A Fit-for-Purpose Method for the Detection of Human Antibodies to Surface-Exposed Components of BMS-986263, a Lipid Nanoparticle-Based Drug Product Containing a siRNA Drug Substance.

ND-L02-s0201/BMS-986263 is a lipid nanoparticle (LNP) drug product containing a heat shock protein 47 (HSP47)-specific small interfering ribonucleic acid (siRNA) and being developed for the treatment of liver and idiopathic pulmonary fibrosis. To address immunogenicity-related issues, we developed a robust, fit-for-purpose (FFP) three-tier electrochemiluminescent (ECL) anti-drug antibody (ADA) assay for the detection of antibodies (Abs) generated to surface-exposed components of BMS-986263. The drug was coated directly on plates, and several Abs specific for polyethylene glycol (PEG) and other surface components were tested for use as positive quality controls (QCs). Following selection of a rabbit monoclonal anti-PEG Ab, the assay was optimized, and various method development challenges specific to the modality and pseudo surrogate rabbit control were addressed. Screening, confirmatory, and titer cut points were validated following a statistical evaluation of 41 individual K2EDTA human plasma samples at a minimum required dilution (MRD) of 100. Assay precision, sensitivity, selectivity, drug tolerance, and hook effect were determined for the rabbit Ab prepared in human K2EDTA plasma matrix. The assay was used to interrogate anti-drug Ab (ADA) responses in normal human subjects who were administered 90 mg of the drug intravenously (IV) once every week for 3 weeks in phase I clinical trials. All pre- and post-dose samples were found to be negative for ADA. Based on these results, we concluded that BMS-986263 is not immunogenic. To the best of our knowledge, this work represents the first ADA method developed and reported for an LNP-based drug product.

Periodontal bacteria and the rheumatoid arthritis-related antigen RA-A47: the cross-reactivity potential.

PURPOSE OF REVIEW: The purpose of this review is to evaluate the mechanisms that underlie the association between periodontal pathogens and rheumatoid arthritis (RA). RECENT FINDINGS: This review focuses on the cross-reactivity hypothesis as a mechanism that might contribute to explain the pathologic evolution of periodontal infections from periodontitis to RA. The scientific rationale is that immune reactions following infection by periodontal bacteria might cross-react with RA autoantigens, in this way eventually leading to autoimmunity. SUMMARY: Using the rheumatoid antigen associated with RA-A47 arthritis as an antigen model and analyzing five periodontal bacteria (eg, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Treponema denticola, Tannerella forsythia and Prevotella intermedia), an extremely varied pattern of peptide sharing was found. In the context of the cross-reactivity hypothesis, the data allow us to glimpse the possibility of distinguishing the periodontal bacteria capable of attacking the periodontal tissue from those that are additionally equipped with a rheumatologic potential by virtue of the sharing of peptide sequences with RA antigens.

CTL responses to HSP47 associated with the prolonged survival of patients with glioblastomas.

OBJECTIVE: To define heat shock protein 47 (HSP47) as a novel glioma-associated antigen and to preliminarily assess the association of cytotoxic T lymphocyte (CTL) responses to HSP47 with clinical outcomes in patients with glioblastomas (GBMs). METHODS: The expression of HSP47 was determined in primary GBM tissues (n = 17) and controlled brain tissues (n = 10) by Western blot. Candidate epitope peptides were predicted using the human leukocyte antigen (HLA) Peptide Binding Predictions Program. The CTL responses to HSP47 were quantified in peripheral blood mononuclear cells from 6 healthy donors and 38 patients (benign tumors = 5, astrocytoma grade II = 7, anaplastic gliomas grade III = 10, GBMs = 16) by stimulation with the mixture of the identified peptides above. Kaplan-Meier survival curves were used to analyze the association between CTL responses and clinical outcomes. RESULTS: Expression of HSP47 was hardly detectable in controlled brain tissues and increased in GBM tissues (p = 0.018). HSP47(184-192) (KLPEVTKDV) and HSP47(3-11) (LLLLSAFCL) were predicted as the most potent candidate epitope peptides with experimentally confirmed binding affinity to the HLA-A0201 molecule. Seven of 26 patients (26.9%) with malignant gliomas had positive CTL responses. Furthermore, patients with GBM with positive CTL responses to HSP47 experienced a prolonged progress-free survival time (12.6 ± 1.3 vs 8.1 ± 3.2 months, p = 0.01) and overall survival (13.4 ± 1.3 vs 10.4 ± 2.7 months, p = 0.035) than those with negative responses. CONCLUSION: Our data demonstrated that HSP47 is a novel glioma-associated antigen. HSP47-based vaccine will likely confer additional survival benefit to patients with GBM after surgical treatment.

High serum concentrations of autoantibodies to HSP47 in nonspecific interstitial pneumonia compared with idiopathic pulmonary fibrosis.

BACKGROUND: The pathological diagnosis of idiopathic interstitial pneumonias (IIP) by surgical lung biopsy is important for clinical decision-making. However, there is a need to use less invasive biomarkers to differentiate nonspecific interstitial pneumonia (NSIP) from other IIP such as usual interstitial pneumonia (UIP). Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagen. HSP47 is increased in various fibrotic diseases. We investigated the autoantibodies to HSP47 in IIPs. METHODS: We measured the serum levels of the autoantibodies to HSP47 in 38 patients with various forms of IIP [16 with idiopathic pulmonary fibrosis (IPF), 15 with idiopathic NSIP, 7 with cryptogenic organizing pneumonia (COP)] and 18 healthy volunteers. RESULTS: The serum levels of autoantibodies to HSP47 in patients with idiopathic NSIP were significantly higher than in patients with IPF (P < 0.01), COP (P < 0.05), and healthy volunteers (P < 0.05). In addition, those in fibrosing NSIP were significantly higher than those of cellular and fibrosing NSIP (p < 0.05). CONCLUSION: We found high levels of anti-HSP47 autoantibody titers in sera of patients with idiopathic fibrosing NSIP compared with other IIPs and healthy volunteers.

Strain differences and the role for HSP47 and HSP70 in adjuvant arthritis in rats.

Because of high sequence homology between microbial and endogenous heat shock proteins (HSP), immunological cross-reactivity to microbial HSP has been suggested as a possible cause of the development of autoimmune diseases, such as rheumatoid arthritis. The present study aimed to determine a potential role of HSP47, a molecular chaperone involved in the synthesis and assembly of collagen molecules, and microbial HSP71 (mHSP71) in adjuvant arthritis (AA) in two rat strains: Dark Agouti (DA), susceptible to AA induction and Albino Oxford (AO), which is resistant to AA induction. Immunization with complete Freund's adjuvant (CFA) induced an increased expression of HSP47 in joints of DA rats, which exhibited severe clinical signs of AA at the time of disease peak, while this protein was not detectable in joints of AO rats. In contrast, no strain differences in HSP72 (rat analogue of mHSP71) expressions in joints were observed. The increased levels of anti-HSP47 antibodies were detected in sera of DA rats during the AA peak, while the immunization with CFA increased levels of anti-mHSP71 antibodies in sera of AO rats. HSP47 and mHSP71 reduced proliferation of draining inguinal lymph node cells (LNC) in resistant AO rat strain, leading to a hypothesis that both HSP participated in AA control. Finally, mHSP71 potentiated the apoptotic response of LNC in susceptible DA rat strain. In conclusion, our findings indicate involvement of HSP47 in the development of AA in the rat, and point out to the regulatory role for both HSP47 and mHSP71.