Abstract B032: PhIP-seq assessment of the serum antibody repertoire before and after immune-related adverse events in four melanoma patients treated with checkpoint blockade immunotherapy
Authors: O’Donnell Timothy J. , Shan Meimi , Merritt Elliott , Gugel Elena Gonzalez , Blasquez Ana B. , Meseck Marcia , Friedlander Phillip A. , Rubinsteyn Alexander , Horowitz Amir , Bhardwaj Nina , Laserson Uri 
Published: February 2019

Abstract
Phage immunoprecipitation sequencing (PhIP-seq) is a technique to profile the epitope specificities of an antibody repertoire by phage display of a peptide library followed by immunoprecipitation and next-generation sequencing (1). We have developed phage libraries corresponding to all 36-mer peptides in the human proteome (approximately 413,000 peptides), as well as libraries of peptides found in viruses, bacteria, and toxins. Here, in a pilot study to understand if PhIP-seq might be used to identify predictive biomarkers for immune-related adverse events (irAE) in the context of checkpoint blockade immunotherapy, we applied the human proteome library to probe the self-directed IgG response in sera from four melanoma patients receiving checkpoint blockade who experienced irAE. In total, 16 serum samples acquired at the pre-treatment, post-treatment / pre-irAE, or post-irAE timepoints were assayed from these patients. We additionally analyzed samples from six melanoma patients who received vaccines targeting the MART-1 and NY-ESO-1 antigens. Using a stringent confidence threshold, we identified a median 31 self-directed antibody specificities (hits) at the pre-immunotherapy timepoint in these cancer patients and 41.5 hits post-therapy, compared to a median 6.5 hits in healthy donors. Patients receiving combination checkpoint blockade showed more hits than those treated with monotherapy. For a patient who received combination nivolumab and ipilimumab and developed a hepatic irAE, a cluster of 25 hits (of 161 total) was detected uniquely in a sample taken within two months subsequent to the adverse event. This cluster included a peptide from the C-Reactive Protein (CRP) gene and other genes expressed in the liver (EHBP1, VSTM2L), or across tissues (SAFB2), but also included genes with low expression in the liver (SPTBN4, DMBT1, IQGAP3, SPTBN4). In a patient who received ipilimumab and nivolumab and developed myalgia, hits against targets associated with autoimmune disease were found including NUMA1 (connective tissue autoimmunity), TRPM1 (melanoma-associated retinopathy), as well as three epitopes in the Mediator Of DNA Damage Checkpoint 1 (MDC1) gene. Finally, for three patients receiving a peptide vaccine targeting MART-1 and NY-ESO-1 with adjuvant poly-ICLC and montanide, we detected an IgG response against NY-ESO-1, but not MART-1, in each patient. No response to either protein was observed in three patients receiving a dendritic cell vaccine against the same antigens.Our small study suggests that PhIP-seq readily detects changes in the epitope specificities of the serum antibody repertoire in the course of immunotherapy. A key limitation of PhIP-seq is that the method can only detect antibodies with specificities for linear (as opposed to conformational) epitopes. Nevertheless, the assay’s low cost (approximately $30 per sample), small sample size requirement (1 µL serum), and ability to work with dried blood spot samples may make it an attractive technology to discover serological predictors of irAE in larger cohorts. References: 1. Larman HB, Zhao Z, Laserson U, Li MZ, Ciccia A, Gakidis MA, Church GM, Kesari S, Leproust EM, Solimini NL, Elledge SJ. Autoantigen discovery with a synthetic human peptidome. Nat Biotechnol 2011;29(6):535–41.
Citation Format: Timothy J. O’Donnell, Meimi Shan, Elliott Merritt, Elena Gonzalez Gugel, Ana B. Blasquez, Marcia Meseck, Phillip A. Friedlander, Alexander Rubinsteyn, Amir Horowitz, Nina Bhardwaj, Uri Laserson. PhIP-seq assessment of the serum antibody repertoire before and after immune-related adverse events in four melanoma patients treated with checkpoint blockade immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B032.