A H3K27M-targeted vaccine in adults with diffuse midline glioma

Abstract

Substitution of lysine 27 to methionine in histone H3 (H3K27M) defines an aggressive subtype of diffuse glioma. Previous studies have shown that a H3K27M-specific long peptide vaccine (H3K27M-vac) induces mutation-specific immune responses that control H3K27M⁺ tumors in major histocompatibility complex-humanized mice. Here we describe a first-in-human treatment with H3K27M-vac of eight adult patients with progressive H3K27M⁺ diffuse midline glioma on a compassionate use basis. Five patients received H3K27M-vac combined with anti-PD-1 treatment based on physician's discretion. Repeat vaccinations with H3K27M-vac were safe and induced CD4⁺ T cell-dominated, mutation-specific immune responses in five of eight patients across multiple human leukocyte antigen types. Median progression-free survival after vaccination was 6.2 months and median overall survival was 12.8 months. One patient with a strong mutation-specific T cell response after H3K27M-vac showed pseudoprogression followed by sustained complete remission for >31 months. Our data demonstrate safety and immunogenicity of H3K27M-vac in patients with progressive H3K27M⁺ diffuse midline glioma.

Fig. 1. Patient characteristics at baseline and individual disease trajectories.

a, Baseline characteristics of eight patients with progressive DMG H3K27M⁺ before initiation of treatment with H3K27M-vac. Age specified in years, tumor size measured as product of maximal orthogonal diameter on contrast-enhanced T1-weighted MRI sequences (mm²); cumulative dose of intensity-modulated radiotherapy measured in Gy; TMZ, temozolomide (75 mg m⁻² body surface area (BSA)) daily during radiotherapy; CCNU, lomustine (110 mg m⁻² BSA d1, TMZ mg m⁻² BSA d2–6, q42d for six cycles); oral dexamethasone intake in mg d⁻¹. Brain illustration taken from Adobe Stock Standard under License ID 222738500. b, Swimmer plot depicting clinical course since initial diagnosis, vaccine administration and time point of first H3K27M-specific immune responses in peripheral blood (n = 8 patients). Source data

Fig. 2. Treatment schedule, safety and immunogenicity of H3K27M-vac.

a, Treatment scheme for H3K27M-vac administration. b, Treatment-related AEs occurring in the observation period graded by CTCAE v.5.0. Two injection site reactions were related to H3K27M-vac and the remaining AEs were either judged to be related to concomitant medication or disease. GGT, gamma-glutamyltransferase; ALT, alanine aminotransferase; AST, aspartate aminotransferase. c, T cell immune responses as a function of time measured by difference in mean spot-forming units (s.f.u.) in IFN-γ ELISpot assay between 4 × 10⁵ peripheral blood mononuclear cells (PBMCs) stimulated with H3-mut and H3-wt control peptide. Source data

Fig. 3. Clinical response to H3K27M-vac.

a, Tumor size in mm² as a function of time in months from start of vaccination. Size determined by product of maximal orthogonal diameters on T1-weighted contrast-enhanced MRI imaging. Dots indicate measurements that are considered measurable by iRANO criteria (cerebral lesion with diameter >10 mm). b,c, PFS (b) and OS (c) since the start of vaccination. d, T1-weighted with contrast enhancement (CE) MRI sequences of PsPD of patient ID 1 at baseline, week 10 and week 34. White arrows indicate tumor lesion with PsPD at week 10. e, T1-weighted with CE MRI series of patient ID 8 with early progression between baseline and week 12 followed by disease stabilization concurrent to first detectable H3K27M-specific immune response in peripheral blood in week 18. Source data

Fig. 4. H3K27M neoepitope colocalizes with HLA class II-DR on tumor cells and myeloid cells.

a–c, PLA of primary tumor tissue of patient ID 1 (top) and ID 8 (bottom) with H3K27M and HLA-DR antibodies (red) in combination with 4′,6-diamidino-2-phenylindol (DAPI) nuclear staining (blue) alone (a), co-staining with GFAP (green) (b) and co-staining with IBA1 (green) (c). All PLAs were repeated independently twice with similar results. Scale bar in white, 30 μm; in gray, 10 μm. d, Pearson correlation of PLA spots per visual field with immunohistochemistry score of HLA-DR expression across seven patients with available FFPE tissue. A two-sided t-test was used. e, Result of automated segmentation following rolling ball background subtraction, filtering with Gaussian blur and maxima detection. Scale bar in white, 30 μm. Source data

Fig. 5. H3K27M-specific immune responses are CD4+ T cell-mediated.

a, Suppression of H3K27M-specific IFN-γ-ELISpot response by anti-MHC class II antibody (anti-MHC II) (n = 2 biologically independent experiments (BIEs)), but not by anti-MHC class I antibody (anti-MHC I) (n = 3 BIE) compared to baseline (n = 3 BIE) 18 (P = 0.002; P = 0.008, top to bottom) and 22 (P = 0.001; P = 0.006, from top to bottom) weeks since start of H3K27M-vac treatment in patient ID 1. Two-sided t-test, not adjusted for multiple comparisons. Dots mark individual data points, bar plots show the mean and error bars indicate the s.d. ** signifies P < 0.01, *** signifies P < 0.001. b–e, Flow cytometry-based intracellular IFN-γ and tumor necrosis factor (TNF)-α detection in H3K27M-peptide expanded PBMCs restimulated with H3-wt (b,d) or H3-mut (c,e), gated on CD4⁺ (b,c) and CD8⁺ (d,e) T cell subsets. f, Difference in percentage of TNF-α-expressing cells among all CD4⁺ T cells between T cells stimulated with H3-mut and H3-wt either directly (ex vivo intracellular cytokine staining (ICS)) or following expansion of T cells with H3K27M peptide (post-expansion ICS). Samples were analyzed from ID 1, ID 4, ID 5, ID 6, ID 7 and ID 8 at the weeks indicated. ELISpot responses in the first column are displayed as in Fig. 1d. g–j, H3K27M-reactive, TNF-α⁺ CD4⁺ T cells (orange) among all CD4⁺ T cells (gray) did not comprise CD25⁺FoxP3⁺ regulatory T cells. Depicted are ex vivo ICS data from patient ID 1 week 18 (g) and week 118 (h) as well as patient ID 8 week 0 (i) and week 18 (j). k, Clonotype proportion of the ten most abundant H3K27M-vac expanded CD4⁺ T cells among all sequenced T cells in primary tissue, CSF and peripheral blood across different time points of patient ID 1. l, Motif plot of sequence similarities of the CDR3β region of top ten TCRs in k after removal of recurring CAS sequence in all ten TCRs. Overlap of CDR3β of TCR3, TCR4 and TCR6 detected in CSF with motif is indicated by color and underline. Source data

Extended Data Fig. 1. IFNγ-ELISpot responses for all 8 patients over time.

Spot forming units (SFU) per 4 × 10⁵ PBMCs after restimulation with MOG, H3-wt and H3-mut. Dots mark individual data points, bar plots the mean and error bars the SD (n = 3 BIE for all patients, conditions and time points except for ID2 week 0, 4, 14, 18, where n = 2 BIE). Yellow background indicates the time period of concomitant anti-PD-1 therapy. Source data

Extended Data Fig. 2. Distribution of tumor size, beginning of vaccination and age between patients with immune response and patients without response at baseline.

Distribution of tumor size in mm² determined by product of maximal orthogonal diameters on T1 weighted contrast enhanced MRI imaging (a) time between histological diagnosis and start of vaccination in days (b) and patient age in years at start of vaccination (c) between patients with H3K27M specific immune response and patients without H3K27M specific immune response. Source data

Extended Data Fig. 3. Longitudinal course of all tumor diameters and pseudoprogression in ID 1.

a, b Tumor size in mm² (a) and relative to baseline before start of vaccination (b) as a function of time in months from start of vaccination. Size determined by product of maximal orthogonal diameters on T1 weighted contrast enhanced (CE) MRI imaging. Dots indicate measurements that are considered measurable by iRANO criteria (that is cerebral lesion with diameters > 10 mm) and stars non-measurable lesions by iRANO criteria. c, Coronary T1-weighted CE sequences (top) and fluid-attenuated inversion recovery sequences (bottom) of PsPD of patient ID 1 at baseline, week 10 and week 34. White arrows indicate tumor lesion with PsPD in week 10. Source data

Extended Data Fig. 4. H3K27M neoepitope co-localization with HLA class II-DR on tumor cells and myeloid cells in patients ID 2, ID 3, ID 4, ID 6 and ID 7.

a–c, Proximity ligation assay (PLA) of primary tumor tissue of patient ID 2, ID 3, ID 4, ID6 and ID7 from top to bottom with H3K27M and HLA-DR antibodies (red) in combination with 4’,6-Diamidino-2-phenylindol (DAPI) nuclear staining (blue) alone (a), co-staining with glial fibrillary acidic protein (GFAP) (green) (b) and co-staining with ionized calcium-binding adapter molecule 1 (IBA1) (green) (c). Scale bar in white = 30 μm; in gray = 10 μm. e, f, Automated segmentation of PLA spots (e) and nuclei (f) following rolling ball background subtraction, filtering with gaussian blur and maxima detection of visual field in a. Scale bar in gray = 10 μm. f, g, PLA as in a and co-staining as in b of H3-wildtype glioblastoma. Scale bar in white = 30 μm. h, Pearson correlation of PLA spots per cell with IHC score of HLA-DR expression across 7 patients with available FFPE tissue. Two-sided t-Test was used. Source data

Extended Data Fig. 5. PLA segmentation and single channels of PLA images of patient ID 1 and ID 8.

a,b,i, Automated segmentation of PLA spots (a) and nuclei (b, i) of ID 1 and ID 8 following rolling ball background subtraction, filtering with gaussian blur and maxima detection. Scale bar in white = 30 μm. c-h, j-o, Single channels of PLA with co-stainings as indicated on the left for ID 1 and ID 8 from images in Fig. 4a–c. Scale bar in gray = 10 μm.). All PLAs were repeated independently two times with similar results. Source data