Neoantigen vaccine generates intratumoral T cell responses in phase Ib glioblastoma trial methods
Aim. Evidence-backed execution summary for Neoantigen vaccine generates intratumoral T cell responses in phase Ib glioblastoma trial methods from Neoantigen vaccine generates intratumoral T cell responses in phase Ib glioblastoma trial.
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human
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
RNA-seq.
reagent used in the protocol.
- Use
- For RNA-seq library construction, RNA was extracted from fresh-frozen sections (patients 1, 2 and 9) or FFPE samples (patients 3, 4, 5, 6, 7, 8 and 10) using the Qiagen AllPrep DNA/RNA/miRNA Universal Kit or Qiagen AllPrep DNA/RNA FFPE Kit, respectively. RNA-seq libraries were prepared using Illumina TruSeq Stranded...
Patient samples and cell lines
reagent used in the protocol.
- Use
- Tumour samples and heparinized blood were obtained from study subjects on IRB-approved protocols at the DFCI. Patient samples were de-identified and assigned a study-specific tracking number. PBMCs were collected via leukapheresis before initiation of external beam radiotherapy (pre-treatment baseline) and approxima...
Antigen formats for immune monitoring
reagent used in the protocol.
- Use
- Assay (ASPs) and predicted class I epitope peptides (EPTs) were synthesized and lyophilized (from either JPT Peptide Technologies or RS Synthesis; >80% purity) in a manner previously described. ASPs were 15-16 amino acids long and overlapped by at least 11 amino acids, covering the immunizing peptide sequence...
Generation and detection of patient neoantigen-specific T cells
reagent used in the protocol.
- Use
- Screening ex vivo IFNγ ELISPOT assays on PBMCs stimulated for 18 h with synthesized screening peptides consisting of ASP and EPT peptides were performed. The averages of responses to DMSO, HIV-GAG or OVA peptide for each time point were subtracted from experimental wells for background correction. If no respon...
Intracellular cytokine staining.
reagent used in the protocol.
- Use
- PBMCs were thawed and rested overnight in DMEM GlutaMAX medium (Gibco, ThermoFisher) supplemented with 10% AB-positive heat-inactivated human serum (Gemini Bioproduct), nonessential amino acids, HEPES, β-mercaptoethanol, sodium pyruvate, penicillin-streptomycin (Gibco, ThermoFisher) and 20 ng ml -1...
Multiplex immunofluorescence
reagent used in the protocol.
- Use
- Staining was performed using BOND RX fully automated stainers (Leica Biosystems). The target antigens, antibody clones and dilutions for markers, as well as the details of controls are listed in. Image acquisition was performed using the Mantra multispectral imaging platform (Vectra 3, PerkinElmer). Areas with non-...
Processing of GBM and PBMC specimens for scRNA-seq
reagent used in the protocol.
- Use
- Surgically resected GBM tissue from patient 7 was obtained on ice within 30 min of lesion excision. The tumour specimen was mechanically disrupted into small pieces with a disposable, sterile scalpel and further dissociated into a single-cell suspension using the enzymatic brain dissociation kit (P) from Miltenyi Bi...
Processing of GBM and PBMC specimens for scRNA-seq
reagent used in the protocol.
- Use
- Single neoantigen-reactive CD4 + and CD8 + T cells were isolated from PBMCs of patient 7, obtained 8-16 weeks after vaccine initiation. For isolation of CD4 + T cells, PBMCs were stimulated in vitro with 10 µg ml -1 of ASP peptide pools overnight, and then subsequently flow cytometrically sorted on...
Generation of personalized neoantigen vaccines
The personalized neoantigen vaccines were prepared based on the analysis of whole-exome sequencing (WES) and RNA-seq data generated from fresh-frozen tumours or tumours that were available as formalin-fixed paraffin-embedded (FFPE) tissue, obtained at the time of diagnostic resection. WES of normal tissue was genera...
- Use
- The personalized neoantigen vaccines were prepared based on the analysis of whole-exome sequencing (WES) and RNA-seq data generated from fresh-frozen tumours or tumours that were available as formalin-fixed paraffin-embedded (FFPE) tissue, obtained at the time of diagnostic resection. WES of normal tissue was genera...
Generation of personalized neoantigen vaccines
CLIA-certified WES was conducted by the Clinical Research Sequencing Platform, Broad Institute (CLIA 22D2055652). Library construction from surgical GBM specimens and matched germline DNA of all 10 patients was performed as previously described. For patients 1, 2 and 9, whole-exome capture was performed using the A...
- Use
- CLIA-certified WES was conducted by the Clinical Research Sequencing Platform, Broad Institute (CLIA 22D2055652). Library construction from surgical GBM specimens and matched germline DNA of all 10 patients was performed as previously described. For patients 1, 2 and 9, whole-exome capture was performed using the A...
RNA-seq.
For RNA-seq library construction, RNA was extracted from fresh-frozen sections (patients 1, 2 and 9) or FFPE samples (patients 3, 4, 5, 6, 7, 8 and 10) using the Qiagen AllPrep DNA/RNA/miRNA Universal Kit or Qiagen AllPrep DNA/RNA FFPE Kit, respectively. RNA-seq libraries were prepared using Illumina TruSeq Stranded...
- Use
- For RNA-seq library construction, RNA was extracted from fresh-frozen sections (patients 1, 2 and 9) or FFPE samples (patients 3, 4, 5, 6, 7, 8 and 10) using the Qiagen AllPrep DNA/RNA/miRNA Universal Kit or Qiagen AllPrep DNA/RNA FFPE Kit, respectively. RNA-seq libraries were prepared using Illumina TruSeq Stranded...
Somatic mutation calling.
Analyses of WES data of tumour and matched PBMCs (as source of normal germline DNA) from the patients were used to identify the specific coding-sequence mutations, including single-, di- or tri-nucleotide variants that lead to single amino acid missense mutations and small insertions/deletions (indels). Output from...
- Use
- Analyses of WES data of tumour and matched PBMCs (as source of normal germline DNA) from the patients were used to identify the specific coding-sequence mutations, including single-, di- or tri-nucleotide variants that lead to single amino acid missense mutations and small insertions/deletions (indels). Output from...
Patient samples and cell lines
Tumour samples and heparinized blood were obtained from study subjects on IRB-approved protocols at the DFCI. Patient samples were de-identified and assigned a study-specific tracking number. PBMCs were collected via leukapheresis before initiation of external beam radiotherapy (pre-treatment baseline) and approxima...
- Use
- Tumour samples and heparinized blood were obtained from study subjects on IRB-approved protocols at the DFCI. Patient samples were de-identified and assigned a study-specific tracking number. PBMCs were collected via leukapheresis before initiation of external beam radiotherapy (pre-treatment baseline) and approxima...
Intracellular cytokine staining.
PBMCs were thawed and rested overnight in DMEM GlutaMAX medium (Gibco, ThermoFisher) supplemented with 10% AB-positive heat-inactivated human serum (Gemini Bioproduct), nonessential amino acids, HEPES, β-mercaptoethanol, sodium pyruvate, penicillin-streptomycin (Gibco, ThermoFisher) and 20 ng ml -1...
- Use
- PBMCs were thawed and rested overnight in DMEM GlutaMAX medium (Gibco, ThermoFisher) supplemented with 10% AB-positive heat-inactivated human serum (Gemini Bioproduct), nonessential amino acids, HEPES, β-mercaptoethanol, sodium pyruvate, penicillin-streptomycin (Gibco, ThermoFisher) and 20 ng ml -1...
Multiplex immunofluorescence
Staining was performed using BOND RX fully automated stainers (Leica Biosystems). The target antigens, antibody clones and dilutions for markers, as well as the details of controls are listed in. Image acquisition was performed using the Mantra multispectral imaging platform (Vectra 3, PerkinElmer). Areas with non-...
- Use
- Staining was performed using BOND RX fully automated stainers (Leica Biosystems). The target antigens, antibody clones and dilutions for markers, as well as the details of controls are listed in. Image acquisition was performed using the Mantra multispectral imaging platform (Vectra 3, PerkinElmer). Areas with non-...
Processing of GBM and PBMC specimens for scRNA-seq
Surgically resected GBM tissue from patient 7 was obtained on ice within 30 min of lesion excision. The tumour specimen was mechanically disrupted into small pieces with a disposable, sterile scalpel and further dissociated into a single-cell suspension using the enzymatic brain dissociation kit (P) from Miltenyi Bi...
- Use
- Surgically resected GBM tissue from patient 7 was obtained on ice within 30 min of lesion excision. The tumour specimen was mechanically disrupted into small pieces with a disposable, sterile scalpel and further dissociated into a single-cell suspension using the enzymatic brain dissociation kit (P) from Miltenyi Bi...
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METHODS
Study eligibility was assessed among patients seen at the Center for Neuro-Oncology, Dana-Farber Cancer Institute and required: age ≥18 years; Karnofsky performance status ≥70; histopathological confirmation of WHO grade IV glioblastoma (GBM) or variant; tumour MGMT promoter unmethylated by CLIA-certified laboratory; supratentorial tumour with no more than 4 cm in maximal diameter of enhancing tumour on post-operative imaging in any plane; and adequate hepatic, renal and bone marrow function. Patients were excluded if: fewer than five actionable neoepitopes were identified for vaccine generation; they developed disease progression following external beam radiotherapy as defined by Response Assessment in Neuro-Oncology (RANO); required more than 4 mg of dexamethasone per day within one week before vaccine initiation; developed active infection; or were pregnant or lactating.
METHODS
Following surgery, patients received conventional radiation therapy administered at 180-200 cGy per fraction daily for five days per week to a total of approximately 60 Gy. Personalized neoantigen vaccines were prepared using information from fresh tumour and normal tissue obtained at the time of diagnostic resection, as described below. Temozolomide chemotherapy was not administered as this adds only nominal benefit for patients with tumours that lack methylation of the MGMT promoter. The vaccine was administered subcutaneously at least four weeks following completion of external beam radiotherapy. Five priming vaccine doses were initially administered over four weeks (days 1, 4, 8, 15 and 22) followed by two booster doses eight and sixteen weeks later. For each dose, vaccine pools were administered within six hours of thawing in a non-rotating fashion to one of up to four ext...
Synthesis of long peptides, pooling and final vaccine preparation.
Good Manufacturing Practice (GMP) peptides 20-30 amino acids in length were synthesized by standard solid-phase synthetic peptide chemistry and purified using reverse phase high performance liquid chromatography (RP-HPLC) (CSBio). Each vaccine consisted of up to 20 distinct peptides that were grouped into four pools (A, B, C and D), each consisting of up to five peptides, with the intent of separating peptides that bind to the same MHC allele into different pools to decrease potential antigen competition at the draining lymph node. For each dose, the vaccine pools were administered within six hours of thawing, in a non-rotating fashion to one of up to four extremities. The final concentration of peptides per pool was 400 µg ml -1, with a goal of administering a final dose of 300 µg of each peptide per vaccine injection. Each vaccine pool was filter-sterilized an...
Generation and detection of patient neoantigen-specific T cells
Screening ex vivo IFNγ ELISPOT assays on PBMCs stimulated for 18 h with synthesized screening peptides consisting of ASP and EPT peptides were performed. The averages of responses to DMSO, HIV-GAG or OVA peptide for each time point were subtracted from experimental wells for background correction. If no responses were observed, additional in vitro stimulation with the peptide pool was performed for up to 21 days. Upon detection of a positive ELISPOT response (defined as at least 55 spot-forming units (SFU) per 10 6 PBMCs or a ≥3-fold increase over baseline), the peptide pools were allocated into sub-pools in order to deconvolute the peptide pool to determine the immunogenic peptide(s) per pool. PBMCs were cultured in DMEM GlutaMAX medium supplemented with HEPES, β-mercaptoethanol, sodium pyruvate, nonessential amino acids, penicillin-streptomycin (Gibco, ThermoF...
Intracellular cytokine staining.
PBMCs were thawed and rested overnight in DMEM GlutaMAX medium (Gibco, ThermoFisher) supplemented with 10% AB-positive heat-inactivated human serum (Gemini Bioproduct), nonessential amino acids, HEPES, β-mercaptoethanol, sodium pyruvate, penicillin-streptomycin (Gibco, ThermoFisher) and 20 ng ml -1 IL-7 (Peprotech). On the next day, PBMCs were stimulated with 10 µg ml -1 vaccine peptide pools, HIV-GAG peptide (negative control) or 1% phytohemagglutinin (PHA) (positive control) overnight. Stimulated PBMCs were treated with GolgiStop (BD Biosciences) according to the manufacturer's recommendations for 8 h the following day. After treatment, PBMCs were stained for 30 min at room temperature with a fixable dead cell stain (Thermofisher), anti-CD3 (BV605), anti-CD8 (PerCP-Cy5.5), anti-CD4 (BV650) (BD Biosciences), anti-CD45RO (APC-Cy7) and anti...
Processing of GBM and PBMC specimens for scRNA-seq
Surgically resected GBM tissue from patient 7 was obtained on ice within 30 min of lesion excision. The tumour specimen was mechanically disrupted into small pieces with a disposable, sterile scalpel and further dissociated into a single-cell suspension using the enzymatic brain dissociation kit (P) from Miltenyi Biotec, following the manufacturer's protocol. Fc receptor blocking was performed on the total cell suspension using Human TruStain FcX (Biolegend). The cell suspension was subsequently stained for flow cytometry using antibodies against CD45 [HI30]-BV605, CD3 [HIT3a]-BV510 from BD Bioscience, CD4 [OKT4]-PE-Cy7, CD8 [HIT8a]-PerCP-Cy5.5, exclusion panel-APC (CD14 [63D3], CD64 [10.1], CD163 [GHI/61], CD15 [HI98] from Biolegend and CD66b [G10F5] from ThermoFisher Scientific). The tumour cell suspension was next spiked with 1 µM calcein AM (ThermoFish...
Processing of GBM and PBMC specimens for scRNA-seq
Single neoantigen-reactive CD4 + and CD8 + T cells were isolated from PBMCs of patient 7, obtained 8-16 weeks after vaccine initiation. For isolation of CD4 + T cells, PBMCs were stimulated in vitro with 10 µg ml -1 of ASP peptide pools overnight, and then subsequently flow cytometrically sorted on the basis of IFNγ secretion (IFNγ Secretion Assay, Miltenyi Biotec) and co-expression of CD3 + and CD4 + into 96-well plates (FACSAria II Cell Sorter, BD Biosciences). Single neoantigen-reactive CD8 + T cells were isolated from PBMCs of patient 7 that were stimulated with 10 µg ml -1 of EPT peptides, corresponding to the ARHGAP35 MUT or SLX4 MUT peptides in DMEM complete medium supplemented with 10% human serum. Three days after stimulation, the cells were supplemented with human IL-2 (20 units) and human IL-7 (20 ng ml -1 ). The medium was replen...
Statistical considerations
This study incorporated co-primary end points of safety and feasibility. Safety was assessed using a standard dose escalation design. With a true probability of dose limiting toxicity (DLT) estimated to be ≤10%, 0 or 1 DLT in a cohort of five patients was associated with a 92% probability of proceeding to the dose expansion phase. Patients were deemed evaluable for DLT if they received at least three study vaccinations or experienced DLT. A stopping rule for unexpected toxicity during the expansion phase was incorporated that required interruption of further accrual to the dose expansion if four or more patients experienced DLT. Feasibility was assessed as the percentage of patients who were able to have at least 10 actionable neoepitope vaccine peptides generated and the percentage of patients who were able to initiate study vaccination within 12 weeks of diagnostic surgery. Fe...
Measurement outputs
What raw and processed outputs should exist?
Mutations in oncogenes were given the highest priority within each ranked group; otherwise epitopes were ranked by predicted mutated peptide affinity. Only sSNVs that demonstrat...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
The personalized neoantigen vaccines were prepared based on the analysis of whole-exome sequencing (WES) and RNA-seq data generated from fresh-frozen tumours or tumours that wer...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analyses of WES data of tumour and matched PBMCs (as source of normal germline DNA) from the patients were used to identify the specific coding-sequence mutations, including sin...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Tumour samples and heparinized blood were obtained from study subjects on IRB-approved protocols at the DFCI. Patient samples were de-identified and assigned a study-specific tr...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
This study incorporated co-primary end points of safety and feasibility.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Mutations in oncogenes were given the highest priority within each ranked group; otherwise epitopes were ranked by predicted mutated peptide affinity. Only sSNVs that demonstrat...; The personalized neoantigen vaccines were prepared based on the analysis of whole-exome sequencing (WES) and RNA-seq data generated from fresh-frozen tumours or tumours that wer...; Analyses of WES data of tumour and matched PBMCs (as source of normal germline DNA) from the patients were used to identify the specific coding-sequence mutations, including sin...; Tumour samples and heparinized blood were obtained from study subjects on IRB-approved protocols at the DFCI. Patient samples were de-identified and assigned a study-specific tr....
from paperNormalization
Normalize image-derived measurements against the matched acquisition or segmentation rules before comparing groups.
inferred from protocolStatistical comparison
This study incorporated co-primary end points of safety and feasibility. Safety was assessed using a standard dose escalation design. With a true probability of dose limiting to...; Changes between pre-treatment and post-vaccination PBMC immune responses were assessed using the Wilcoxon signed-rank test. All P values were two-tailed and reported without adj...; Investigations of changes in the T cell infiltrate between initial and relapse tumour resection specimens from patients 3, 4, 5, 7 and 8 were based on repeated-measures linear m...; Rates of detection of ASP35 and ASP34 specificity among single T cells in the brain at relapse and in the PBMCs collected at week 16 (patient 7) were compared using a Poisson te...
from paperReporting output
Report representative outputs alongside summary comparisons for Mutations in oncogenes were given the highest priority within each ranked group; otherwise epitopes were ranked by predicted mutated peptide affinity. Only sSNVs that demonstrat..., The personalized neoantigen vaccines were prepared based on the analysis of whole-exome sequencing (WES) and RNA-seq data generated from fresh-frozen tumours or tumours that wer..., Analyses of WES data of tumour and matched PBMCs (as source of normal germline DNA) from the patients were used to identify the specific coding-sequence mutations, including sin..., Tumour samples and heparinized blood were obtained from study subjects on IRB-approved protocols at the DFCI. Patient samples were de-identified and assigned a study-specific tr....
inferred from protocolStructured statistical methods
This study incorporated co-primary end points of safety and feasibility. Safety was assessed using a standard dose escalation design. With a true probability of dose limiting to...; Changes between pre-treatment and post-vaccination PBMC immune responses were assessed using the Wilcoxon signed-rank test. All P values were two-tailed and reported without adj...; Investigations of changes in the T cell infiltrate between initial and relapse tumour resection specimens from patients 3, 4, 5, 7 and 8 were based on repeated-measures linear m...; Rates of detection of ASP35 and ASP34 specificity among single T cells in the brain at relapse and in the PBMCs collected at week 16 (patient 7) were compared using a Poisson te...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Study eligibility was assessed among patients seen at the Center for Neuro-Oncology, Dana-Farber Cancer Institute and required: age ≥18 years; Karnofsky performance status ≥70; histopathological confirmation of WHO grade IV glioblastoma (GBM) or variant; tumour MGMT promoter unmethylated by CLIA-certified laboratory; supratentorial tumour with no more than 4 cm in maximal diameter of enhancing tumour on post-operative imaging in any plane; and adequate hepatic, renal and bone marrow function. Patients were excluded if: fewer than five actionable neoepitopes were identified for vaccine generation; they developed disease progression following external beam radiotherapy as defined by Response Assessment in Neuro-Oncology (RANO); required more than 4 mg of dexamethasone per day within one week before vaccine initiation; developed active infection; or were pregnant or lactating.
Following surgery, patients received conventional radiation therapy administered at 180-200 cGy per fraction daily for five days per week to a total of approximately 60 Gy. Personalized neoantigen vaccines were prepared using information from fresh tumour and normal tissue obtained at the time of diagnostic resection, as described below. Temozolomide chemotherapy was not administered as this adds only nominal benefit for patients with tumours that lack methylation of the MGMT promoter. The vaccine was administered subcutaneously at least four weeks following completion of external beam radiotherapy. Five priming vaccine doses were initially administered over four weeks (days 1, 4, 8, 15 and 22) followed by two booster doses eight and sixteen weeks later. For each dose, vaccine pools were administered within six hours of thawing in a non-rotating fashion to one of up to four extremities. Concomitant medications deemed necessary for adequate patient care were allowed, including concomitant corticosteroids for symptoms associated with cerebral oedema, but the study vaccine was held for patients requiring more than 4 mg per day of dexamethasone within seven days of vaccine ad...
Good Manufacturing Practice (GMP) peptides 20-30 amino acids in length were synthesized by standard solid-phase synthetic peptide chemistry and purified using reverse phase high performance liquid chromatography (RP-HPLC) (CSBio). Each vaccine consisted of up to 20 distinct peptides that were grouped into four pools (A, B, C and D), each consisting of up to five peptides, with the intent of separating peptides that bind to the same MHC allele into different pools to decrease potential antigen competition at the draining lymph node. For each dose, the vaccine pools were administered within six hours of thawing, in a non-rotating fashion to one of up to four extremities. The final concentration of peptides per pool was 400 µg ml -1, with a goal of administering a final dose of 300 µg of each peptide per vaccine injection. Each vaccine pool was filter-sterilized and frozen at -80 °C. Following final testing for identity, sterility and endotoxins, vaccine pools were released for clinical use. On the day of vaccine administration, each pool was thawed and 0.75 ml was mixed with 0.25 ml (0.5 mg) of polyinosinic and polycytidylic acid, stabilized with...
Screening ex vivo IFNγ ELISPOT assays on PBMCs stimulated for 18 h with synthesized screening peptides consisting of ASP and EPT peptides were performed. The averages of responses to DMSO, HIV-GAG or OVA peptide for each time point were subtracted from experimental wells for background correction. If no responses were observed, additional in vitro stimulation with the peptide pool was performed for up to 21 days. Upon detection of a positive ELISPOT response (defined as at least 55 spot-forming units (SFU) per 10 6 PBMCs or a ≥3-fold increase over baseline), the peptide pools were allocated into sub-pools in order to deconvolute the peptide pool to determine the immunogenic peptide(s) per pool. PBMCs were cultured in DMEM GlutaMAX medium supplemented with HEPES, β-mercaptoethanol, sodium pyruvate, nonessential amino acids, penicillin-streptomycin (Gibco, ThermoFisher) and 10% AB-positive heat-inactivated human serum (Gemini Bioproduct). For in vitro expansion (pre-stimulation) of antigen-specific T cells, PBMCs were stimulated in 24-well cell-culture plates at 5 × 10 6 cells per well with individual (10 µg ml -1 ) or pooled peptides (each a...
PBMCs were thawed and rested overnight in DMEM GlutaMAX medium (Gibco, ThermoFisher) supplemented with 10% AB-positive heat-inactivated human serum (Gemini Bioproduct), nonessential amino acids, HEPES, β-mercaptoethanol, sodium pyruvate, penicillin-streptomycin (Gibco, ThermoFisher) and 20 ng ml -1 IL-7 (Peprotech). On the next day, PBMCs were stimulated with 10 µg ml -1 vaccine peptide pools, HIV-GAG peptide (negative control) or 1% phytohemagglutinin (PHA) (positive control) overnight. Stimulated PBMCs were treated with GolgiStop (BD Biosciences) according to the manufacturer's recommendations for 8 h the following day. After treatment, PBMCs were stained for 30 min at room temperature with a fixable dead cell stain (Thermofisher), anti-CD3 (BV605), anti-CD8 (PerCP-Cy5.5), anti-CD4 (BV650) (BD Biosciences), anti-CD45RO (APC-Cy7) and anti-PD-1 (PE) (Biolegend). Cells were fixed and permeabilized (Fixation/Permeabilization Solution Kit, BD Biosciences). Intracellular cytokines were stained with anti IFNγ (PE-Cy7), IL-2 (APC) and TNF (AF700) (BD Biosciences) for 1 h at 4 °C. Cells were washed with permeabilization buff...
Surgically resected GBM tissue from patient 7 was obtained on ice within 30 min of lesion excision. The tumour specimen was mechanically disrupted into small pieces with a disposable, sterile scalpel and further dissociated into a single-cell suspension using the enzymatic brain dissociation kit (P) from Miltenyi Biotec, following the manufacturer's protocol. Fc receptor blocking was performed on the total cell suspension using Human TruStain FcX (Biolegend). The cell suspension was subsequently stained for flow cytometry using antibodies against CD45 [HI30]-BV605, CD3 [HIT3a]-BV510 from BD Bioscience, CD4 [OKT4]-PE-Cy7, CD8 [HIT8a]-PerCP-Cy5.5, exclusion panel-APC (CD14 [63D3], CD64 [10.1], CD163 [GHI/61], CD15 [HI98] from Biolegend and CD66b [G10F5] from ThermoFisher Scientific). The tumour cell suspension was next spiked with 1 µM calcein AM (ThermoFisher) to enable gating of live cells and incubated in the dark at room temperature for 10 min. Live, single T cells (gating: calcein AM +, exclusion -, CD45 +, CD3 +, CD8 + or CD4 + ) were sorted into individual wells of a 96-well twin.tec PCR plate (Eppendorf), which contained 10 µl pe...
Single neoantigen-reactive CD4 + and CD8 + T cells were isolated from PBMCs of patient 7, obtained 8-16 weeks after vaccine initiation. For isolation of CD4 + T cells, PBMCs were stimulated in vitro with 10 µg ml -1 of ASP peptide pools overnight, and then subsequently flow cytometrically sorted on the basis of IFNγ secretion (IFNγ Secretion Assay, Miltenyi Biotec) and co-expression of CD3 + and CD4 + into 96-well plates (FACSAria II Cell Sorter, BD Biosciences). Single neoantigen-reactive CD8 + T cells were isolated from PBMCs of patient 7 that were stimulated with 10 µg ml -1 of EPT peptides, corresponding to the ARHGAP35 MUT or SLX4 MUT peptides in DMEM complete medium supplemented with 10% human serum. Three days after stimulation, the cells were supplemented with human IL-2 (20 units) and human IL-7 (20 ng ml -1 ). The medium was replenished as necessary over three weeks of culture, after which CD8 + T cells were enriched (CD8 magnetic beads, Miltenyi Biotec), rested overnight in cytokine-free medium and then stimulated overnight with autologous APCs (CD4- and CD8-depleted PBMCs) loaded with peptide (10 µg). Subsequently, IFN...
This study incorporated co-primary end points of safety and feasibility. Safety was assessed using a standard dose escalation design. With a true probability of dose limiting toxicity (DLT) estimated to be ≤10%, 0 or 1 DLT in a cohort of five patients was associated with a 92% probability of proceeding to the dose expansion phase. Patients were deemed evaluable for DLT if they received at least three study vaccinations or experienced DLT. A stopping rule for unexpected toxicity during the expansion phase was incorporated that required interruption of further accrual to the dose expansion if four or more patients experienced DLT. Feasibility was assessed as the percentage of patients who were able to have at least 10 actionable neoepitope vaccine peptides generated and the percentage of patients who were able to initiate study vaccination within 12 weeks of diagnostic surgery. Feasibility was defined as at least 50% of the patients (1) having at least 10 actionable peptides for vaccine production and (2) initiating vaccine therapy within 12 weeks of diagnostic surgery.
Machine-readable layer
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"text": "Study eligibility was assessed among patients seen at the Center for Neuro-Oncology, Dana-Farber Cancer Institute and required: age ≥18 years; Karnofsky performance status ≥70; histopathological confirmation of WHO grade IV glioblastoma (GBM) or variant; tumour MGMT promoter unmethylated by CLIA-certified laboratory; supratentorial tumour with no more than 4 cm in maximal diameter of enhancing tumour on post-operative imaging in any plane; and adequate hepatic, renal and bone marrow function. Patients were excluded if: fewer than five actionable neoepitopes were identified for vaccine generation; they developed disease progression following external beam radiotherapy as defined by Response Assessment in Neuro-Oncology (RANO); required more than 4 mg of dexamethasone per day within one week before vaccine initiation; developed active infection; or were pregnant or lactating."
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"text": "Following surgery, patients received conventional radiation therapy administered at 180-200 cGy per fraction daily for five days per week to a total of approximately 60 Gy. Personalized neoantigen vaccines were prepared using information from fresh tumour and normal tissue obtained at the time of diagnostic resection, as described below. Temozolomide chemotherapy was not administered as this adds only nominal benefit for patients with tumours that lack methylation of the MGMT promoter. The vaccine was administered subcutaneously at least four weeks following completion of external beam radiotherapy. Five priming vaccine doses were initially administered over four weeks (days 1, 4, 8, 15 and 22) followed by two booster doses eight and sixteen weeks later. For each dose, vaccine pools were administered within six hours of thawing in a non-rotating fashion to one of up to four ext..."
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"name": "Generation and detection of patient neoantigen-specific T cells",
"text": "Screening ex vivo IFNγ ELISPOT assays on PBMCs stimulated for 18 h with synthesized screening peptides consisting of ASP and EPT peptides were performed. The averages of responses to DMSO, HIV-GAG or OVA peptide for each time point were subtracted from experimental wells for background correction. If no responses were observed, additional in vitro stimulation with the peptide pool was performed for up to 21 days. Upon detection of a positive ELISPOT response (defined as at least 55 spot-forming units (SFU) per 10 6 PBMCs or a ≥3-fold increase over baseline), the peptide pools were allocated into sub-pools in order to deconvolute the peptide pool to determine the immunogenic peptide(s) per pool. PBMCs were cultured in DMEM GlutaMAX medium supplemented with HEPES, β-mercaptoethanol, sodium pyruvate, nonessential amino acids, penicillin-streptomycin (Gibco, ThermoF..."
},
{
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"name": "Intracellular cytokine staining.",
"text": "PBMCs were thawed and rested overnight in DMEM GlutaMAX medium (Gibco, ThermoFisher) supplemented with 10% AB-positive heat-inactivated human serum (Gemini Bioproduct), nonessential amino acids, HEPES, β-mercaptoethanol, sodium pyruvate, penicillin-streptomycin (Gibco, ThermoFisher) and 20 ng ml -1 IL-7 (Peprotech). On the next day, PBMCs were stimulated with 10 µg ml -1 vaccine peptide pools, HIV-GAG peptide (negative control) or 1% phytohemagglutinin (PHA) (positive control) overnight. Stimulated PBMCs were treated with GolgiStop (BD Biosciences) according to the manufacturer's recommendations for 8 h the following day. After treatment, PBMCs were stained for 30 min at room temperature with a fixable dead cell stain (Thermofisher), anti-CD3 (BV605), anti-CD8 (PerCP-Cy5.5), anti-CD4 (BV650) (BD Biosciences), anti-CD45RO (APC-Cy7) and anti..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Processing of GBM and PBMC specimens for scRNA-seq",
"text": "Surgically resected GBM tissue from patient 7 was obtained on ice within 30 min of lesion excision. The tumour specimen was mechanically disrupted into small pieces with a disposable, sterile scalpel and further dissociated into a single-cell suspension using the enzymatic brain dissociation kit (P) from Miltenyi Biotec, following the manufacturer's protocol. Fc receptor blocking was performed on the total cell suspension using Human TruStain FcX (Biolegend). The cell suspension was subsequently stained for flow cytometry using antibodies against CD45 [HI30]-BV605, CD3 [HIT3a]-BV510 from BD Bioscience, CD4 [OKT4]-PE-Cy7, CD8 [HIT8a]-PerCP-Cy5.5, exclusion panel-APC (CD14 [63D3], CD64 [10.1], CD163 [GHI/61], CD15 [HI98] from Biolegend and CD66b [G10F5] from ThermoFisher Scientific). The tumour cell suspension was next spiked with 1 µM calcein AM (ThermoFish..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Processing of GBM and PBMC specimens for scRNA-seq",
"text": "Single neoantigen-reactive CD4 + and CD8 + T cells were isolated from PBMCs of patient 7, obtained 8-16 weeks after vaccine initiation. For isolation of CD4 + T cells, PBMCs were stimulated in vitro with 10 µg ml -1 of ASP peptide pools overnight, and then subsequently flow cytometrically sorted on the basis of IFNγ secretion (IFNγ Secretion Assay, Miltenyi Biotec) and co-expression of CD3 + and CD4 + into 96-well plates (FACSAria II Cell Sorter, BD Biosciences). Single neoantigen-reactive CD8 + T cells were isolated from PBMCs of patient 7 that were stimulated with 10 µg ml -1 of EPT peptides, corresponding to the ARHGAP35 MUT or SLX4 MUT peptides in DMEM complete medium supplemented with 10% human serum. Three days after stimulation, the cells were supplemented with human IL-2 (20 units) and human IL-7 (20 ng ml -1 ). The medium was replen..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Statistical considerations",
"text": "This study incorporated co-primary end points of safety and feasibility. Safety was assessed using a standard dose escalation design. With a true probability of dose limiting toxicity (DLT) estimated to be ≤10%, 0 or 1 DLT in a cohort of five patients was associated with a 92% probability of proceeding to the dose expansion phase. Patients were deemed evaluable for DLT if they received at least three study vaccinations or experienced DLT. A stopping rule for unexpected toxicity during the expansion phase was incorporated that required interruption of further accrual to the dose expansion if four or more patients experienced DLT. Feasibility was assessed as the percentage of patients who were able to have at least 10 actionable neoepitope vaccine peptides generated and the percentage of patients who were able to initiate study vaccination within 12 weeks of diagnostic surgery. Fe..."
}
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"headline": "Neoantigen vaccine generates intratumoral T cell responses in phase Ib glioblastoma trial",
"datePublished": "2018",
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