Autoimmunity is a consequence of both genetic and environmental factors, occurring in genetically susceptible hosts with environmental triggers. While genome-wide association studies have revealed a number of susceptible genes contributing to etiology, the environmental triggers remain poorly understood. Primary biliary cholangitis, formally known as primary biliary cirrhosis, is considered a model autoimmune disease for which our group has extensively evaluated environmental factors involved in its etiology. Bacterial infection and xenobiotics have been proposed as candidate environmental factors that may explain tolerance breakdown and production of primary biliary cholangitis-specific antimitochondrial autoantibodies. Large-scale case-control studies have consistently detected an association of primary biliary cholangitis with urinary tract infections caused by Escherichia coli, as E. coli PDC-E2 is molecularly similar to human PDC-E2, the immunodominant target of AMAs. Another bacterium of interest is Novosphingobium aromaticivorans, a ubiquitous xenobiotic-metabolizing bacterium that produces lipoylated proteins, which are highly reactive with sera from primary biliary cholangitis patients. Regarding xenobiotics, case-control studies have suggested that frequent use of nail polish is associated with an increased susceptibility to primary biliary cholangitis. We found that 2-octynamide, the conjugate derived from 2-octynoic acid present in cosmetics, lipsticks, and some chewing gums, was unique in both its quantitative structure-activity relationship analysis and reactivity with primary biliary cholangitis sera. 2-nonyamide is another xenobiotic that also has the optimal chemical structure for xenobiotic modification of the PDC-E2 epitope, as demonstrated by the enhanced epitope recognition with AMA-positive PBC sera. Moreover, we found that C57BL/6 mice immunized with 2-octynoic acid-BSA possess many of the features characteristic to primary biliary cholangitis. Impact statement Autoimmunity is believed to develop in genetically susceptible hosts with triggers from the environment. Researchers have recently demonstrated that bacteria and xenobiotics commonly present in our environment are potential triggers of tolerance breakdown against autoantigens and autoimmunity, particularly in primary biliary cholangitis (PBC). The link between xenobiotics and PBC has been further confirmed with the establishment of PBC model mice by immunizing mice with xenobiotics.

Background

Primary sclerosing cholangitis (PSC) is a chronic progressive liver disease leading to biliary fibrosis and cirrhosis. Cilofexor is a nonsteroidal farnesoid X receptor agonist that demonstrated significant improvements in liver biochemistry and markers of cholestasis in patients with PSC in a phase 2 study. We describe here the rationale, design, and implementation of the phase 3 PRIMIS trial, the largest placebo-controlled trial in PSC.

Methods

Adults with large-duct PSC without cirrhosis are randomized 2:1 to receive oral cilofexor 100 mg once daily or placebo for up to 96 weeks during the blinded phase. Patients completing the blinded phase are eligible to receive open-label cilofexor 100 mg daily for up to 96 weeks. The primary objective is to evaluate whether cilofexor reduces the risk of fibrosis progression compared with placebo. Liver biopsy is performed at screening and Week 96 of the blinded phase for histologic assessment of fibrosis. The primary endpoint-chosen in conjunction with guidance from the U.S. Food and Drug Administration-is the proportion of patients with ≥ 1-stage increase in fibrosis according to Ludwig histologic classification at week 96. Secondary objectives include evaluation of changes in liver biochemistry, serum bile acids, liver fibrosis assessed by noninvasive methods, health-related quality of life, and safety of cilofexor.

Conclusion

The phase 3 PRIMIS study is the largest randomized, double-blind, placebo-controlled trial in PSC to date and will allow for robust evaluation of the efficacy and safety of cilofexor in noncirrhotic patients with large-duct PSC.

Trial registration

ClinicalTrials.gov NCT03890120; registered 26/03/2019.

There is considerable interest in expanding B cell-targeted therapies in human autoimmune diseases. However, clinical trials in human primary biliary cholangitis (PBC) using a chimeric antibody against human CD20 (hCD20) have showed limited efficacy. Two potential explanations for these disappointing results are the appearance of anti-drug antibodies (ADAs) and the high frequency of patients with moderate PBC or patients who had failed ursodeoxycholic acid treatment. Here, we studied a novel humanized IgG1 antibody against hCD20 and explored its efficacy in early stage PBC using a well-defined murine model. We developed a unique murine model consisting of dnTGF-βRII mice expressing hCD20 and human Fcγ receptors (hFcγRs). Beginning at 4-6 weeks of age, equivalent to stage I/II human PBC, female mice were given weekly injections of an anti-hCD20 antibody (TKM-011) or vehicle control, and monitored for liver histology as well as a broad panel of immunological readouts. After 16 weeks' treatment, we observed a significant reduction in portal inflammation, a decrease in liver-infiltrating mononuclear cells as well as a reduction in liver CD8⁺ T cells. Importantly, direct correlations between numbers of liver non-B cells and B cells (r = 0.7426, p = 0.0006) and between numbers of liver memory CD8⁺ T cells and B cells (r = 0.6423, p = 0.0054) were apparent. Accompanying these changes was a dramatic reduction in anti-mitochondrial antibodies (AMAs), interleukin (IL)-12p40 and IL-5, and elevated levels of the anti-inflammatory chemokine CXCL1/KC. In mice that developed ADAs, clinical improvements were less pronounced. Sustained treatment with B cell-targeted therapies may broadly inhibit effector pathways in PBC, but may need to be administered early in the natural history of PBC.

Most eukaryotes harbor two distinct pre-mRNA splicing machineries: the major spliceosome, which removes >99% of introns, and the minor spliceosome, which removes rare, evolutionarily conserved introns. Although hypothesized to serve important regulatory functions, physiologic roles of the minor spliceosome are not well understood. For example, the minor spliceosome component ZRSR2 is subject to recurrent, leukemia-associated mutations, yet functional connections among minor introns, hematopoiesis and cancers are unclear. Here, we identify that impaired minor intron excision via ZRSR2 loss enhances hematopoietic stem cell self-renewal. CRISPR screens mimicking nonsense-mediated decay of minor intron-containing mRNA species converged on LZTR1, a regulator of RAS-related GTPases. LZTR1 minor intron retention was also discovered in the RASopathy Noonan syndrome, due to intronic mutations disrupting splicing and diverse solid tumors. These data uncover minor intron recognition as a regulator of hematopoiesis, noncoding mutations within minor introns as potential cancer drivers and links among ZRSR2 mutations, LZTR1 regulation and leukemias.