High-resolution epitope mapping of anti-Hu and anti-Yo autoimmunity by programmable phage display

Abstract

Paraneoplastic neurological disorders are immune-mediated diseases understood to manifest as part of a misdirected anti-tumor immune response. Paraneoplastic neurological disorder-associated autoantibodies can assist with diagnosis and enhance our understanding of tumor-associated immune processes. We designed a comprehensive library of 49-amino-acid overlapping peptides spanning the entire human proteome, including all splicing isoforms and computationally predicted coding regions. Using this library, we optimized a phage immunoprecipitation and sequencing protocol with multiple rounds of enrichment to create high-resolution epitope profiles in serum and cerebrospinal fluid (CSF) samples from patients suffering from two common paraneoplastic neurological disorders, the anti-Yo (n = 36 patients) and anti-Hu (n = 44 patients) syndromes. All (100%) anti-Yo patient samples yielded enrichment of peptides from the canonical anti-Yo (CDR2 and CDR2L) antigens, while 38% of anti-Hu patients enriched peptides deriving from the nELAVL (neuronal embryonic lethal abnormal vision like) family of proteins, the anti-Hu autoantigenic target. Among the anti-Hu patient samples that were positive for nELAVL, we noted a restricted region of immunoreactivity. To achieve single amino acid resolution, we designed a novel deep mutational scanning phage library encoding all possible single-point mutants targeting the reactive nELAVL region. This analysis revealed a distinct preference for the degenerate motif, RLDxLL, shared by ELAVL2, 3 and 4. Lastly, phage immunoprecipitation sequencing identified several known autoantigens in these same patient samples, including peptides deriving from the cancer-associated antigens ZIC and SOX families of transcription factors. Overall, this optimized phage immunoprecipitation sequencing library and protocol yielded the high-resolution epitope mapping of the autoantigens targeted in anti-Yo and anti-Hu encephalitis patients to date. The results presented here further demonstrate the utility and high-resolution capability of phage immunoprecipitation sequencing for both basic science and clinical applications and for better understanding the antigenic targets and triggers of paraneoplastic neurological disorders.

Keywords: anti-Hu; anti-Yo; autoimmunity; paraneoplastic; phage display.

Graphical Abstract

Figure 1

Design and characterization of PhIP-Seq library. (A) All human protein isoforms, variants and computationally predicted coding regions were downloaded from the NCBI Protein database. Full-length sequences were clustered on 99% sequence identity (CD-HIT v4.6) to remove duplicate and partial sequences. Each protein was computationally divided into 49-amino-acid peptides using a 24-AA sliding window. Redundant or duplicate sequences were removed by further clustering on 95% sequence identity. The resulting 731 724 sequences were synthesized and cloned into the T7 select vector (Millipore, Burlington, MA, USA). (B) Assessment of the library quality before (blue) and after (yellow) cloning and packaging.

Figure 2

Validation of library by commercial antibody IPs. Phage library was subjected to three rounds of selection by polyclonal commercial antibodies to GFAP and GPHN. (A) Replicates show high correlation of gene-level counts for IPs performed on separate days. Final libraries are dominated by the commercial antibody target. The GPHN antibody also consistently enriched a single peptide from CHGA. (B) Scatterplots of peptide-level enrichments show that 18 unique GFAP peptides and 4 GPHN peptides were identified as antibody binders (red markers). Peptides sharing a motif with both GPHN and CHGA (white, see D). (C) Alignment of GFAP peptides to the full-length protein reveals two distinct regions of antibody affinity corresponding to regions of high solvent exposure and B-cell antigenicity as predicted by IEDB tools Emini surface exposure and Bepipred algorithms. (D) Motif analysis of the significant peptides in the GPHN IP reveals a short, discontiguous motif shared by 144 significantly enriched peptides in the final population (from 94 unique genes/proteins), including the most abundant CHGA peptide. The motif is highlighted on the crystal structure of GPHN (top), and peptides sharing this motif are marked by a white circle in B. CHGA = chromogranin A; GFAP = glial fibrillary acid protein; IEDB = Immune Epitope Database.

Figure 3

Specific enrichment of CDR2/CDR2L peptides by PhIP-Seq. (A) Representative data from three patients demonstrating the robust enrichment of CDR2/CDR2L peptides. CDR2/CDR2L peptides were the top five most abundant peptides in 44/51 samples. (B) While CDR2/CDR2L peptides represented 21% of all phage, CDR2L enrichment is more pronounced and complex (more unique peptides per sample) than CDR2.

Figure 4

Anti-Yo cohort PhIP-Seq results. (A) Epitope map showing normalized coverage of each samples’ significant CDR2L peptides aligned to the full-length gene (coverage is divided into five amino acid bins). Patient-binding signatures vary, though a majority converge on residues 322–346. (B) Patient antibodies targeting CDR2L bind to regions of least homology with CDR2, suggesting that the antibody responses to each protein are independent. (C) Epitope map showing normalized peptide coverage across the full-length CDR2 sequence. Patient antibodies enrich for peptides primarily restricted to the first 100 amino acids at the N-terminus. CDR2 = cerebellar degeneration-related protein 2; CDR2L = CDR2-like.

Figure 5

Anti-Hu PhIP-Seq results. (A) Antibody-binding signatures for samples that enriched nELAVL peptides. A majority of patients show a markedly convergent binding preference for a short 17-residue sequence at the exon 6/7a junction of ELAVL4. (B) Mean fold-change values for peptides spanning the unique regions defining ELAV4 splice variants and those from ELAVL2 and ELAVL3, indicating lack of enrichment beyond the exon 6/7a junction. (C) Mutational scan using smaller phage library with all possible point mutations at each location spanning the motif highlighted in B. Mutational tolerance is calculated for each position, defined as the fold-change over unselected (AG-bead only IP) relative to the reference sequence. Most telling are the mutations encoding stop codons (denoted *) revealing that Patient 01’s antibodies require a minimal sequence up to residues 255 (RLDDNLL) with the subsequence QRFRLDNLL least amenable to mutation. Patient 38’s antibodies bind to residues up to and including position 260 (RLDNLLNMAYGV), with highest affinity for RLDNLLN-AYG.

Figure 6

Immunodominant nELAVL motif in Patient 01 CSF sample is that identified by mutational scanning. The short sequence, dominated by RLDxLL, is present in 34 of the 36 peptides identified in both replicates. Apart from nELAVL proteins, the patient’s antibodies enrich for peptides from 16 additional genes containing the motif. The biological consequences of such promiscuous autoantibody binding are unknown.

Figure 7

Anti-CRMP5 antibody epitope mapping with PhIP-Seq. Two anti-Hu PND patient (Patients 51 and 52) CSF samples also enriched CRMP5 peptides. CRMP5 is a well-established clinical biomarker for SCLC and malignant thymoma and anti-CRMP5 antibodies are often found concurrently with anti-Hu antibodies in patients with a PND. Though the peptides identified in each patient were discontiguous on the primary amino acid sequence, they are in close proximity in the 3D protein structure of CRMP5 at the region surrounding the C-terminus and the interface of the CRMP5 homodimer. SCLC = small-cell lung cancer.