Discovery of autoimmune antigens from non-coding sequences

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7. Project Summary. This project addresses an acute clinical need for the many neuroimmune diseases with no known causative antigen, and those with variation that remains unexplained by single defining antigens. Antibodies in NMDA-receptor encephalitis (α-NMDAR) have revolutionized diagnosis and treatment of that paraneoplastic disease (PND), while other PNDs, like Opsoclonus Myoclonus Syndrome (OMS), await these groundbreaking discoveries. Several methods, such as Phage immunoprecipitation and sequencing (PhIP-seq) have been used successfully to pan for biomarkers and defining autoantigens. However, for some diseases, these methods have failed. One possibility is that defining antigens have been missed by conventional screens because of unconventional origins. We hypothesize that some important autoantigens may reside within “non-coding” sequences. These include the vast landscape of small open reading frames (sORFs), or “micropeptides”, that originate in regions of the genome, like UTRs and long “non-coding” RNAs, that may have been erroneously overlooked in past screens. Importantly for this work, their proven translation is temporally and spatially regulated, they can be tumor specific antigens and are presented to the immune system. A second source of untested proteins in these assays is transposable elements. The proteins they naturally encode, and those that arise from their transposition into active genes, are also potential sources of immunogenic material. While few are active, their expression in embryos, adult brain and in tumor suggest their potential relevance for paraneoplastic autoimmunity. Cryptic peptides generated by a host of mechanisms remain a further challenge to screens that rely on defined target reference peptidomes. Our study aims to improve upon previous PhIPseq searches, by designing and building new libraries that cover this unconventional set of peptides. In Aim 1, we will design and build two new T7 PhIP-seq libraries covering this defined “non-coding” peptidome: one with targets represented as arrays of oligos encoding overlapping peptides, and a second in which full-length targets are expressed, to mitigate risk of disrupting epitopes by sequence fragmentation. In Aim 2, we will screen samples from pediatric neuroimmune disease patients (and controls) against these gene-specific libraries, to identify distinctive signatures of reactivity. To capture information about cryptic antigenic peptides, Aim 3 will test the same samples against an extant random peptide library, which reports on convergent feature specificity of patient samples from each disorder. This high-risk approach, if successful, will lead quickly to revolutionary new diagnostic paradigms for the neuroimmune diseases tested, as it did for NMDAR encephalitis, with paradigm-shifting potential for other autoimmune diseases, as well.