PBMCs collected before vaccination show low/no response to candidate vaccine peptides. (A) The nomenclature of the N-peptides and D-neopeptides. I or II indicate if the peptides were chosen/designed for HLA class I or -II binding; MUT/RNA/GBM indicates a naturally mutated peptide (MUT), derivation from the over-/highly expressed genes (RNA) or from known glioblastoma (GBM) targets; X indicates the number of the peptide; 0 or 1 indicate if the peptides had been artificially mutated (1) or not (0); (’) indicates that peptides contained the naturally mutated amino acid. (B) PBMCs collected before vaccination were stimulated with 4 μg/mL Tetanus toxoid or 2 μM CEF II peptide pool for 7 days, and proliferation was measured by ³H-thymidine incorporation assay. The proliferation strength is depicted as counts per minute (cpm). In each group, the N-peptides and their corresponding D-neopeptides are included, and the designed mutated positions are highlighted in the red box. The blue dotted line indicates the mean value of the no peptide control. The red dotted line indicates the mean value plus three standard deviations of the no peptide control. Values above the red dotted line were considered positive. (C) PBMCs collected before vaccination were stimulated with 5 μM predicted class I-peptides, including N-peptides and D-neopeptides, for 7 days, and proliferation was measured by ³H-thymidine incorporation assay. 10-15 replicate wells were tested for proliferation, and responses were depicted as cpm. (D) PBMCs collected before vaccination were stimulated with 5 μM predicted class II-peptides, including N-peptides and D-neopeptides, for 7 days, and proliferation was measured by ³H-thymidine incorporation assay. 10-15 replicate wells were tested for proliferation, and responses were depicted as cpm.

Designed mutations increase the stimulatory strength of D-neopeptides above N-peptides. (A) TILs were stimulated with 5 μM class I-peptides, including N-peptides and D-neopeptides, for 5 days. 5 replicate wells were tested for proliferation, and proliferation was measured by 3H-thymidine incorporation assay. In each group, the N-peptides and their corresponding D-neopeptides are included, and the designed mutated positions are highlighted in the red box. The blue dotted line indicates the mean value of the no peptide control. The red dotted line indicates the mean value plus three standard deviations of the no peptide control. Values above the red dotted line were considered positive. (#) indicates the peptide that was used in the 1st to 4th vaccinations. (B) TILs were stimulated with 5 μM class II-peptides, including N-peptides and D-neopeptides, for 5 days. 5 replicate wells were tested for proliferation, and proliferation was measured by 3H-thymidine incorporation assay.

Over-/highly expressed genes in the primary glioblastoma when compared with glioblastoma cohort in TCGA Database.

Schematic outline of the personalized peptide vaccination. (A) Timeline of clinical events for a patient with glioblastoma in whom we applied a highly personalized peptide vaccination. I. Vaccine peptide design. Surgically resected primary glioblastoma and PBMCs were collected. Somatic mutants and over-/highly expressed genes in glioblastoma were identified as TSAs and TAAs by WES and RNA-seq, and the HLA genotype were identified with PBMCs by DNA sequencing. Potential vaccine peptides were predicted and selected from TSAs and TAAs based on HLA binding prediction. Peptide cocktails containing ~25 peptides for HLA class I or II molecules were prepared for vaccination. II. Peptide cocktail vaccination and testing. The patient was s.c. vaccinated with the peptide cocktails, and Imiquimod or XS15 + MontanideTM were used as the adjuvant. Meanwhile, the patient received treatment with pembrolizumab and bevacizumab. No dexamethasone was given throughout the periods of vaccination. PBMCs were collected after vaccination at the indicated time points and tested with vaccine peptides to examine the effect of immunotherapy. Surgically resected recurrent glioblastoma was collected after four vaccinations, and tumor-infiltrating lymphocytes (TILs) were isolated and tested with vaccine peptides. (B) HLA genotypes of the patient with glioblastoma, including HLA-A, HLA-B, HLA-DR, HLA-DP, and HLA-DQ alleles. n.a. indicates not available.

TILs do not respond to N-class I-peptides. TILs were stimulated with 5- or 10 μM N-class I-peptides for 5 days. 5 replicate wells were tested for proliferation, and proliferation was measured by 3H-thymidine incorporation assay. The proliferation strength is depicted as SI. (#) indicates the peptide that was used in the 1st to 4th vaccinations. Data are expressed as mean ± SEM.

Activated T lymphocytes in recurrent tumor interact with macrophages. (A) Expression of T cell activating markers HLA-DR and Tim-3 in the perivascular areas and peritumoral areas of the primary and recurrent tumor was detected by IMC. (B) CD3-CD7+ NK cells infiltrated in the perivascular areas and peritumoral areas of the primary and recurrent tumor was detected by IMC. (C) Pro-inflammatory CD11c+ CD68+ macrophages, which had infiltrated the perivascular areas and peritumoral areas of the primary and recurrent tumor, were detected by IMC. (D) Colocalization analysis of CD68+ macrophages and CD3+ T cells in the perivascular areas and peritumoral areas of the primary and recurrent tumor were detected by IMC. Scale bars are indicated in the figures.

Increased T lymphocyte infiltration in the recurrent compared to the primary tumor. (A) CD3 immunohistochemical staining in the primary and recurrent tumor. (B) Three fields of view were selected and counted CD3+ T cells in both perivascular and peritumoral areas of the primary and recurrent tumors using ImageJ. Scale bars are indicated in the figures.

Peripheral blood T cell responses to v-peptides increase with repetitive vaccination. (A) PBMCs were collected 3 weeks after the 2nd peptide cocktail vaccination, and CD45RA-negative PBMCs were stimulated with 4 μg/mL Tetanus toxoid, 2 μM CEF II peptide pool or 5 μM v-peptides for 7 days. 5-10 replicate wells were tested for proliferation, and proliferation measured by 3H-thymidine incorporation assay. CD45RA-negative PBMCs collected before vaccination were used as reference. The proliferation strength is depicted as stimulation index (SI). The red dotted line indicates a stimulatory response of SI = 2, and values above the red dotted line were considered positive. (#) indicates the peptide that was used in the vaccinations. (B) PBMCs were collected 5 weeks after the 3rd peptide cocktail vaccination, and CD45RA-negative PBMCs were stimulated with 4 μg/mL Tetanus toxoid, 2 μM CEF II peptide pool or 5 μM v-peptides for 7 days. 5-10 replicate wells were tested for proliferation, and proliferation was measured by 3H-thymidine incorporation assay. CD45RA-negative PBMCs collected before vaccination were used as the control. Data are expressed as mean ± SEM, and p values were determined using an unpaired t-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Supplementary Table 1. Somatic mutations of the patient's glioblastoma; Supplementary Table 2. See Excel File; Supplementary Table 3. Literature-derived, glioblastoma-associated TAAs; Supplementary Table 4. See Excel File; Supplementary Table 5. Over-/highly expressed genes that were chosen for the design of vaccine peptides; Supplementary Table 6. Peptide cocktails for vaccination; Supplementary Table 7. Antibodies for imaging mass cytometry.

Predicted TSA/TAA-derived N-peptides and D-neopeptides

Vaccinated D-neopeptides activate tumor-infiltrating antitumor T cells. (A) Circos plots provide an overview of the frequencies of Vβ-Jβ pairing in the primary and recurrent tumors. (B) Surface TCR β chain expression of the D-neopeptide-specific CD4+ TCCs was analyzed using FACs.

AbstractPurpose: The low mutational load of some cancers is considered one reason for the difficulty to develop effective tumor vaccines. To overcome this problem, we developed a strategy to design neopeptides through single amino acid mutations to enhance their immunogenicity. Experimental Design: Exome and RNA sequencing as well as in silico HLA-binding predictions to autologous HLA molecules were used to identify candidate neopeptides. Subsequently, in silico HLA-anchor placements were used to deduce putative T-cell receptor (TCR) contacts of peptides. Single amino acids of TCR contacting residues were then mutated by amino acid replacements. Overall, 175 peptides were synthesized and sets of 25 each containing both peptides designed to bind to HLA class I and II molecules applied in the vaccination. Upon development of a tumor recurrence, the tumor-infiltrating lymphocytes (TIL) were characterized in detail both at the bulk and clonal level. Results: The immune response of peripheral blood T cells to vaccine peptides, including natural peptides and designed neopeptides, gradually increased with repetitive vaccination, but remained low. In contrast, at the time of tumor recurrence, CD8+ TILs and CD4+ TILs responded to 45% and 100%, respectively, of the vaccine peptides. Furthermore, TIL-derived CD4+ T-cell clones showed strong responses and tumor cell lysis not only against the designed neopeptide but also against the unmutated natural peptides of the tumor. Conclusions: Turning tumor self-peptides into foreign antigens by introduction of designed mutations is a promising strategy to induce strong intratumoral CD4+ T-cell responses in a cold tumor like glioblastoma.