Detection of neoantigen-specific T cells following a personalized vaccine in a patient with glioblastoma

Abstract

Neoantigens represent promising targets for personalized cancer vaccine strategies. However, the feasibility of this approach in lower mutational burden tumors like glioblastoma (GBM) remains unknown. We have previously reported the use of an immunogenomics pipeline to identify candidate neoantigens in preclinical models of GBM. Here, we report the application of the same immunogenomics pipeline to identify candidate neoantigens and guide screening for neoantigen-specific T cell responses in a patient with GBM treated with a personalized synthetic long peptide vaccine following autologous tumor lysate DC vaccination. Following vaccination, reactivity to three HLA class I- and five HLA class II-restricted candidate neoantigens were detected by IFN-γ ELISPOT in peripheral blood. A similar pattern of reactivity was observed among isolated post-treatment tumor-infiltrating lymphocytes. Genomic analysis of pre- and post-treatment GBM reflected clonal remodeling. These data demonstrate the feasibility and translational potential of a therapeutic neoantigen-based vaccine approach in patients with primary CNS tumors.

Keywords: Neoantigen; clonal evolution; glioblastoma; immunogenomics; personalized vaccine.

Figure 1.

Schematic representation of treatment course. Note: cycle 3 of temozolomide was delayed due to thrombocytopenia, and cycle 4 was given at a reduced dose (100 mg/m² down from 150 mg/m²) due to intolerance. Abbreviations: STR = subtotal resection; CCRT = concurrent chemoradiation therapy; TMZ = temozolomide; DCVax = autologous tumor lysate-dendritic cell vaccine.

Figure 2.

Design and response of a personalized neoantigen-based peptide vaccine for a patient with glioblastoma. (a) Schematic diagram of GBM.PVax design. Non-synonymous missense mutations are identified by comparing patient-matched PBMC (normal) and tumor DNA whole exome sequencing, then mutations are filtered through in silico neoantigen discovery pipelines to identify high-affinity HLA class I and/or class II candidates. Top candidates were selected for peptide synthesis as long peptides (SLPs). Soluble SLPs are incorporated into GBM.PVax. (b) Table listing the 8 SLPs encompassing 7 neoantigens included in GBM.PVax. The location of the mutated amino acid is enlarged and bolded. (c) Representative brain MRI axial images at indicated time points during treatment with GBM.PVax. (d) Representative H&E sections from initial tumor resection (day 0) and post-treatment tumor resection (day 347). Black line denotes 200 microns.

Figure 3.

Immunogenicity of GBM.PVax in peripheral blood. (a-d) Bar graphs from IFN-γ ELISPOT data of positively selected CD8⁺ (left column) and CD4⁺ (right column) PBMC stimulated with indicated peptide. Media = negative control (no peptide). (a) Reactivity screen to GBM.PVax neoantigens. (b) Comparison of reactivity between mutated and wild-type GBM.PVax neoantigens that demonstrated positive reactivity in Figure 3(a). (c) Comparison of reactivity between pre- and post-GBM.PVax PBMC. (d) Reactivity screen of non-GBM.PVax neoantigens from Table 1. A, b, and d represent a minimum of two independent experiments done in duplicate. (c) represents a single experiment done in duplicate due to limited sample. (*) represents p < 0.05 compared to negative control unless otherwise indicated.

Figure 4.

Clonal evolution of neoantigens after treatment. (a) Bar graphs from IFN-γ ELISPOT data of positively selected CD8⁺ (left column) and CD4⁺ (right column) ex vivo expanded TIL isolated from the post-GBM.PVax specimen and stimulated with indicated peptide. Media = negative control (no peptide). Graphs represent a single experiment done in duplicate due to limited sample. (*) represents p < 0.05 compared to negative control. (b) Representative immunohistochemistry sections of anti-CD3 and anti-CD8 staining in the post-treatment resection specimen. Black arrow identifies perivascular lymphocytes. Black line denotes 200 microns. (c) SciClone plot of clonal and subclonal populations present in pre- and post-treatment tumor specimens. VAF = variant allele fraction. Cluster 1 = founder clone mutations. Bolded neoantigens represent those with observed reactivity by ELISPOT. (d) Venn diagrams of missense mutations (left) and predicted high-affinity HLA class I neoantigens (right) from the pre- and post-treatment tumor specimens. Numbers indicate respective private or shared missense mutations or neoantigens. Parentheses represent the number of neoantigens with confirmed expression by RNA-seq.

Figure 5.

Immune landscape of the pre- and post-treatment tumor microenvironment. (a-d) Bar graphs demonstrate the relative gene expression level (log2 fold change) of indicated immune cell subsets (a, b) or immune checkpoint molecules (c, d) derived from bulk RNA-seq. Dotted line indicates +1.5 and −1.5 log2 fold changes.

Figure 6.

Modified immunophenoscore diagrams derived from Charoentong et al²¹ for pre-treatment (top) and post-treatment (bottom) specimens. The gene or gene set comprising each point around the circle is labeled and grouped in corresponding quadrants. Red to blue color scale represent averaged z-score generated from 166 TCGA GBM samples. Grayscale represents weighted z-score associated with the composite score of each quadrant corresponding to the genes or gene sets within the quadrant. MHC = Major histocompatibility complex-associated and antigen presentation genes; CP = checkpoint molecules; EC = effector cells; SC = suppressor cells.