Article Text

Download PDFPDF

144 Novel public and tumor-wide neoantigens arising from clonal aberrant splicing events drive tumor-specific T-cell responses across diverse cancer types
  1. Darwin Kwok1,
  2. Nicholas Stevers1,
  3. Takahide Nejo1,
  4. Jangham Jung1,
  5. Kaori Okada1,
  6. Lee Chen1,
  7. Senthilnath Lakshmanachetty1,
  8. Inaki Etxeberria2,
  9. Marco Gallus1,
  10. Akane Yamamichi1,
  11. Emilio Ramos1,
  12. Chibo Hong1,
  13. Maggie Colton Cove1,
  14. Gary Chan1,
  15. Aidan Du1,
  16. James Woo1,
  17. Arun Wiita1,
  18. Christopher A Klebanoff2,
  19. Joseph Costello1 and
  20. Hideho Okada1
  1. 1University of California San Francisco (UCSF), San Francisco, CA, USA
  2. 2Memorial Sloan Kettering Cancer Center, New York, NY, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Immunotherapy in gliomas is limited by tumor heterogeneity and low mutational burden. We developed a novel comprehensive in silico pipeline for detecting tumor-specific splicing events (neojunctions) across multiple cancer types, and in gliomas, we successfully identified tumor-wide, public, alternatively spliced neoantigens (ASNs) that elicit CD8+ T-cell-mediated immune responses.

Methods Our pipeline identified recurring public neojunctions expressed in TCGA RNA-seq (positive sample rate (PSR) > 10%) and not in GTEx normal tissue RNA-seq data (PSR < 1%) across six cancer types. To characterize intratumorally-conserved neojunctions, we utilized available multi-site RNA sequencing across diverse cancer types. With gliomas, our in-house dataset comprised of 56 patients with approximately 10 maximally-distanced intratumoral biopsy sites per patient (n=535). Tumor-wide public neojunction expression was subsequently validated in RNA-sequencing and mass spectrometry (MS) data from patient-derived cell lines (n=68) and samples (n=99). Two independent algorithms then predicted peptide processing likelihood and HLA-binding affinity of ASN candidates. In vitro sensitization (IVS) of healthy donor-derived CD8+ T-cells against high-confidence ASN candidates, followed by 10x VDJ scRNA-seq, was performed to identify ASN-specific TCR sequences. TCRs were transduced into triple-reporter Jurkat76s and co-cultured with ASN and HLA-expressing COS7 cells and glioma cell lines to evaluate TCR functionality.

Results Pan-cancer analysis identified large subsets of neojunctions that were interpatiently and intratumorally conserved. In particular, our glioma-specific analysis identified 249 public neojunctions with varying intratumoral heterogeneity. 4 ASNs were concurrently identified in transcriptomic and proteomic glioma data and predicted to be presented by HLA-A*02:01 with high confidence. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) performed on COS7 cells transfected with HLA-A*02:01 and full-length mRNA containing the neojunction validated proper translation and processing of the full-length mutant peptide into its HLA-bound n-mer. IVS and subsequent 10x VDJ scRNA-seq on expanded PBMC-derived CD8+ populations cultured against ASN-pulsed dendritic cells identified TCR clonotypes reactive against neojunctions in RPL22 (n=7) and GNAS (n=1), the latter being highly intratumorally-conserved (detected in > 90% of spatially-mapped biopsies across 17/56 patients (26.78%)). TCR-transduced T-cells demonstrated recognition and immunogenic activation against endogenously processed and presented neoantigens in both glioma and transfected COS7 cells. Pan-cancer analysis revealed the detection of both RPL22 and GNAS neojunctions in various tumor types beyond gliomas.

Conclusions Our unique integrative pipeline identified novel public tumor-wide splice-derived neoantigen candidates and ASN-specific TCRs, offering a promising off-the-shelf immunotherapy approach for diverse cancer types. Furthermore, characterization of novel intratumorally-conserved neoantigens addresses the critical challenge of intratumoral heterogeneity in immunotherapy resistance.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

Statistics from

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.