Neoepitope Peptide Research Articles

Neo-epitope Peptides as Biomarkers of Disease Progression for Muscular Dystrophies and Other Myopathies.

For several decades, serological biomarkers of neuromuscular diseases as dystrophies, myopathies and myositis have been limited to routine clinical biochemistry panels. Gauging the pathological progression is a prerequisite for proper treatment and therefore identifying accessible, easy to monitor biomarkers that can predict the disease progression would be an important advancement. Most muscle diseases involve accelerated muscle fiber degradation, inflammation, fatty tissue substitution and/or fibrosis. All these pathological traits have been shown to give rise to serological peptide biomarkers in other tissues, underlining the potential application of existing biomarkers of such traits in muscle disorders. A significant quantity of tissue is involved in these pathological mechanisms alongside with qualitative changes in protein turnover in myofibrillar, extra-cellular matrix and immunological cell protein fractions accompanied by alterations in body fluids. We propose that protein and peptides can leak out of the afflicted muscles and can be of use in diagnosis, prediction of pathology trajectory and treatment efficacy. Proteolytic cleavage systems are especially modulated during a range of muscle pathologies, thereby giving rise to peptides that are differentially released during disease manifestation. Therefore, we believe that pathology-specific post-translational modifications like cleavages can give rise to neoepitope peptides that may represent a promising class of peptides for discovery of biomarkers pertaining to neuromuscular diseases.

Abstract 4900: Increased IgG antibody responses to neoepitope and native peptides containing high affinity domains for MHCI following combination cancer immunotherapy

Abstract The importance of anti-tumor immunity by CD8+ T cells is well defined, but antigen-specific immune monitoring for CD8+ responses across many potential tumor antigens is expensive and difficult. This difficulty is compounded by increasing evidence that every tumor has unique mutant neoepitopes. We hypothesized that new technologies in peptide printing might allow us to partially overcome this obstacle by instead characterizing antibody responses against a paired array of neoepitope and native peptides. We designed a 15-mer peptide array of mutated and native peptides from the murine mammary carcinoma 4T1 and used it to characterize the serum IgG antibody responses to vaccination with 4T1 autophagosome-enriched vaccine (DRibbles), poly-IC adjuvant, the combination of both, or naïve animals across 4 independent experiments. This array was printed by JPT peptides and included 75 pairs of single-nucleotide polymorphic and native peptides identified by both our own whole exome sequencing and the current 4T1 literature. We also included other sequences of interest to 4T1 immunity such as the retrovirus fragment AH1. MHCI peptide predictions were robust and used the top NetMHCpan v2.8 score from all possible windowed 8,9,10, and 11-mers for H2-Kd,Ld, and Dd surrounding the mutation site in its native protein context. The results of this work identify a linear correlation between higher predicted peptide MHCI binding affinity and the IgG antibody signals increased in combination treated groups versus controls. Linear regression analysis demonstrates that normalized antibody responses to mutated and native 4T1 peptides are more likely to be higher in sera from autophagosome-enriched vaccine plus poly-IC treated animals than naïve animals if these peptides contain higher affinity domains for MHCI (p<.0001). This correlation is not significant in the groups treated with vaccine or adjuvant alone. Animals pre-treated with this combination therapy also benefit from a significant delay in tumor growth upon challenge with 4T1 versus naïve animals (p<.001). These results identify a previously unknown link between predicted MHCI binding affinity and the anti-tumor antibody response following combination immunotherapy - suggesting some antigen-specific interaction between B cells and CD8+ T cells that might be exploited to improve the immune monitoring of cancer therapies. Citation Format: Tyler W. Hulett, Shawn Jensen, Christopher Dubay, Carlo Bifulco, Michael Afentoulis, Ashok Reddy, Larry David, Bernard A. Fox. Increased IgG antibody responses to neoepitope and native peptides containing high affinity domains for MHCI following combination cancer immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4900.

I-27 TARGETING NF-KAPPAB SIGNALING IN OA: IS IT ONLY ABOUT INFLAMMATION?

I-27 TARGETING NF-KAPPAB SIGNALING IN OA: IS IT ONLY ABOUT INFLAMMATION?

Clinical validation of an immunoaffinity LC–MS/MS assay for the quantification of a collagen type II neoepitope peptide: A biomarker of matrix metalloproteinase activity and osteoarthritis in human urine

The degradation of type II collagen has been associated with the pathology of osteoarthritis (OA). Matrix metalloproteinases (MMPs) are enzymes that are responsible for catalyzing the degradation of collagen and, therefore, are pursued as potential targets for the treatment of OA. Collagen-derived peptides identified as a reflection of in vivo MMP activity have been investigated as target biomarkers of MMP activity as well as potential biomarkers of OA disease state and/or progression. An immunoaffinity liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay developed for the quantification of the most abundant urinary type II collagen neoepitope (uTIINE) peptide, a 45-mer with 5 HO-proline residues resulting from MMP-13-catalyzed degradation, was validated for clinical use. Validation experiments were designed with attention to specific challenges related to quantification of endogenous analytes. The validated method is sensitive, selective, accurate (<15% relative error) and precise (<15% coefficient of variation) over a linear range of 0.156–7.50 ng/ml. Sample stability and inter- and intrasubject variability were evaluated in the urine of normal and OA populations. The method was applied to analyze human urine samples from clinical studies investigating the utility of uTIINE as a potential biomarker for OA.

Discovery and development of a type II collagen neoepitope (TIINE) biomarker for matrix metalloproteinase activity: From in vitro to in vivo

Destruction of cartilage by matrix metalloproteinases (MMPs) plays a significant role in the pathology of osteoarthritis (OA). A translatable biomarker of MMP activity would enable development of MMP inhibitors for the treatment of OA and potentially the improved diagnosis of OA. A directed approach to identifying specific MMP cleavage products as potential biomarkers has been undertaken. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) was used to identify peptides generated by MMP-driven degradation of human articular cartilage (HAC) in vivo. It was shown that a 45-mer peptide fragment of collagen type II with five hydroxyprolines (OH) can be selectively produced by the activity of collagenase, an enzyme purported to be involved in the pathology of OA. This 45-mer is the most abundant neoepitope peptide found in biological fluids such as urine and synovial fluid. An immunoaffinity LC–MS/MS assay has been developed to quantify collagen type II neoepitope peptides as biomarkers of collagenase modulation. The lower limit of quantification for this assay was established to be 0.035 nM. The assay was used to measure the levels of collagen type II peptides in the urine of both clinical (healthy human subjects) and preclinical species. The urinary levels of the most abundant peptides are reported for rat, rabbit, guinea pig, dog, and healthy human adult subjects. The utility of this peptide to monitor collagenase activity in vivo has been demonstrated through its detailed characterization in HAC explants as well as in the urine of human and other preclinical species.

Development and characterization of a highly specific and sensitive sandwich ELISA for detection of aggrecanase-generated aggrecan fragments

To develop an enzyme linked immunosorbent assay (ELISA) to quantify the levels of specific aggrecan fragments generated by aggrecanase-mediated cleavage at the 373Glu-374 Ala bond within the aggrecan interglobular domain. The ELISA employs a commercially available monoclonal antibody to capture aggrecan fragments containing keratan sulfate (KS). Aggrecan fragments generated by cleavage at the Glu-Ala bond were then detected using a monoclonal neoepitope antibody (mAb OA-1) that specifically recognizes the N-terminal sequence 'ARGSVIL'. The mAb OA-1 antibody was highly specific for the immunizing neoepitope peptide since neither peptides spanning the cleavage site nor mutated peptides were detected. Aggrecan fragments generated by ADAMTS-4 digested human aggrecan monomers and from IL-1-stimulated human cartilage explants were quantified by the ELISA, and we observed increased sensitivity of the ELISA compared to mAb OA-1 Western analysis. We also observed that the basal, as well as IL-1-stimulated production of ARGS aggrecan fragments from human articular cartilage explants was blocked by a selective aggrecanase inhibitor, consistent with generation of the ARGS neoepitope in human articular cartilage being mediated by aggrecanase. Using purified human aggrecan digested by ADAMTS-4 as standard to quantify ARGS aggrecan fragments in human synovial fluids, we determined that the calculated amount of ARGSVIL-aggrecan fragments by ELISA measurement is in agreement with the published levels of these fragments, supporting its potential utility as a biomarker assay for osteoarthritis. We have developed an assay that detects and quantifies specific aggrecan fragments generated by aggrecanase-mediated cleavage. Because aggrecanase mediates degradation of human articular aggrecan in joint disease, the KS/mAb OA-1 ELISA may serve as a biomarker assay for evaluation of preclinical and clinical samples.