative PBC therapy. In various embodiments, administration of TIMP-PBC reduces the use of an alternative PBC therapy. In various embodiments, the therapy is a steroid, a corticosteroid, a nonsteroidal immunosuppressant, an immunomodulatory agent, a monoclonal antibody, a cytokine and chemokine targeted therapy, a JAK inhibitor, a chimeric antigen receptor (CAR) T cell therapy, a regulatory T cell (Treg) therapy, a B cell targeted therapy, an antiviral drug, or a surgical treatment.

[0107] In various embodiments, the therapy is selected from the group consisting of ursodeoxycholic acid (UDCA), obeticholic acid (OCA), fibrates, peroxisome proliferator-activated receptor delta (pparδ) agonists, ileal bile acid transporter (IBAT) inhibitors, tauroursodeoxycholic acid, 6α-ethylchenodeoxycholic acid, setanaxib, moexipril, abatacept, fibroblast growth factors, statins, corticone, ustekinumab, CD20 targeted therapy, CD19 targeted therapy, CD40 / CD40L targeted therapy, CD80 / CD86 targeted therapy, and the like. Therapeutic options include chemotherapy, B-cell targeting factor (BAFF) targeted therapy, B-cell maturation antigen (BCMA) targeted therapy, cytokine / chemokine inhibitors, anti-IL6 therapy, anti-IFN therapy, amifampridine, cyclosporine, cyclophosphamide, batoclimab, inebilizumab, nipocalimab, pozelimab, rituximab, satralizumab, seladelpar, pentoxifylline, tocilizumab, tofacitinib, trebrutinib, abolstatin, fenofibrate, bezafibrate, linelixibat, methotrexate, or liver transplant.

[0108] In various embodiments, the steroid or corticosteroid is selected from the group including beclomethasone, cyclosonide, fluticasone furoate, mometasone, budenoside, fluticasone, triamcinolone, loteprednol, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, betamethasone, or hydrocortisone.

[0109] In various embodiments, administration of TIMP-PBC reduces use of alternative PBC therapies by 1%-100% (e.g., about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges therebetween), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% relative to a subject's baseline measurement and / or relative to a healthy subject. In various embodiments, the use of alternative PBC therapy is reduced or eliminated 1, 2, 3, 4, 5, 6, or 7 days after administration of TIMP-PBC. In various embodiments, the use of alternative PBC therapy is reduced or eliminated 1, 2, 3, or 4 weeks after administration of TIMP-PBC. In various embodiments, the use of alternative PBC therapy is reduced or eliminated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration of TIMP-PBC. In various embodiments, the use of alternative PBC therapy is reduced or eliminated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years after administration of TIMP-PBC.

[0110] The cells assayed from the biological sample include immune cells, non-immune cells, and / or combinations thereof. The immune cells include innate immune cells, adaptive immune cells, and / or combinations thereof. The innate immune cells assayed from the biological sample are antigen-presenting cells (APCs). Exemplary innate immune cells assayed from the biological sample include monocytes, macrophages, neutrophils, granulocytes, dendritic cells, mast cells, eosinophils, basophils, and / or combinations thereof. The adaptive immune cells assayed from the biological sample include effector immune cells such as CD4+ T cells, CD8+ T cells, B cells, NK cells, NK-T cells, and / or combinations thereof. In various embodiments, the T cells are Th1 cells, Th2a cells, Treg cells, and Tr1 cells.

[0111] In certain embodiments, the cells assayed from the biological sample are epithelial cells, stromal cells, endothelial cells, fibroblasts, pericytes, adipocytes, mesenchymal stem cells, hematopoietic stem cells, hematopoietic progenitor cells, hepatocytes, hepatic sinusoidal endothelial cells (LSECs), and / or Kupffer cells.

[0112] Combination therapy Concomitant administration of two therapeutic agents does not require that the agents be administered at the same time or by the same route, so long as there is an overlap in the period during which the agents are exerting their therapeutic effect. Prior, simultaneous or sequential administration is contemplated, as is administration on different days or weeks.

[0113] It is contemplated that the TIMP-PBC and the PBC therapeutic agent may be administered concomitantly or simultaneously, in the same formulation or in separate formulations. It is further contemplated that the TIMP-PBC and the PBC therapeutic agent may be administered in separate formulations, concomitantly, where concomitant refers to the agents being administered within 30 minutes to 12 hours of each other.

[0114] In another embodiment, the TIMP-PBC and PBC therapeutic are administered prior to administration of the other composition, where pre-administration refers to administration of the TIMP-PBC and PBC therapeutic within one week prior to treatment with the other therapy and up to 30 minutes prior to administration of the other therapy.

[0115] In another embodiment, the TIMP-PBC and PBC therapeutic are administered subsequent to administration of the other composition, where subsequent administration refers to administration of the TIMP-PBC and PBC therapeutic within one week of treatment with the other therapy, and up to 30 minutes after administration of the other therapy.

[0116] It is further contemplated that other adjunctive or alternative therapies may be administered as appropriate.

[0117] In various embodiments, TIMP-PBC is administered alone or in combination with a PBC therapeutic agent. In various embodiments, the therapeutic agent is administered prior to, concomitantly with, or subsequent to administration of TIMP-PBC.

[0118] In various embodiments, the therapeutic agent is selected from the group consisting of steroids, corticosteroids, nonsteroidal immunosuppressants, immunomodulatory agents, monoclonal antibodies, cytokine and chemokine targeted therapies, JAK inhibitors, chimeric antigen receptor (CAR) T cell therapies, regulatory T cell (Treg) therapies, B cell targeted therapies, antiviral agents, and surgical treatments. In various embodiments, the therapeutic agent is selected from the group consisting of ursodeoxycholic acid (UDCA), obeticholic acid (OCA), fibrates, peroxisome proliferator-activated receptor delta (pparδ) agonists, ileal bile acid transporter (IBAT) inhibitors, ustekinumab, tauroursodeoxycholic acid, 6α-ethylchenodeoxycholic acid, setanaxib, moexipril, abatacept, fibroblast growth factors, statins, corticone, CD20 targeted therapy, CD19 targeted therapy, CD40 / CD40L targeted therapy, CD80 / CD86 targeted therapy, B cell targeting factor (BAFF) targeted therapy, B cell maturation antigen (BCMA) targeted therapy, anti-IL6 therapy, anti-IFN therapy, amifamycin, cetanaxib ... aztreonam, cyclosporine, cyclophosphamide, batoclimab, inebilizumab, nipocalimab, pozelimab, rituximab, satralizumab, seladelpar, pentoxifylline, tocilizumab, tofacitinib, trebrutinib, abolstatin, fenofibrate, bezafibrate, linelixibat, methotrexate, liver transplant, beclomethasone, ciclesonide, fluticasone fluritol, mometasone, budenoside, fluticasone, triamcinolone, loteprednol, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, betamethasone, and hydrocortisone.

[0119] In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of TIMP-PBC. In various embodiments, the therapeutic agent is administered 1, 2, 3, or 4 weeks prior to administration of TIMP-PBC. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months prior to administration of TIMP-PBC. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years prior to administration of TIMP-PBC. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, or 7 days after administration of TIMP-PBC. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, or 7 weeks after administration of TIMP-PBC. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration of TIMP-PBC.In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years after administration of TIMP-PBC.

[0120] Pharmaceutical preparations The pharmaceutical composition of the present disclosure containing the TIMP-PBC described herein as an active ingredient may contain a pharma- ceutically acceptable carrier or additive depending on the route of administration. Examples of such carriers or additives include water, pharma- ceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacryl, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, pharma-ceutically acceptable surfactants, etc. The additives used may be selected from the above or combinations thereof as necessary depending on the dosage form of the present disclosure, but are not limited thereto.

[0121] The formulation of pharmaceutical compositions will vary according to the route of administration (e.g., solution, emulsion) selected. The appropriate composition containing the therapeutic agent to be administered can be prepared in a physiologically acceptable vehicle or carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's oil or fixed oils. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers.

[0122] A variety of aqueous carriers include, for example, sterile phosphate buffered saline, bacteriostatic water, water, buffered water, 0.4% saline, 0.3% glycine, and the like, and may contain other proteins to enhance stability, such as albumin, lipoproteins, globulins, etc., that have been mildly chemically modified.

[0123] Therapeutic formulations of the inhibitors are prepared for storage by mixing the inhibitors having the desired purity, in the form of a lyophilized formulation or aqueous solution, with any physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride); hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin, or other soluble amines. or proteins such as immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0124] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0125] Aqueous suspensions may contain the active compound in a mixture with excipients suitable for the manufacture of aqueous suspensions.Such excipients are suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents are naturally occurring phosphatides, such as lecithin, or condensation products of alkylene oxides with fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide with long-chain aliphatic alcohols, such as heptadecaethyleneoxycetanol, and hexitols, such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids, and hexitol anhydrides, such as polyethylene sorbitan monooleate.Aqueous suspensions may also contain one or more preservatives, such as ethyl or n-propyl, p-hydroxybenzoate.

[0126] The TIMP-PBCs described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use.

[0127] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the modified particles are mixed with at least one inert pharma- ceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants, such as glycerol, d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents, such as paraffin, f) absorption accelerators, such as quaternary ammonium compounds, g) humectants, such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents, such as kaolin and bentonite clay, and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0128] kit As a further aspect, the present disclosure includes a kit comprising one or more compounds or compositions packaged in a manner that facilitates their use to practice the methods of the present disclosure. In one embodiment, such a kit comprises a compound or composition described herein (e.g., a composition comprising a TIMP alone or in combination with a second agent) packaged in a container, such as a sealed bottle or container, with a label affixed to the container or included in a package that describes the use of the compound or composition in practicing the method. Preferably, the compound or composition is packaged in a unit dosage form. The kit may further comprise a device suitable for administering the composition according to a particular route of administration or for carrying out a screening assay. Preferably, the kit comprises a label that describes the use of the inhibitor composition.

[0129] In a further embodiment, the disclosure provides an article of manufacture or unit dosage form comprising: (a) a composition of matter comprising TIMP-PBC as described herein; (b) a container containing the composition; and (c) a label affixed to, or package insert included with, the container, referencing use of the TIMP-PBC in the treatment of PBC as described herein.

[0130] Additional aspects and details of the present disclosure will be apparent from the following examples, which are intended to be illustrative rather than limiting. EXAMPLES

[0131] Example 1: TIMP-PBCs inhibit antigen-specific T cell responses in animal models PBC antigen PDC-E2 155~185 The efficacy of TIMP-PBC (CNP-104) encapsulating (KVGEKLSEGDLLAEIETDKATIGFEVQEEGY) (SEQ ID NO: 1) in inducing T cell tolerance was examined in a therapeutic model of PBC.

[0132] In rodents, the PBC model can be induced with the chemical xenobiotic 2OA BSA, which induces a PBC-like cholangitis in C57BL / 6 mice. In the PBC model, mice develop PDC-E2-specific anti-mitochondrial antibodies (AMA) in the serum as well as infiltration of pathogenic T cells in the hepatic bile duct mimicking the human disease. The addition of poly(I:C) treatment to this model enhances CD8 T cell infiltration and leads to liver fibrosis, a hallmark of human PBC.

[0133] On the day of the experiment (day 0), naïve 6-week-old C57BL / 6 mice were primed with 2OA-BSA by intraperitoneal (ip) injection. The 2OA-BSA ip priming was repeated with incomplete Freund's adjuvant (IFA) on day 14.

[0134] On days 0 and 2, mice were injected ip with 100 ng of pertussis toxin (PTX). On days 0 and 14, mice were injected subcutaneously (sc) with 5 mg / kg of poly(I:C). On day 7, mice were injected with PDC-E2 155~185 On days 7 and 14, mice were treated with CNP-104 (2.5 mg dose / mouse or approximately 10 mg / kg HED) or control nanoparticles.

[0135] On day 22, mice from each treatment group (N=5 / group) were sacrificed and their spleens were harvested to assess antigen-specific T cell responses and serum anti-mitochondrial antibody (AMA) titers.

[0136] Antigen-specific T cell response from splenocytes: 5×10 5 Total splenocytes were incubated with anti-CD3 antibody (0.5 μg / mL) and anti-CD28 (1 μg / mL) (positive control), or with T cell-specific PDC-E2 155~185 (titrated to 1.5 μg / mL, 15 μg / mL, 75 μg / mL) were plated in triplicate wells in 96-well flat bottom plates with complete RPMI medium containing 10 μg / mL IgG1.

[0137] Cells were harvested 72 hours after culture setup and cell proliferation levels were assayed via WST-1 cell proliferation reagent (Roche, Cat. No. 11644807001) according to the manufacturer's protocol.

[0138] As shown in Figure 1, CNP-104 treatment significantly reduced the expression of PDC-E2 compared to control unloaded nanoparticle treatment. 155~185 There was a significant inhibition of T cell responses (p<0.0001).

[0139] Antimitochondrial antibody titers from blood: Whole blood was collected from mice via retro-orbital route and placed in serum collection tubes. Blood was allowed to clot for 2 hours at room temperature. Serum was collected by centrifugation (3,000 g for 5 minutes at 4°C). AMA levels were assayed using the QUANTA Lite® M2 EP (MIT3) ELISA kit using PDC-E2 coated microwells / strips according to the manufacturer's protocol. Mouse serum samples were added to wells in duplicate (100 μL sample / well). Plates were incubated for 1 hour at room temperature and then washed 4 times with 1× PBS + 0.05% Tween-20.

[0140] IgG AMA was detected by adding anti-mouse IgG HRP secondary antibody (1:4000 dilution in 1× PBS+5% BSA) to each well, followed by incubation for 1 hour at room temperature.

[0141] The plates were washed 4 times with 1x PBS + 0.05% Tween-20. 100 μL of TMP one-component HRP microwell substrate was added to each well and developed at room temperature until sufficient color development occurred. 100 μL of stop reagent was added to stop the reaction and the plates were read at 450 nm using an ELISA plate reader.

[0142] Optical density readings were recorded for all samples tested. As shown in Figure 2, treatment with CNP-104 resulted in a decrease in serum IgG AMA titers when compared to control unloaded nanoparticles.

[0143] Delayed type hypersensitivity: C57BL / 6 mice were primed with 200 μg PDC-E2-peptide emulsified in CFA (100 μL injection volume) on day 0. On days 0 and 7 after priming, mice were treated with CNP104 lot representative of a GLP manufacturing process administered via IV infusion at a dose of 2.5 mg dose / mouse (10 mg / kg HED). On day 14 after priming, mice were challenged intradermally with 10 μg PDC-E2-peptide (right ear, 10 μL injection volume) or ovalbumin (left ear, 10 μL injection volume). Pinna thickness was measured for each ear immediately after and 24 hours after challenge with PDC-E2-peptide and OVA. The change in pinna thickness (ΔT) for both ears of each mouse was calculated to assess the DTH response. CNP-104 significantly inhibited the DTH response compared to unloaded control particles at 2.5 mg / dose (10 mg / kg HED).

[0144] In follow-up DTH experiments using CNP-104 representative of a GMP manufacturing process, CNP-104 significantly inhibited the DTH response compared to unloaded control particles at 2.5 mg / dose (10 mg / kg HED) (Figures 3A, 3B).

[0145] Example 2: TIMP-PBC inhibits leukocyte infiltration in the liver in a PBC animal model. PBC antigen PDC-E2 in inhibiting leukocyte infiltration in the liver 155~185 The efficacy of encapsulating TIMP-PBC (CNP-104) was investigated in a therapeutic model of PBC.

[0146] On the day of the experiment (day 0), naïve 6-week-old C57BL / 6 mice were primed with 2OA-BSA by intraperitoneal injection (ip). The 2OA-BSA ip priming was repeated on day 14 with incomplete Freund's adjuvant (IFA).

[0147] On days 0 and 2, mice were injected ip with 100 ng of pertussis toxin (PTX). On days 0 and 14, mice were injected subcutaneously (sc) with 5 mg / kg of poly(I:C). On day 7, mice were injected with PDC-E2 155~185 On days 7 and 14, mice were treated with CNP-104 or control nanoparticles (N=6 / group).

[0148] Another group of animals (N=3) was left unprimed and served as untreated controls.

[0149] On day 55, animals were sacrificed and livers were harvested. Livers were processed to isolate leukocytes using gradient separation and the level of leukocyte infiltration in each group was assessed by flow cytometry by gating on live CD45+ cells. As shown in Figure 4, treatment with CNP-104 resulted in reduced levels of leukocyte infiltration in the liver when compared to control nanoparticle treatment. As shown in Figure 5, treatment with CNP-104 significantly increased Treg (CD4 + CD25 + Foxp3 + ) resulting in an increase in

[0150] Example 3: Phase 2a study of TIMP-PBC in patients with PBC Product-specific repeat dose-ranging findings and pivotal GLP toxicity studies were initially conducted with CNP-104 (Study CNP-104-2.001 and Study CNP-104-2.002). These studies encompass and support the dose levels, dosing frequency, and route of administration for the CNP-104 Phase 2a study.

[0151] A non-GLP repeated dose study was conducted with CNP-104 administered by intravenous (IV) on days 1, 8, and 15 at dose levels of 0 mg / kg, 25 mg / kg, 75 mg / kg, and 125 mg / kg (Study CNP-104-2.001). No drug-related effects were noted on weight gain, food intake, ophthalmologic examination, physical examination, clinical observations, functional observation battery, body temperature, and serum cytokine levels. Transient non-adverse clinical pathology observations resolved quickly. None of these findings were considered adverse. The highest dose used in this study, 125 mg / kg IV (administered on days 1, 8, and 15), had no observed adverse effects and was therefore determined to be the NOAEL.

[0152] A pivotal GLP repeat dose study was conducted with CNP-104 administered by intravenous infusion (IV) on days 1, 8, and 15 at dose levels of 0 mg / kg, 25 mg / kg, 75 mg / kg, and 125 mg / kg (Study CNP-104-2.002). There were no drug-related effects in clinical observations or changes in body weight, food intake, temperature, or physical necropsy findings. Increases in cytokine parameters IL-5, IL-1β, IL-6, and IFN-γ in some female rats were observed but were not considered to be adverse. The highest dose used in this study, 125 mg / kg IV (administered on days 1, 8, and 15), had no observed adverse effects and was therefore determined to be the NOAEL.

[0153] Conduct a Phase 2a, double-blind, placebo-controlled study to evaluate the safety, tolerability, pharmacodynamics, and efficacy of CNP-104 in subjects 22 to 72 years of age with primary biliary cholangitis unresponsive to UDCA and / or OCA.

[0154] CNP-104 is PDC-E2 155~185It consists of PLGA nanoparticles that encapsulate peptides. CNP-104 particles have an average diameter of approximately 400-800 nm and a negative zeta potential of -30 mV to -60 mV. CNP-104 is supplied as a lyophilized formulation. CNP-104 is reconstituted in sterile water for injection and diluted in sterile saline (0.9% sodium chloride, USP) prior to administration.

[0155] Subjects aged 22-72 and 18-75 years with primary biliary cholangitis will be screened within 14 days prior to enrollment in the study. If a subject is currently receiving standard of care therapy, they will continue their current standard of care therapy for the duration of the study, at the discretion of the investigator, regardless of treatment arm (CNP-104 or placebo).

[0156] Screening is completed to assess eligibility, obtain vital signs, collect laboratory samples and pharmacodynamic (PD) measurements, receive a fibrosis scan for liver fibrosis, and collect a liver core biopsy for inflammation / fibrosis assessment.

[0157] Subjects will complete an initial PBC-40 assessment and begin the Itch Diary, a questionnaire and scoring system to be completed by patients every morning and evening for the first 120 days of the trial, and weekly for the duration of the trial. The PBC-40 will be completed monthly from study day 1 through study end. Subjects who meet all inclusion criteria and no exclusion criteria after completing the screening visit will be enrolled in the clinical trial.

[0158] Prior to administration of study drug on Day 1, subjects will have a second set of liver laboratory samples collected. The results of these tests will be averaged with those obtained at the screening visit to provide patient-specific baseline ALT, total and direct bilirubin, and ALP values ​​for monitoring hepatic safety and detection of drug-induced liver injury (DILI) throughout the course of the study.

[0159] Subjects are then randomized on Day 1 in a 1:1 ratio into the currently open dose level cohort to receive two separate doses of intravenous CNP-104 or placebo on Days 1 and 8 (Figure 6). The study drug will be administered by IV infusion (3.25 mg / ml) over approximately 3-4 hours using the following escalating infusion rates: 20mL / hour for the first 15 minutes, then 40 mL / hour for the next 15 minutes, then The remainder of the infusion is 80 mL / hour.

[0160] Subjects will be under medical observation in the clinic for acute adverse events for 4 hours after infusion on Days 1 and 8. Subjects will be discharged 4 hours after infusion if all scheduled evaluations are completed on that visit, vital signs (sitting or supine blood pressure, heart rate, temperature) measured 4 hours after infusion are within expected ranges for the subject, and no other health concerns are noted by the investigator.

[0161] Subjects return for clinic visits 2 days after each infusion (Days 3 and 10) for collection of safety laboratory tests, drug review, and adverse event assessment, and are followed daily via telephone visits during the infusion (Days 4-7 and 11-14) to assess and document any adverse events and drug changes.

[0162] During the post-dosing period, subjects return to the clinic for immune safety laboratory tests, PD measurements, PBC-40 assessments, adverse event and drug change assessments (Days 15, 60, 90, 120, 180, 270, 365, 450, 540, 630, 700, and / or 730), fibro scans (Days 60, 90, 120, 180, 270, 365, 450, 540, 630, 700, and / or 730), and core liver biopsy (Day 60) (Figure 6). Subjects continue to complete twice-daily itch diaries for the duration of the study through Day 730. Subjects return to the clinic for the end of study visit on Day 730 for safety laboratory test collections, PD measurements, PBC-40 assessments, fibro scans, and final assessment of adverse events and drug changes.

[0163] The study will enroll two cohorts at two dose levels. Subjects will be randomized in a 1:1 ratio to receive either CNP-104 or placebo (0.9% Sodium Chloride Injection, USP) as a 200 mL intravenous infusion on days 1 and 8. The planned dose levels are as follows: 1. Cohort 1: 4 mg / kg (N=6 subjects randomized 1:1) 2. Cohort 2: 8 mg / kg (N=max 34 subjects randomized 1:1)

[0164] Doses for subjects within a given dose cohort will be separated by at least 48 hours. After all subjects in a dose cohort have completed the Day 15 visit (7 days after the second dose), the DMC will convene to review all available safety data and determine whether it is acceptable to proceed to the next dose cohort, whether cohort expansion is justified, or whether other clinical recommendations should be made.

[0165] Participation criteria: • Subjects willing and able to provide Institutional Review Board (IRB) approved written informed consent and privacy language in accordance with national regulations. ●Includes men and non-pregnant, non-breastfeeding women, aged 22-27 or 18-75. Subjects with a confirmed diagnosis of primary biliary cholangitis based on at least two of the following: - positive AMA titer (e.g., >1:40) or, if AMA negative or low titer (<1:40), positive PBC-specific antibodies (anti-GP210 and / or anti-SP100 and / or antibodies against major M2 components [PDC-E2, 2-oxo-glutarate dehydrogenase complex] - Alkaline phosphatase >1.5 x ULN (upper limit of normal) for at least 6 months -Liver biopsy findings consistent with PBC • Subjects with class A Child-Pugh score. - Subjects who are unresponsive to or intolerant to UDCA and / or OCA after 6 months of treatment at a stable dose as measured by ALP > 1.5 x ULN ●Subjects with ALP>1.5×ULN. ●Subjects with AST and ALT ≤ 10 × ULN. • Subjects positive for antigen-specific CD8+ T cells. ●Male and female subjects of non-childbearing potential or women of childbearing potential who agree, from the start of the initial screening and continuing throughout the study through Day 90, to not become pregnant during the study, have a negative pregnancy test at the screening visit and before each dose, and agree to practice highly effective contraception, including but not limited to abstinence, sexual intercourse with persons of the same sex only, monogamous relationship with a vasectomized partner, vasectomy, hysterectomy, bilateral tubal ligation, approved hormonal methods, or intrauterine device (IUD). ●Women who agree to abstain from breastfeeding from the first screening until day 120 throughout the study. -Female subjects who agree not to donate OVA from the start of initial screening through day 90 throughout the study. Male subjects who agree not to donate sperm until Day 90 at screening and throughout the study.

[0166] Exclusion Criteria: ●Subjects with early stage 3 or higher primary biliary cholangitis. Subjects with Class B or Class C Child-Pugh scores. - Subjects with concomitant liver disease including viral hepatitis, PSC, alcoholic liver disease, Wilson's disease, hemochromatosis, or Gilbert's syndrome. ●Subjects who have previously undergone a liver transplant. Subjects with decompensated liver disease, defined as the presence or history of any of the following: MELD score > 15 Hepatic encephalopathy ○Ascites Hepatorenal syndrome or serum creatinine > 2mg / dL Total bilirubin > 3.0 mg / dL ○INR>1.8 (except when using anticoagulants such as Coumadin) History of variceal bleeding -Subjects with a history of cerebrovascular disease within the past 12 months Subjects with a history of myocardial infarction, as defined by any of the following criteria: Occurrence of pathological Q waves, with or without symptoms Imaging evidence of lost areas of viable myocardium that thin and do not contract in the absence of non-ischemic causes Pathological findings of healed or healed myocardium Glomerular filtration rate (GFR) < 60 mL / min / 1.73 m for at least 3 months, with GFR calculated based on the CKD-EPI equation 2 Subjects with chronic kidney disease as defined by: eGFR = 142 × min(Scr / κ,1) α ×max(Scr / κ,1) -1.200 ×0.9938 年齢 × 1.018 [for women], or ○GFR = 141 × min(Scr / κ,1)α × max(Scr / κ,1)-1.209 × 0.993 age × 1.018 [for men] × 1.159 [for blacks], where Scr is serum creatinine in mg / dL. ○ κ is 0.7 for women and 0.9 for men ○ α is -0.329 for women and -0.411 for men ○Min indicates the minimum value of Scr / κ or 1 ○Max indicates the maximum value of Scr / κ or 1 • Subjects with uncontrolled diabetes, defined by HbA1c>7%. -Subjects who have used biologic agents, including anti-cellular and anti-cytokine therapies, within 12 months from Day 0 or prior to screening. • Subjects with a history of tuberculosis or a positive PPD skin test. -Subjects who have received any live vaccine (excluding intranasal influenza vaccine) within 28 days or a subunit vaccine within 14 days prior to Screening 1, or who are scheduled to receive any vaccination within 90 days prior. Subjects who have received a COVID-19 vaccine (first or second dose) within 14 days prior to screening. Subjects who have received the first dose of the COVID-19 vaccine will not be screened for this study until 14 days after receiving the second dose of the vaccine, if applicable. -Subjects who used systemic steroids within 3 months prior to screening. Subjects with laboratory test results at screening or pre-dose that are outside normal limits and are considered clinically significant by the investigator. This criterion does not apply to liver function tests. In addition, clinically significant laboratory test results at screening related to the condition (PBC) are accepted. • Subjects with a positive test result for Hepatitis B surface antigen (HBsAg), Hepatitis C virus (HCV) antibody, or Human Immunodeficiency Virus (HIV) antigen / antibody, as determined at screening. Subjects with a history of or currently active immune disease (including autoimmune disease) other than PBC (unless, after discussion with the medical monitor, it is determined that the subject's participation in this study is acceptable) Subjects with a history of or current active disease requiring immunosuppressive medication (including azathioprine, prednisone, prednisolone, budesonide, cyclosporine, tacrolimus, methotrexate, or mycophenolate mofetil) (unless, after discussion with the Medical Monitor, it is determined that the subject's participation in this study is acceptable) Subjects with a clinical history of significant cardiovascular disease as determined by the investigator. At the investigator's option, any medical history of comorbidities or malignancies within the past 5 years will make the subject ineligible for study participation. • Subjects who, in the opinion of the investigator, are unable to comply with study procedures. -Subjects who have received any investigational treatment other than CNP-104 within 28 days or 5 half-lives prior to screening, whichever is longer. Subjects who, in the opinion of the Investigator, have any condition that makes the subject unsuitable for participation in the study. • Known sensitivity to any component of CNP-104.

[0167] The primary objectives of this study include evaluating the safety and tolerability of CNP-104, as well as evaluating changes in serum alkaline phosphatase (ALP) levels in patients treated with CNP-104 or placebo.

[0168] Secondary endpoints include evaluating the change in levels of bilirubin, albumin, ALT, AST, IFN-γ, GGT, Gp210, Sp100, soluble CD14, serum IgM, serum kynurenine, intrahepatic inflammation, and liver fibrosis in patients receiving CNP-104 compared to placebo, and evaluating the change in T cell infiltration in the liver by core biopsy between patients treated with CNP-104 or placebo. Patients' ELF scores, daily worst itch scores, and PBC-40 scale ratings will also be evaluated.

[0169] Subjects randomized to study product will receive either 4 mg / kg or 8 mg / kg (up to a maximum dose of 650 mg per day) of CNP-104 as a 200 mL intravenous infusion on days 1 and 8.

[0170] Throughout the course of the study, subjects are closely monitored for possible signs of both hepatocellular and cholestatic drug-induced liver injury (DILI). Liver test monitoring is divided into three categories: subjects with normal baseline (subject-specific mean baseline) ALT values ​​with hepatocellular DILI signal, subjects with elevated baseline (subject-specific mean baseline) ALT values ​​with hepatocellular DILI signal, and subjects with cholestatic DILI signal. All liver test values ​​are compared to subject-specific mean baseline values ​​("baseline") from two studies obtained prior to exposure to the suspected drug.

[0171] For subjects with normal baseline ALT values, the following monitoring for hepatocyte DILI signal is performed: [Table 2]

[0172] For subjects with elevated baseline ALT levels, hepatocyte DILI signal will be monitored as follows: [Table 3]

[0173] For cholestatic DILI signals, the following monitoring is performed: [Table 4]

[0174] Blood tests (ALT, total and direct bilirubin, and ALP) should be repeated within 2 to 5 days if hepatocellular DILI is suspected and within 7 to 10 days if cholestatic DILI is suspected to confirm reproducibility of initial values ​​and direction of change from initial values.

[0175] The safety assessment includes: Complete screening and physical examinations on days 1 and 8, and symptom-driven physical examinations on days 3, 10, 15, 60, 90, 120, 180, 270, 365, 450, 540, 630, 700, and 720; ● Screen and vital signs on days 1, 3, 8, 10, 15, 60, 90, 120, 180, 270, 365, 450, 540, 630, 700, and 720; optionally followed by a symptom-driven physical examination on days 15, 60, 90, 120, 180, 270, 365, 450, 540, 630, 700, and 720; • 12-lead electrocardiogram (ECG) at screening and on days 1, 3, 8, and 10; • AEs at screen, followed by days 1, 3, 4–7 (remote), 8, 10, 11–14 (remote), 15, 60, 90, 120, 180, 270, 365, 450, 540, 630, 700, and 720; • Safety laboratory tests (chemistry, hematology, coagulation, and urinalysis) at screen, followed by days 1, 3, 8, 10, 15, 60, 90, 120, 180, 270, 365, 450, 540, 630, 700, and 720; • Cytokine (IL-1β, TNF-α, IL-6, MCP-1, MIP-1α, IFN-γ, IL-4, IL-10) profiling at screen and subsequently on days 1, 3, 8, 10, and 15; • Antimitochondrial antibodies at screen, followed by days 1, 3, 8, 10, 15, 60, 90, 120, 180, 270, 365, 450, 540, 630, 700, and 720.

[0176] Laboratory / PD evaluation includes: • T-cell infiltration (from liver core biopsy) at screening, followed by day 60; • Antigen-specific CD4+ and CD8+ T cells at screening, followed by days 1, 8, 15, 60, and 90; • Serum alkaline phosphatase (ALP) at screening, followed by days 1, 8, 15, 60, 90, 120, 180, 270, 365, 450, 540, 630, 700, and 720; • Liver chemistry tests (bilirubin, albumin, ALT, AST) at screening, followed by days 1, 8, 15, 60, 90, 120, 180, 270, 365, 450, 540, 630, 700, and 720; • Antinuclear autoantibodies (Gp210 and Sp100) at screening, followed by days 1, 8, 15, 60, 90, 120, 180, 270, 365, 450, 540, 630, 700, and 720; • Serum IgM at screening, followed by days 1, 8, 15, 60, 90, 120, 180, 270, 365, 450, 540, 630, 700, and 720; • Inflammation / fibrosis (from liver core biopsy) at screening followed by day 60; • Serum soluble CD14 at screening, followed by days 1, 8, 15, 60, 90, 120, 180, 270, 365, 450, 540, 630, 700, and 730; ● Serum kynurenine on day 1, followed by days 15, 60, and optionally days 1, 8, 90, 120, 180, 270, 365, 450, 540, 630, 700, and 720.

[0177] Clinical efficacy evaluations included: • Liver stiffness at screening, followed by days 60, 90, 120, 180, 270, 365, 450, 540, 630, 700, and 720 (FibroScan); • Screen, followed by ELF scores at days 60, 90, 120, 180, 270, 365, 450, 540, 630, 700, and 720; ●PBC-40 assessment on day 1, followed by days 8, 15, 60, 90, 120, 180, 270, 365, 450, 540, 630, 700, and 720. ● Itch diary on Day 1, followed by all subsequent test days up to Day 120, then weekly test days up to Day 730.

[0178] Example 4: Patient Selection Criteria for CNP104 Treatment PBMCs were obtained from 17 PBC patients to determine whether there was a correlation between AMA positivity, HLA positive restriction of PDC-E2, and ALP levels. 70% of patients in this study expressed either HLA-A*02 or HLA-DRB4*01, or both HLA restrictions for PDC-E2. Because CNP104 encompasses PDC-E2 subjects with HLA-A*02 or HLA-DRB4*01, the status could be a criterion for inclusion in CNP104 clinical trials. 75% of patients were AMA positive. Four of 17 patients were AMA negative, while three of four had a positive HLA phenotype for CNP-104. 70% of PBC patients had elevated ALP levels in their blood. Selection criteria for CNP104 treatment could be based on HLA haplotype restriction, AMA positivity, and ALP levels. Selection criteria could identify patients who are more likely to achieve efficacy with CNP104. (Figure 7). Patients with PBC, whose disease is driven by autoreactivity against the PDC-E2 antigen, are eligible for treatment with CNP104 and can be identified based on HLA haplotype, antimitochondrial antibodies, and alkaline phosphatase levels in the blood.

[0179] Liver biopsies from PBC patients were evaluated for the presence of infiltrating T cells as a possible criterion for inclusion in clinical trials of CNP104. The severity of liver damage was scored on a scale of 1 to 4, with 4 having the most severe liver pathology. The cell counts of CD4+ T cells (Figure 8A), CD8+ T cells (Figure 8B), and biliary epithelial cells PanCK+ (Figure 8C) were detected in stage 1, 3, and 4 PBC livers via isotype controls. Selection criteria for CNP104 treatment can be based on the presence of PDC-E2-specific CD4+ and / or CD8+ T cells in the blood or liver (Figure 8).

[0180] It is understood that each embodiment of the present disclosure described herein may be optionally combined with any one or more of the other embodiments described herein. All patent and non-patent literature cited herein is hereby incorporated by reference in its entirety.

[0181] It is to be understood, therefore, that the invention is not limited to the particular embodiments disclosed, but is intended to cover all modifications that are within the spirit and scope of the invention as defined by the appended claims, the above specification, and / or as illustrated in the accompanying drawings. Accordingly, only such limitations as appear in the appended claims should be placed on this disclosure. References 1.Lu M, Zhou Y, Haller IV, et al.Increasing Prevalence of Primary Biliary Cholangitis and Reduced Mortality With Treatment.Clin Gastroenterol Hepatol.2018;16(8):1342-1350.e1341. 2. Webb GJ, Siminovitch KA, Hirschfield GM. The immunogenetics of primary biliary cirrhosis: A comprehensive review. J Autoimmun.2015;64:42-52. 3.Gershwin ME, Ansari AA, Mackay IR, et al.Primary biliary cirrhosis: an orchestrated immune response against epithelial cells.Immunol Rev.2000;174:210-225. 4.Jones DEJ.Autoantigens in primary biliary cirrhosis.Journal of Clinical Pathology.2000;53(11):813-821. 5.Hu CJ,Zhang FC,Li YZ,Zhang X.Primary biliary cirrhosis:what do autoantibodies tell us? World J Gastroenterol.2010;16(29):3616-3629. 6.Bowlus CL,Kenney JT,Rice G,Navarro R.Primary Biliary Cholangitis: Medical and Specialty Pharmacy Management Update.J Manag Care Spec Pharm.2016;22(10-a-s Suppl):S3-s15. 7.Aguilar MT,Chascsa DM.Update on Emerging Treatment Options for Primary Biliary Cholangitis.Hepat Med.2020;12:69-77. 8.Lindor KD,Bowlus CL,Boyer J,Levy C,Mayo M.Primary Biliary Cholangitis: 2018 Practice Guidance from the American Association for the Study of Liver Diseases.Hepatology.2019;69(1):394-419. 9.Hiramatsu K,Aoyama H,Zen Y,Aishima S,Kitagawa S,Nakanuma Y.Proposal of a new staging and grading system of the liver for primary biliary cirrhosis.Histopathology.2006;49(5):466-478. 10.Kakuda Y,Harada K,Sawada-Kitamura S,et al.Evaluation of a new histologic staging and grading system for primary biliary cirrhosis in comparison with classical systems.Hum Pathol.2013;44(6):1107-1117.

Claims

1. 1. A pharmaceutical composition for treating primary biliary cholangitis (PBC) in a subject, comprising administering to the subject tolerizing immunomodulating particles (TIMP-PBC) encapsulating one or more PBC antigens, wherein the TIMP-PBC is administered at a dose level of 0.01 mg / kg to 12 mg / kg.

2. 2. The pharmaceutical composition of claim 1, wherein the PBC antigen is pyruvate dehydrogenase complex E-2 (PDC-E2).

3. The PBC antigen is PDC-E2 amino acid residues 155 to 185 (PBC １５５～１８５ 3. The pharmaceutical composition of claim 1 or 2, comprising:

4. 10. The pharmaceutical composition according to any one of the preceding claims, wherein the TIMP-PBC particles have an average diameter of from 100 nm to 1500 nm.

5. 10. The pharmaceutical composition of any one of the preceding claims, wherein the TIMP-PBC particles have a negative zeta potential.

6. 10. The pharmaceutical composition according to any one of the preceding claims, wherein the particles have a negative zeta potential of between -30mV and -100mV.

7. TIMP-PBC is administered at a concentration of 0.005 mg / mL to 50 mg / mL, optionally 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 3.25 mg / mL, 3.5 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12.5 mg / mL, 15 mg / mL, 17.5 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, or 50 mg / mL; or TIMP-PBC is administered at a dose level of 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 6 mg / kg, 8.0 mg / kg, 10 mg / kg, or 12 mg / kg; 10. A pharmaceutical composition according to any one of the preceding claims.

8. TIMP-PBC is administered at a dosage level of 1 mg to 800 mg; or TIMP-PBC is administered at a dose of 1 mg, 2 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, or 800 mg; 10. A pharmaceutical composition according to any one of the preceding claims.

9. TIMP-PBC is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every six months, or once a year; or TIMP-PBC is administered in two doses, one week apart; 10. A pharmaceutical composition according to any one of the preceding claims.

10. 10. The pharmaceutical composition according to any one of the preceding claims, wherein the TIMP-PBC is administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally or orally.

11. 10. The pharmaceutical composition of any one of the preceding claims, wherein administering TIMP-PBC to a subject reduces or ameliorates one or more symptoms of PBC, and optionally, the one or more symptoms of PBC are selected from the group consisting of hepatitis, cirrhosis, cholestasis, liver dysfunction, liver failure, liver fibrosis, increased liver immune infiltration, elevated bile acid levels, elevated liver enzyme levels (ALT, ALP, AST, gamma-glutamyl transpeptidase), elevated bilirubin levels, circulating antimitochondrial antibodies (AMA), circulating antinuclear antibodies (ANA), fatigue, itchy skin, pruritus, dry eyes and mouth, abdominal pain, splenomegaly, musculoskeletal pain, edema, fluid accumulation, skin xanthomas, jaundice, hyperpigmentation, osteoporosis, high cholesterol, diarrhea, steatorrhea, hypothyroidism, and weight loss.

12. i) administering TIMP-PBC to a subject reduces the duration and / or severity of an inflammatory immune response to a PBC antigen. ii) the inflammatory immune response is a T cell, B cell, or myeloid cell response; and / or iii) the inflammatory immune response is assayed from one or more biological samples obtained from the subject; 10. A pharmaceutical composition according to any one of the preceding claims.

13. 13. The pharmaceutical composition of claim 12, wherein the biological sample is selected from the group consisting of whole blood, peripheral blood, peripheral blood mononuclear cells (PBMC), serum, plasma, urine, cerebrospinal fluid (CSF), stool, tissue biopsy, and / or bone marrow biopsy.

14. administering TIMP-PBC to a subject, i) reducing the levels of activated antigen-specific T cells; ii) reducing the level of T cell infiltration in the liver; iii) reducing the level of antimitochondrial antibodies in the blood; iv) reducing the level of antinuclear antibodies in the blood; v) reducing the level of IgM in the blood; vi) reducing the level of anti-Gp210 antibodies in the blood; vii) reducing the level of anti-Sp100 antibodies in the blood; viii) reducing the level of kynurenine in the blood; ix) reducing the level of soluble CD14 in the blood; x) reducing liver fibrosis, and / or xi) altering the coagulation profile; 10. A pharmaceutical composition according to any one of the preceding claims.

15. 12. The pharmaceutical composition of claim 11, wherein the improvement in one or more symptoms of PBC is determined using the PBC-40 scale, the Improved Liver Fibrosis (ELF) score, the PBC-27 scale, the GLOBE scale, the UK-PBC scale, the Average Daily Worst Itch Scale, the 5-D Itch Scale, the Visual Analogue Scale (VAS), the Scheuer staging system, the Nakanuma staging system, the fibrosis score, the bile duct loss score, the FIB-4 index, or the APRI.

16. 10. The pharmaceutical composition of any one of the preceding claims, wherein administering TIMP-PBC to a subject reduces liver fibrosis.

17. Administering TIMP-PBC reduces the use of alternative therapies, and optionally: the therapy is selected from the group consisting of steroids, corticosteroids, nonsteroidal immunosuppressants, immunomodulators, monoclonal antibodies, cytokine and chemokine targeted therapies, JAK inhibitors, chimeric antigen receptor (CAR) T cell therapy, regulatory T cell (Treg) therapy, B cell targeted therapy, antiviral drugs, synthetic bile acids, and surgical treatment; or The therapy is selected from the group consisting of ursodeoxycholic acid (UDCA), obeticholic acid (OCA), fibrates, peroxisome proliferator-activated receptor delta (ppaRδ) agonists, ileal bile acid transporter (IBAT) inhibitors, tauroursodeoxycholic acid, 6α-ethylchenodeoxycholic acid, cetanaxib, moexipril, abatacept, fibroblast growth factors, statins, corticone, ustekinumab, CD20 targeted therapy, CD19 targeted therapy, CD40 / CD40L targeted therapy, CD80 / CD86 targeted therapy, B-cell targeting factor (BAFF) targeted therapy, B-cell maturation antigen (BCMA) targeted therapy, anti-IL6 therapy, anti-IFN therapy, amifampridine, cyclosporine, and cyclosporine. porin, cyclophosphamide, batoclimab, inebilizumab, nipocalimab, pozeliimab, rituximab, satralizumab, seladelpar, pentoxifylline, tocilizumab, tofacitinib, trebrutinib, abolstatin, fenofibrate, bezafibrate, linelixibat, methotrexate, butyrate, palmitate, liver transplant, beclomethasone, ciclesonide, fluticasone furoate, mometasone, budenoside, fluticasone, triamcinolone, loteprednol, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, betamethasone, or hydrocortisone.

10. A pharmaceutical composition according to any one of the preceding claims.

18. TIMP-PBC is administered alone or in combination with a PBC therapeutic, optionally the therapeutic agent is selected from the group consisting of steroids, corticosteroids, nonsteroidal immunosuppressants, immunomodulators, monoclonal antibodies, cytokine and chemokine targeted therapies, JAK inhibitors, chimeric antigen receptor (CAR) T cell therapy, regulatory T cell (Treg) therapy, B cell targeted therapy, antiviral agents, synthetic bile acids, and surgical treatment; or The therapeutic agent may be ursodeoxycholic acid (UDCA), obeticholic acid (OCA), fibrates, peroxisome proliferator-activated receptor delta (ppaRδ) agonists, ileal bile acid transporter (IBAT) inhibitors, ustekinumab, tauroursodeoxycholic acid, 6α-ethylchenodeoxycholic acid, setanaxib, moexipril, abatacept, fibroblast growth factors, statins, corticone, CD20 targeted therapy, CD19 targeted therapy, CD40 / CD40L targeted therapy, CD80 / CD86 targeted therapy, B-cell targeting factor (BAFF) targeted therapy, B-cell maturation antigen (BCMA) targeted therapy, anti-IL6 therapy, anti-IFN therapy, amifampridine, cyclosporine, selected from the group consisting of cyclophosphamide, batoclimab, inebilizumab, nipocalimab, pozeliimab, rituximab, satralizumab, seladelpar, pentoxifylline, tocilizumab, tofacitinib, trebrutinib, abolstatin, fenofibrate, bezafibrate, linelixibat, methotrexate, butyrate, palmitate, liver transplant, beclomethasone, ciclesonide, fluticasone furoate, mometasone, budenoside, fluticasone, triamcinolone, loteprednol, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, betamethasone, and hydrocortisone; 10. A pharmaceutical composition according to any one of the preceding claims.

Images