In-depth characterization of vaccine-induced neoantigen-specific T cells in patients with IDH1-mutant glioma undergoing personalized peptide vaccination

Abstract

Isocitrate dehydrogenase (IDH) mutant glioma is a malignant primary brain tumor diagnosed in adults. In recent years, there has been significant progress in understanding the molecular pathogenesis and biology of these tumors. The first targeted IDH-inhibitor was approved by the US Food and Drug Administration in August 2024 for grade 2 gliomas, in light of results of a phase III trial which showed significant advantages in progression-free survival. However, biologic therapy is not curative, and subsequent treatment options offer only limited clinical benefit and often result in long-term toxicities. In addition, targeted treatment options for grade 3 and grade 4 IDH-mutant gliomas are still missing. In this study, we present n=52 patients with glioma (grade 2, 3 and 4) with confirmed IDH1 mutation (mutIDH1) in the newly diagnosed and recurrent setting who, in addition to standard-of-care, received a personalized neoantigen-targeting peptide vaccine. Each tumor was initially analyzed for somatic mutations by whole exome sequencing, and a peptide vaccine containing potential neoepitopes was designed, manufactured and vaccinated. Each vaccine consisted of peptides derived from numerous somatic mutations, including at least one peptide targeting the mutIDH1.Vaccine immunogenicity was determined by intracellular cytokine staining and simultaneous measurement of four T-cell activation markers (Interferon-γ, Tumor Necrosis Factor, Interleukin-2, CD154) after 12-day in vitro expansion of pre and post vaccination peripheral blood mononuclear cells. Extracellular CD154 staining was used to sort mutIDH1-specific CD4+T cells.Immunomonitoring revealed that the vaccines were immunogenic and induced mainly CD4 but also CD8 T cell responses. Vaccine-induced immune responses were robust and polyfunctional. Immunogenicity against mutIDH1 was high (89%). We implemented an assay which allowed us to isolate functional antigen-specific CD4+T cells in an HLA-independent manner. Subsequent T cell receptor (TCR) repertoire sequencing revealed that CD4+T cells reacting on mutIDH1 stimulation were polyclonal. Strikingly, we detected two mutIDH1-specific TCRβ candidate sequences in three different patients. These three patients had the same human leukocyte antigen (HLA) DQA-DQB alleles. The obtained TCRβ sequences could be tracked in autologous ex-vivo single-cell transcriptomic data. Our results provide a rationale for pursuing vaccination and T cell transfer strategies targeting IDH1. Furthermore, our findings indicate that personalized neoantigen-targeting vaccines might be considered for the treatment of IDH1-mutant gliomas.

Keywords: T cell; T cell Receptor - TCR; Vaccine.

Figure 1. General immunogenicity data of the cohort. List of all study participants. Displayed are the number of total vaccinated peptides and the phenotype of the vaccine-induced neoantigen-specific T cells. Solely CD4 response (blue), solely CD8 response (orange), combined CD4 and CD8 response (“Both”; green), or no response (gray).

Figure 2. Immunogenicity of vaccinated mutIDH1 neoantigens. Percentage of patients that developed a solely CD4 response (blue), solely CD8 response (orange), a combined CD4 and CD8 response ("Both"; green) or no response (grey) against the HLA class II (right) or the HLA class I (left) mutIDH1-derived peptides. IDH1, isocitrate dehydrogenase 1; mutIDH1, mutated IDH1; HLA, Human leukocyte antigen.

Figure 3. Multicytokine profile of CD4 T cell responses against vaccinated mutIDH HLA class II peptides Heat map showing the frequencies of each distinct multicytokine profile. Hierarchical clustering of patients was performed according to indicated frequencies of CD154, IFN-γ, IL-2, and TNF expressing CD4+T cells as determined by flow cytometry analysis and after square root transformation (color gradient from dark blue=0 to dark red=5 of the parent cell subset). Clinical parameters depicted on the left (glioma grade; presence of IDH1 mutation, ATRX mutation, or TP53 mutation; recurrence before start; receipt of an HLA class I peptide targeting the same IDH1 mutation, immune responder) were not included in the clustering. ATRX, alpha thalassemia/mental retardation syndrome X-linked gene; astro, astrocytoma; HLA, Human leukocyte antigen; IDH1, isocitrate dehydrogenase 1; IFN-γ, Interferon-γ; IL-2, Interleukin-2; mutIDH1, mutated IDH1; TNF, Tumor necrosis factor; TP53, Tumor protein p53.

Figure 4. TCRβ sequences tracked in single-cell transcriptomic data UMAP plot showing 79,662 cells from a PBMC sample of patient ID14 after vaccination. Cells were clustered and annotated using the R package Seurat as seen in the lower UMAP plot. Paired V(D)J information was added to the single-cell data with scRepertoire. The amino acid sequence of the TCR beta chain that was identified via mutIDH1-specific immunogenicity assay was used to highlight the corresponding TCR clone in the single-cell data and is shown highlighted in blue in the upper UMAP plot in contrast to the other cells (gray). mutIDH1, mutated isocitrate dehydrogenase 1; PBMC, peripheral blood mononuclear cell; TCR, T cell receptor; UMAP, Uniform manifold approximation and projection.