Detecting Intratumoral T Cell Responses After Personal Neoantigen Vaccine in Glioblastoma Using a Novel T Cell Receptor Cloning and Expression System

Abstract

Recent studies have highlighted the promise of targeting tumor neoantigens to generate potent anti-tumor immune responses. Our group, together with others, recently reported that personalized neoantigen vaccines for patients with melanoma generated de novo polyfunctional CD4+ and CD8+ T cell responses. To assess the feasibility of this strategy for tumors with lower mutation rate and a cold tumor microenvironment, we conducted a phase I/Ib study of personalized neoantigen vaccines for newly diagnosed methylguanine methyltransferase (MGMT)-unmethylated glioblastoma patients. The study was designed to incorporate vaccination into standard of care, with 8 patients receiving a personalized neoantigen vaccine that consisted of up to 20 synthetic long peptides encoding predicted neoepitopes, and poly-ICLC adjuvant, following surgery and radiation. Many questions arise about the impact of these novel vaccines, including their ability to generate circulating neoantigen-specific T cell responses, the functionality of the T cells elicited, and their potential to transform the tumor microenvironment from cold to inflamed. While some of these questions can be addressed using conventional approaches, novel tools for probing the specificity and functionality of T cell receptors (TCR), could allow us to analyze the intratumoral TCR repertoire, and determine the extent to which T cells specific for neoantigens are able to migrate to the intracranial site of disease. In the current study, we describe a streamlined approach for matching TCR sequences with cognate antigen through on-demand cloning and expression of TCRs and screening against candidate antigens. This system can be applied to monitor antigen-specific T cell responses, and potentially guide the design of effective T cell-based immunotherapies. We first demonstrate the system’s capacity to identify viral-antigen specific TCRs and compare the functional avidity of TCRs specific for a given antigen target. We then applied this pipeline to study the intra-tumoral TCR repertoire of a patient with glioblastoma treated with personal neoantigen vaccine. We demonstrate that patients who did not receive dexamethasone, a highly potent corticosteroid frequently prescribed to glioblastoma patients for cerebral edema, generated circulating polyfunctional neoantigen-specific CD4+ and CD8+ T-cell responses, and increased tumor-infiltrating T-cells. Utilizing single-cell T-cell receptor analysis and our cloning and expression pipeline, we provide evidence that neoantigen-specific T-cells from the peripheral blood can migrate into an intracranial glioblastoma tumor. However, infiltrating T cells, including those with neoantigen-specificity, expressed multiple co-inhibitory receptors consistent with a severe exhaustion phenotype, underscoring the potential for synergy in combination with checkpoint blockade. Neoantigen-targeting vaccines thus have the potential to favorably alter the immune milieu of glioblastoma, and hold great promise for use in combination immunotherapy strategies.