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 (a-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.