Abstract PR02: Identification of breast cancer neoantigens exposed by radiation therapy
Authors: Lhuillier Claire , Rudqvist Nils , Demaria Sandra 
Published: February 2019

Abstract
Tumor-specific mutations can generate neoantigens, a class of major histocompatibility complex (MHC)-binding peptides that may be highly immunogenic because they are not expressed in healthy tissues and thus not subject to central thymic tolerance. Recent studies have highlighted the key role for neoantigens in cancer immunotherapy.
We have previously shown in the 4T1 mouse model (syngeneic with BALB/c mice) of immune checkpoint blockade (ICB)-resistant metastatic breast cancer that local radiation therapy (RT) combined with CTLA-4 blockade induces the CD8+ T cell-mediated regression of irradiated tumors and limits metastatic lung colonization. Preliminary analysis of the T-cell receptor (TCR) repertoire by deep sequencing of CDR3 regions of TCRβ indicated that unique clonotypes are expanded in 4T1 tumors treated with RT and CTLA-4 blockade, suggesting that tumor rejection involves T cells reactive to a set of tumor antigens that are made available to the immune system by RT. Therefore, we hypothesize that RT, by changing the transcriptional profile of cancer cells, may expose antigenic mutations not transcribed at sufficient levels in untreated tumors, and hence promote priming of T cells to these unique mutated antigens.
We performed whole-exome sequencing (WES) and RNA sequencing (RNA-seq) of 4T1 cells irradiated (8GyX3) or not (0Gy) in vitro to identify tumor-specific mutations, including nonsynonymous and frameshift mutations (insertions or deletions). Genetic variants in WES data were called using the mm10 mouse reference genome, and genomic DNA from BALB/c mouse tail was used to identify somatic mutations. We then used several algorithms (NetMHC 3.4; SMM; SYFPEITHI) to predict MHC-I (H2-Kd, -Dd, -Ld)-binding epitopes from these mutated genes, and we selected those with a predicted affinity &lt;500 nM. In order to focus on the mutations likely to be expressed in a majority of tumor cells, we used the RNA-seq data to select for variants present at a minimum of 40% allelic frequency in both conditions (irradiated or not), but we prioritized mutated peptides that were upregulated by radiation.
Using these genomic and computational analyses, we selected 17 predicted MHC-I restricted neo-epitopes, out of 309 total mutations initially identified in the 4T1 cell line. These candidate peptides were synthesized and tested in vitro for binding to H2-Ld or H2-Kd in a MHC stabilization assay using RMA-S cells expressing the appropriate MHC- I allele.
We identified two mutated peptides that bind H2-Ld with high affinity. One of these neoepitopes arises from the Dhx58 gene which is significantly upregulated (fold change=3.4, p&lt;0.001) in 4T1 cells irradiated with 8GyX3. Vaccination experiments are ongoing to test the immunogenicity of these peptides, and their ability to elicit a tumor-specific CD8+ T cell response in BALC/c mice.
We demonstrate here that the use of an integrative approach combining WES, RNA-seq and in vitro binding assays allows for the identification of potential neoantigens that may be selectively unveiled by RT. Although the immunogenicity of the RT-exposed neoantigens identified here needs to be confirmed in vivo, these data provide initial proof-of-principle evidence that RT can uncover existing neoantigens to the immune system.
This abstract is also being presented as Poster B85.
Citation Format: Claire Lhuillier, Nils Rudqvist, Sandra Demaria. Identification of breast cancer neoantigens exposed by radiation therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr PR02.