Abstract B126: Robust vaccination response to proteasome catalyzed peptide spliced peptides in brain tumor models— CD8+ T cell epitopes are composed of quantal antigens assembled to make quantitopes
Authors: Gupta Puja , Clark Jason J , Shanker Varun J , Skirboll Stephen J , Wong Albert J 
Published: February 2025

Abstract
pepVIII, an anticancer vaccine targeting EGFRvIII, has been tested in randomized Phase II and III trials for glioblastoma. In ACT IV, a randomized Phase III trial for patients with newly diagnosed tumors, one arm for &lt;2 cm3 residual tumors showed equivocal results but patients with &gt;2 cm3 residual disease showed statistically significant increase in survival. In ReACT, a study for recurrent tumors, the arm with vaccine plus bevacizumab also showed significant results. Available data suggests that pepvIII can be made more robust. Despite its essential role in antigen presentation, enhancing proteasomal processing is an unexploited strategy for improving vaccines. We examined 20 peptides in silico and experimentally, which showed that a tyrosine substitution (Y6-pepVIII) maximizes proteasome cleavage and survival in a subcutaneous tumor model. In an intracranial glioma model, Y6-pepVIII showed a 62 and 31% improvement in median survival compared to control animals and pepVIII-vaccinated mice. Y6-pepVIII vaccination altered tumor-infiltrating lymphocyte subsets and expression of PD-1 on intratumoral T cells. Combination with anti–PD-1 therapy cured 45% of the Y6-pepVIII–vaccinated mice but was ineffective for pepVIII-treated mice. LC-MS/MS analysis of proteasome-digested pepVIII and Y6-pepVIII revealed that most fragments were similar but more abundant inY6-pepVIII digests and 77% resulted from proteasome-catalyzed peptide splicing (PCPS). In PCPS, the intact peptide undergoes excision/splicing to join two distal pieces to create the CD8+ T cell epitope. We identified 10 peptides that bound human and murine MHC class I. Nine were PCPS products and only one peptide was colinear with EGFRvIII, indicating that PCPS fragments are a major component of MHC class I recognition. Despite not being colinear with EGFRvIII, two of three PCPS products tested were capable of increasing survival when administered independently as vaccines. Analysis of peptides eluted from JY/vIII cells, an HLA-A*0201 restricted dendritic cell line expressing EGFRvIII, showed that 15 of 18 peptides are derived from PCPS and only 3 are colinear. Our work suggests a strategy for improving vaccines is through increasing proteasomal processing to increase PCPS, thus augmenting the immune response and survival in mice. While canonical CD8+ T cell epitopes are colinear peptides derived from the parental protein, PCPS-derived epitopes are from two minimal units, or quanta, joined to make the final antigen. We propose these units be called quantal antigens, or quantigens, and the final product be called quantitopes.
Citation Format: Puja Gupta, Jason J Clark, Varun J Shanker, Stephen J Skirboll, Albert J Wong. Robust vaccination response to proteasome catalyzed peptide spliced peptides in brain tumor models— CD8+ T cell epitopes are composed of quantal antigens assembled to make quantitopes [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B126.