Characterization of the T Cell Repertoire Following Personalized Neoantigen-Based Vaccination in Patients with Melanoma

Abstract

Neoantigens represent a class of tumor specific antigens that are encoded by somatic mutations which arise in cancer cells. Neoantigens are particularly attractive for the development of personalized cancer vaccines as they are expected to be both highly specific to tumor cells and highly immunogenic, as they bypass central tolerance. Like other cellular gene products, neoantigens have the potential to be presented on human leukocyte antigen, thus serving as targets for immune recognition, particularly by T cells. The Wu lab conducted a phase I clinical trial to determine whether a personalized, multi-epitope, neoantigen-based vaccine could mount an anti-tumor immune response in patients with high-risk melanoma. The objective of this work is to determine whether vaccination can expand T cell populations that preferentially recognize and kill cells presenting neoantigens, particularly through endogenous processing and presentation. Furthermore, a novel cloning and expression system is used as proof-of-concept to determine the identity of neoantigen-reactive T cell receptors (TCRs) and their cognate antigens which can be used to better understand the diversity of the patient-specific T cell repertoire post-vaccination. This thesis is divided into two parts, the first focusing on the characterization of the immune response after vaccination, and the second focusing on the identification of TCR-neoantigen pairs. Computational pipelines were used to predict immunogenic neoantigens from whole exome sequencing data collected from a patient’s tumor and healthy tissue. Up to 20 neoantigens per patient were packaged into long peptides and were administered to patients using a prime-boost strategy. Post-vaccination peripheral blood mononuclear cells (PBMCs) were analyzed for reactivity against neoantigens using exogenous peptide pulsing, and neoantigen-reactive T cell lines were identified. Autologous B cells isolated from pre-vaccination PBMCs of the 6 patients were nucleofected with tandem minigene constructs encoding neoantigens or their wild-type counterparts. Interferon (IFN)-γ enzyme linked immunospot was performed by co-culturing these B cells with expanded cell lines of the identified neoantigen-reactive T cells. Indeed, mutant peptide-reactive CD8+ T cells were reactive against autologous antigen presenting cells (APCs) expressing 15 of 15 (100%) of predicted mutant minigenes, but not their corresponding wild-type minigenes. Mutant peptide-reactive CD4+ T cells were reactive against APCs expressing 14 of 15 (93%) of mutant minigenes with 13 of 14 recognizing only the mutant version. Additionally, elevated CD107αβ expression, a marker of T cell degranulation and a proxy for cytotoxicity, was observed in T cells following exposure to APCs presenting mutant minigene peptides for both neoantigen-reactive CD4+ and CD8+ T cell lines, providing evidence of cytotoxic capacity in the neoantigen-reactive T cells. A novel cloning and expression system developed in the Wu lab was used to establish the identity and functional avidity of TCR-neoantigen pairs. IFN-γ+ T cells from the PBMCs of two patients after pooled neoantigen stimulation underwent single-cell sequencing. The dominant T cell clonotypes were cloned and expressed in TCR-deficient reporter T cell lines which were screened against APCs loaded with candidate antigens. One of 6 cloned CD4+ TCRs was successfully matched to mutant RUSC2 peptide, while 3 of 7 CD8+ TCRs were each matched to the same neoantigen, mutant VPS16. Functional avidity of the cloned TCRs was established at ~10^6 pg/ml, which was lower than that observed for identified control TCR-viral peptide pairs. Each TCR successfully discriminated between mutant and wild-type peptides. These findings suggest that neoantigens can be endogenously processed and presented by autologous APCs and that neoantigen-based vaccination successfully expands T cell populations which preferentially recognize APCs expressing neoantigens. Efforts to deconvolute the breadth of the T cell responses elicited after vaccination will further elucidate the role of neoantigen-specific T cell diversity in the setting of an anti-tumor immune response. These results provide strong evidence to support further investigation of this vaccination strategy, both alone and in combination with checkpoint blockade or other immunotherapies.