Strange India All Strange Things About India and world

Patients and trial design

NOA16 was a non-controlled, open-label, single-arm, multicentre, first-in-humans phase I trial to assess the safety, tolerability and immunogenicity of eight repeated doses of IDH1-vac in patients with IDH1(R132H)+, non-1p/19q co-deleted, ATRX WHO grade 3 and 4 gliomas. The study ran from May 2015 to November 2018 at seven trial centres in Germany (Supplementary Table 1). Follow-up to evaluate the duration of response, survival, and late adverse events is ongoing. The study was approved by the national regulatory authority (Paul-Ehrlich Institut) and the institutional review board (Ethikkommission) at each study site, namely: Ethikkommission der Medizinischen Fakultät Heidelberg (Heidelberg), Ethik-Kommission Albert-Ludwigs-Universität Freiburg (Freiburg), Ethik-Kommission des Landes Berlin (Berlin), Ethik-Kommission der Medizinischen Fakultät der Universität Duisburg-Essen (Essen), Ethik-Kommission der Medizinischen Fakultät “Carl Gustav Carus” (Dresden), Ethikkommission des Fachbereichs Medizin der Goethe-Universität Frankfurt am Main (Frankfurt), Ethikkommission der Medizinischen Fakultät der Ludwig-Maximilians-Universität München (Munich), Ethik-Kommission an der Medizinischen Fakultät der Eberhard-Karls-Universität und am Universitätsklinikum Tübingen (Tübingen). The study was conducted in accordance with the Good Clinical Practice guidelines of the International Conference on Harmonisation. All participants provided written signed informed consent. We complied with all relevant ethical regulations. The trial population comprised three treatment groups (TGs) based on the SOC treatment that patients had received before enrollment: radiotherapy alone (RT, TG1), three cycles of chemotherapy with TMZ alone (mono-TMZ, TG2) or combined radiochemotherapy with TMZ (RT + cTMZ, TG3). In TG1, vaccination was done alone starting 4–6 weeks after radiotherapy. In TG2 and TG3, vaccination was done in parallel with TMZ starting on day 10 of the fourth cycle of the TMZ monotherapy (TG2) or on day 10 of the first adjuvant (a)TMZ cycle after concomitant radiotherapy (TG3). Treatment consisted of eight vaccinations with IDH1-vac in weeks 1, 3, 5, 7, 11, 15, 19 and 23 (visits (V) 03–10; Extended Data Fig. 1b). For immunogenicity assessment, peripheral T cell and B cell immune responses were assessed at six time points: V03 (baseline), V05, V07, V10, V12, and V13 (Extended Data Fig. 1b). Eligibility criteria included the presence of a histologically confirmed IDH1(R132H)+ glioma (with or without measurable residual tumour after resection or biopsy) with absence of chromosomal 1p/19q co-deletion and loss of nuclear ATRX expression in the tumour tissue, thus limiting inclusion in this first-in-humans trial to the subgroup of molecular astrocytoma without positive prognostic factors22. Exclusion criteria included concomitant treatment with dexamethasone (or equivalent) >2 mg/day, Karnofsky performance status (KPS) < 70, and progressive (including PsPD14) or recurrent disease after SOC. The matched control cohort was built from patients treated at the centre in Heidelberg outside the trial between 2007 and 2018 with sufficient clinical and MRI information available to assess PsPD. Matching was done according to the first treatment phase of a histologically confirmed IDH1(R132H)+ glioma (with or without measurable residual tumour after complete or partial resection or biopsy) without 1p/19q co-deletion or loss of nuclear ATRX expression in the tumour tissue, and according to WHO grade 3 or 4 as well as frequency of treatment adaptions (RT + cTMZ versus mono-TMZ or RT; Supplementary Table 3). No statistical methods were used to predetermine sample size. Sample size estimation was primarily based on the accuracy requirements for the primary endpoint immune response (responder rate) to the IDH1 peptide vaccine. Sample size was adjusted for non-evaluable patients. It was estimated that 70% of patients who would be evaluable for immunogenicity testing would be evaluable for all time points. Because 21 patients were sufficient for immunogenicity testing with all time points, 30 evaluable patients had to be enrolled. Owing to an expected dropout rate of 20% (due to progression or other reasons), 39 patients had to be recruited. All patients received the trial-related intervention; the trial was not randomized and investigators were not blinded concerning trial related intervention during experiments and outcome assessment.

IDH1 vaccination

IDH1-vac consisted of 300 μg of an IDH1(R132H) 20-mer peptide (p123–142) manufactured by the GMP facility of the University of Tübingen, Germany and emulsified in Montanide (ISA50) as described earlier23 by the GMP core facility at the University Hospital Heidelberg, Germany, a maximum of one day in advance. It was administered subcutaneously in combination with topical imiquimod (5%, Aldara). Quality controls for content, sterility and absence of endotoxin were performed for each emulsion at Labour LS s.e. & Co. KG, Germany.


The primary endpoints were safety and immunogenicity. The safety endpoint was the RLT, which was defined as one of the following that was related to IDH1-vac administration: any injection site reaction of National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 grade 4; any injection site reaction of CTCAE grade 3 that persisted after two weeks; any other hypersensitivity, anaphylaxis or local allergic reaction of at least CTCAE grade 3; brain oedema (CTCAE grade 4); autoimmunity of CTCAE grade 3 or more; CTCAE grade 3 or more toxicity to organs other than the bone marrow, but excluding grade 3 nausea, grade 3 or 4 vomiting in patients who had not received optimal treatment with anti-emetics, grade 3 or 4 diarrhoea in patients who had not received optimal treatment with anti-diarrheals, and grade 3 fatigue; and death. Adverse events were counted as treatment-related if the relationship to treatment was ‘certain’, ‘related’, ‘probable’, ‘possible’, or not reported. For safety assessment, patients were medically reviewed at each visit. To exclude unexpected IDH1-vac-induced immunological tolerance against IDH1(R132H), shortening of progression-free survival (PFS), defined as an observed decrease in the estimated 12-months PFS rate of at least 10% compared to the anticipated value of 70.7% derived from previous studies, was defined as a safety criterion for early trial termination. The safety analysis was based on all enrolled patients who received one or more administration(s) of IDH1-vac. The immunogenicity endpoint was defined as the presence of an IDH1(R132H)-specific T cell and/or antibody response at any time point during the trial. IDH1(R132H)-specific T cell and antibody responses were measured on PBMCs using IFNγ ELISpot and on serum using peptide-coated ELISA, respectively. For IFNγ ELISpot, a cut-off of 50 IFNγ spots after subtraction of negative control was defined as positive. For ELISA, the cut-off for positivity was defined as optical density related to negative control ≥5.

Disease assessment

Disease assessment, including overall response rate and diagnosis of PsPD, was performed using standardized three-monthly MRI according to the RANO criteria by central neuroradiology review14. In NOA16 and the molecularly matched control cohort, PsPD, which may mainly indicate an intratumoral inflammatory reaction24, was defined as an increase in the size of the tumour on T2-FLAIR MRI sequences and/or the novel appearance or enlargement of contrast-enhancing lesions followed by stabilization or regression on follow-up MRI up to three months after initiation of SOC and/or immunotherapy14.

Preparation of peptides for analyses

Lyophilized peptides were reconstituted in 100% DMSO and diluted to a final concentration of 10 mg ml−1 with aqua ad iniectabilia (Braun). The final DMSO concentration was 10%.

Isolation of serum

Serum tubes were kept standing upright at room temperature for 15 min before isolation. Serum tubes were centrifuged at 1,000g for 10 min at room temperature. Supernatant was aliquoted on ice and frozen at –80 °C.

Isolation of PBMCs

PBMCs were isolated from heparinized blood from patients with glioma by density-gradient centrifugation (800g without brake at room temperature) by loading onto Biocoll Separation Solution (Biochrom) after dilution with phosphate-buffered saline (PBS) and using Leucosep tubes (Greiner Bio-One). PBMCs were frozen in 50% freezing medium A (60% X-Vivo 20, 40% fetal calf serum (FCS)) and 50% medium B (80% FCS, 20% DMSO) and stored in liquid nitrogen at −140 °C until analysis.

Isolation of LILs

Lesion tissue was dissected into small pieces (2 × 2 mm) and transferred into 24-well tissue culture-treated plates at three pieces per well in 2 ml human tumour-invading lymphocyte (TIL) medium (RPMI1640 (Pan Biotec) with 10% human serum (Sigma Aldrich), 2 mM l-glutamine, 1.25 μg/ml amphotericin B (both Gibco), 1,000 U/ml IL-2 (Proleukin)) containing 30 ng/ml anti-human CD3 (clone OKT-3, eBioscience). Medium was exchanged every 2–3 days and tissue pieces removed on day 7. LILs that migrated out of the tumour into the medium were further expanded until day 14 and cryopreserved as above.

Generation of patient REP cells

To enable HLA-autologous testing for antigen-specific reactivity of LILs and TCR-transgenic cells, patient-autologous rapidly expanded PBMCs (REP cells), which express high levels of MHC molecules and can serve as antigen-presenting cells (APCs), were generated. PBMCs (1 × 105) were co-cultured in a high-density culture with 3 × 107 irradiated (40 Gy) feeder cells (PBMCs from non-autologous donors) in X-vivo15 medium supplemented with 2% human AB serum (Sigma-Aldrich) and 30 ng/ml OKT-3 antibody (Invitrogen) in T-25 flasks in 25 ml total volume. After 24 h, cells were supplemented with 300 IU/ml hIL-2. Medium was replaced every 5 days with hIL-2 supplementation and cells were split as needed. Cells were collected after 14 days of co-culture and cryopreserved.


ELISpot white-bottom multiscreen HTS plates (MSIPS4W10, Millipore) were coated with anti-human IFNγ (1-D1K, Mabtech) and blocked with X-Vivo-20 (Lonza) containing 2% human albumin (HA). PBMCs were thawed, rested overnight in X-Vivo medium and seeded at 4 × 105 cells per well and stimulated with 2 μg peptides per well in 100 μl volume. PBMCs were stimulated with IDH1(R132H) (p123–142), wild-type IDH1 (p123–142), or MOG (p35–55) at equal concentrations or with peptide diluent aqua ad iniectabilia (Braun) with 10% DMSO (vehicle) at equal volume as negative controls, or with 1 μg staphylococcal enterotoxin B (Sigma-Aldrich) per well and 0.05 μg CMV with 0.05 μg AdV per well (both in 100 μl volume) as positive controls. After 40 h, IFNγ-producing cells were detected with biotinylated anti-human IFNγ antibodies (7-B6-1), streptavidin-ALP (both Mabtech) and ALP colour development buffer (Bio-Rad) and quantified using an ImmunoSpot Analyzer (Cellular Technology Ltd). Quality control was performed and reviewed by a second person. For categorization of T cell responses, transient T cell responses were defined as a spot count above 50 followed by a spot count of less than 50 at EOS. Sustained T cell responses were defined as a spot count above 50 followed by a spot count of more than 50 at EOS.

IFNγ ELISpot of LILs

To generate dendritic cells (DCs) to serve as antigen-presenting cells, autologous patient PBMCs were thawed in X-Vivo-20 medium and plated on tissue-culture-treated plates at a density of 5 × 106 cells per ml for 1 h. The supernatant was removed and adherent monocytes were differentiated into DCs by culturing in X-Vivo-20 medium containing 500 U/ml hIL-4 (Miltenyi) and 560 U/ml human granulocyte-macrophage colony-stimulating factor (hGM-CSF) (Genzyme) for 7 days. DCs were collected and purified using magnetic-activated cell sorting (MACS). Anti-CD56 antibodies coupled to pan mouse IgG Dynabeads, CD19 pan B Dynabeads and CD3 Dynabeads (all Invitrogen) were used to remove contaminating cell populations according to the manufacturer’s protocol. To enrich LILs for antigen-reactive T cells, DCs were seeded at a density of 2 × 105 cells per ml in RPMI1640 medium containing 10% AB serum, 100 U/ml penicillin, and 100μg/ml streptomycin, and loaded with 10 μg/ml IDH1(R132H) (p123–142) for 4 h. They were then co-cultured with LILs, which had been thawed and rested overnight in X-VIVO-20 medium, at a ratio of 1:5 (DCs:LILs). For proliferation of T cells, from day 3 onwards, co-culture medium was supplemented with 40 U/ml IL-2 (Proleukin) and 20 ng/ml IL-7 (Peprotech) and refreshed every 2 to 4 days. LILs were collected after 24 days of co-culture, rested overnight in RPMI1640 medium containing 10% AB serum, 100 U/ml penicillin, and 100μg/ml streptomycin, and used for ELISpot in co-culture with freshly isolated autologous DCs as above, which had been loaded with 2 μg/100 μl IDH1(R132H) peptide (p123–142) or MOG peptide (p35–55) as negative control overnight in the same medium, at a ratio of 1:6 (1 × 104 DCs:6 × 104 LILs) for 40 h. ELISpot was performed as described above.

Flow cytometry

For peripheral immune monitoring, 3 × 105 PBMCs were stained with the following antibodies targeting surface proteins: anti-CD3-FITC (clone UCHT1, cat # 300452, 1:100), anti-CD4-Alexa Fluor700 (clone RPA-T4, cat # 300526, 1:100), anti-CD8-PerCP (clone RPA-T8, cat # 301030, 1:100), anti-CD11b-BV510 (clone M1/70, cat # 101263, 1:20), anti-HLA-DR-PE-Cy7 (clone L243, cat # 307616, 1:50), anti-CD14-BV711 (clone M5E2, cat # 301838, 1:100), anti-CD16-PE/Dazzle594 (clone 3G8, cat # 302054, 1:10), anti-CD25-BV605 (clone BC96, cat # 302632, 1:20), anti-CD33-APC (clone P67.6, cat # 366606, 1:50), and anti-CD127-BV421 (clone A019D5, cat # 351310, 1:20) (all BioLegend); and fixable viability dye-eFluor780 (1:1,000, Invitrogen), followed by intracellular staining with anti-FOXP3-PE (clone 206D, cat # 320108, 1:100, BioLegend) using the Fixation and Permeabilization Buffer Set (ebioscience). Antibody amounts were titrated previously. In all experiments, corresponding fluorescence minus one (FMO) controls were used (Extended Data Fig. 9). As many events as possible were measured on an Attune NxT Flow Cytometer using Attune Nxt software version 2.7 (ThermoFisher Scientific).

For analysis of IDH1(R132H)-reactive T cell subsets, we performed an ex vivo peptide recall assay. PBMCs were thawed, rested for 4 h in X-Vivo 20 medium, and seeded into 96-well U-bottom plates. PBMCs (1.5–2 × 106) were stimulated with 2 μg peptide per well using IDH1(R132H) (p123–142), MOG (p35–55) as negative control, or CEFT peptide pool (0.05 µg/ml per peptide, jpt) as positive control for 3 h before adding 10 μg/ml brefeldin A (Sigma-Aldrich, order no. B6542) and 1× GolgiStop (BD Bioscience). Cells were incubated for an additional 12 h and subsequently stained with the following surface antibodies: anti-CD3-BV510 (clone HIT3a, cat # 564713, 1:20), anti-CD4-BV605 (clone SK3, cat # 566908, 1:50), anti-CD8-APC-H7 (clone SK1, cat # 560179, 1:10) (panels 1 and 2), anti-CD25-BV711 (clone 2A3, cat # 563159, 1:10), and anti-CD127-FITC (clone HIL-7R-M21, cat # 560549, 1:2.5) (panel 2) (all BD Biosciences); and fixable viability dye-APC-R700 (1:1,000, Invitrogen), followed by intracellular staining with anti-IFNγ-BV421 (clone 4S.B3, cat # 564791, 1: 20, BD Biosciences), anti-TNF-APC (clone MAb11, cat # 502912, 1:20, Biolegend), anti-IL17-PE (clone N49-653, cat # 560486, 1:5), and anti-IL4-PerCP-Cy5.5 (clone 8D4-8, cat # 561234, 1:20) (panel 1), or anti-FOXP3-PE (clone 259D/C7, cat # 560046, 1:5) and anti-IL10-APC (clone JES3-19F1, cat # 554707, 1:50) (all BD Biosciences), using the Foxp3/Transcription Factor Staining Buffer Set (ebioscience). Antibody amounts were titrated previously or used according to manufacturer’s instructions, and scaled up according to cell numbers at time of seeding. In all experiments, corresponding FMO controls were used (Extended Data Fig. 4). As many events as possible were measured on a Lyric Flow Cytometer (BD Bioscience) using BD FACSuite sotware version 1.3.

For fluorescence-activated cell sorting (FACS) of LILs, patient tissue was dissected into small pieces, transferred to HBSS (Sigma Aldrich) and strained successively through 100-μm, 70-μm and 40-μm cell strainers with intermittent washes with HBSS to obtain a single-cell suspension. Cells were stained with the following antibodies targeting surface proteins: anti-CD45-eFluor450 (clone 2D1, cat # 48-9459-42, 1:50, ebioscience) and anti-CD3-PE (clone HIT3a, cat # 300308, 1:50, BioLegend); and fixable viability dye-eFluor780 (1:1,000, Invitrogen). Cells were gated for lymphocytes, single cells and live cells, and sorted into CD45+CD3+ and CD45+CD3 cell populations (Extended Data Fig. 12) on a FACSAria IIu with FACSDiva software version 8.0 (BD Biosciences).

For ex vivo testing of the reactivity of CD8+ LILs to IDH1(R132H), cryopreserved LILs expanded from tumour pieces and patient-specific REP cells were thawed in X-vivo 15 medium containing 50 U/ml Benzonase (Sigma Aldrich), and rested for 12 h in X-vivo 15 medium with 2% Human AB serum (Sigma-Aldrich) and 20 IU/ml hIL-2 (Proleukin). REP cells were irradiated (30 Gy), seeded in 96-well U-bottom plates at 1 × 105 cells per well and loaded with 10 μg/ml IDH1(R132H) (p123–142) or MOG (p35–55) peptide for 2 h. In the meantime, LILs were labelled with CFSE (ThermoFisher) according to the manufacturer’s protocol to help distinguish them during flow cytometry, and co-cultured with peptide-loaded REP cells at a 1:1 ratio. After 12 h, 10 μg/ml Brefeldin A was added to the co-culture for an additional 5 h. Positive control cells were stimulated with 20 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 μg/ml ionomycin (Sigma-Aldrich). Cells were subsequently stained with the following surface antibodies: anti-CD3-BV510 (clone HIT3a, cat # 564713, 1:20, BD Biosciences) and anti-CD8-PerCP-Cy5.5 (clone RPA-T8, cat # 45-0088-42, 1:100, ebioscience); and fixable viability dye-eFluor780 (1:1,000, Invitrogen), followed by intracellular staining with anti-TNF-APC (clone MAb11, cat # 17-7349-82, 1:50) and anti-IFNγ-eFluor450 (clone 4S.B3, cat # 48-7319-42, 1:50) (all ebioscience) using the IC Fixation buffer kit (eBioscience). Corresponding FMO controls were used (Extended Data Fig. 11) and events were measured on a FACSCanto II flow cytometer with FACSDiva software version 9.0 (BD Biosciences).

Data analysis for all experiments was done using FlowJo software v.10.5.0.


ELISA polysorp plates (Nunc) were coated with human IDH1(R132H) and human wild-type IDH1 (p122–136 and p123–142) for patient IgG detection, and with negative control MOG (p35–55) (10 μg per well in PBS). Wells were washed with PBS 0.05% Tween 20, and blocked with 3% FBS in PBS 0.05% Tween 20. The positive control for patient serum was tetanus toxoid (Millipore) with EBNA-1 (RayBiotech) (each 0.5 ng per well). Patient and healthy control sera were obtained from serum tubes by centrifugation. Patient serum was used at the following dilutions: 1:10, 1:100, 1:333, 1:1,000 and 1:3,333. Healthy control serum was used undiluted. Mouse anti-IDH1(R132H) (1:1,000, H09, Dianova) was used as peptide coating control. HRP-conjugated secondary antibodies were sheep anti-mouse IgG-HRP (1:5,000, Amersham) and goat anti-human IgG-Fc-HRP (1:10,000, Bethyl Laboratories, Inc.). The substrate was tetramethylbenzidine (ebioscience) and the reaction was stopped with 1 M H2SO4. Optical density was measured at 450 nm.

Detection of cytokines in serum

Serum was analysed using multiplex bead technology (Bio-Plex Pro Human Cytokine 27-plex panel, order no. M500KCAFOY, Bio-Rad, Hercules, CA) according to the manufacturer´s instructions. Serum was diluted 1:2. Standard curves were generated by using the reference cytokine sample supplied in the kit and were used to calculate the cytokine concentrations in the samples. Acquisition and data analysis were performed by bio-plex Manager.

Proximity ligation assay

PLA was performed on baseline paraffin-embedded glioma tissues as described previously5. For image acquisition, a nonlinear adjustment (gamma changes) was used for visualization purposes.

TCRB deep sequencing

Genomic DNA was isolated from patient EDTA blood using the DNeasy Blood and Tissue Kit (Qiagen). TCR beta chain (TCRB) deep sequencing was performed to detect rearranged TCRβ gene sequences using hsTCRB Kit (Adaptive Biotechnologies) according to the manufacturer’s protocol. The prepared library was sequenced on an Illumina MiSeq by the Genomics & Proteomics Core Facility, German Cancer Research Center (DKFZ). Data processing (demultiplexing, trimming, gene mapping) was done using the Adaptive Biotechnologies proprietary platform. Data were visualized using the Treemap Visualization package version 2.4.2 ( TCRB sequencing data are available at

Next-generation HLA typing

Genomic DNA was isolated from patient EDTA blood using the QIAamp DNA Blood Mini Kit (Qiagen). Subsequently, peptide-binding domains were sequenced as described previously25.

850k methylation arrays

850k methylation arrays were performed as described previously26.

Panel sequencing

DNA from FFPE tissue was extracted on the Promega Maxwell device (Promega) following the manufacturer’s instructions. Extracted DNA was then sheared on a Covaris M220 (Covaris). DNA integrity and fragment size were determined on a Bioanalyzer 2100 (Agilent). Sequencing was performed on a NextSeq 500 instrument (Illumina) with an average coverage of 550-fold27.

Single-cell RNA and TCR sequencing

Single-cell capturing and downstream library constructions of FACS-sorted cells were performed using Chromium Single Cell V(D)J Reagent kit v1 chemistry (10x Genomics; PN-1000006, PN-1000020, PN-1000005, PN-120262) according to the manufacturer’s protocol. The constructed scVDJ library and scGEX libraries were sequenced on HiSeq2500 rapid and HiSeq4000 platforms (Illumina), respectively.

Single-cell RNA data were processed using cellranger pipeline (version 3.1.0) with GRCh38 genome assembly (version 3.0.0, 10x Genomics) with default setting. The filtered matrices were then analysed using Seurat28. Cells with fewer than 2,000 unique molecular identifiers, fewer than 900 genes, and/or more than 10% mitochondrial gene expression were excluded from the analysis. Genes detected in fewer than three cells were excluded. Gene expression was transformed and normalized using regularized negative binomial regression as implemented in sctransform29. VDJ genes were removed from the variable genes to prevent clustering of cells on the basis of TCR clones. Highly variable genes were selected using principal component analysis, and 40 principal components were selected on the basis of inflection point in the elbow plot. Cells were clustered using graph-based clustering with Louvain modularity of 0.45 and UMAPs were plotted for visualization. Differential gene expression analysis was performed using MAST30 to determine the identity of each cluster and highly upregulated genes were used to label each cluster. Clusters with upregulated heat shock proteins and CD3 cells were excluded and cells were re-normalized and re-clustered as described above. Single-cell VDJ data were processed similarly using cellranger pipeline. Barcodes of individual top TCRs were then mapped onto single-cell RNA data to determine the distribution of TCR clones in the clusters.

Single-cell sequencing data have been deposited in the NCBI Sequence Read Archive with the accession codes SRR12880623 and SRR12880624.

TCR cloning

Synthetic alpha and beta VDJ fragments of the variable region of the TCR compatible with BsaI-mediated Golden Gate Assembly cloning were obtained from Twist Biosciences. An S/MAR sequence-bearing expression vector (pSMARTer) that allows extrachromosomal replication of the vector in eukaryotic cells was used and designed to harbour mouse alpha and beta constant TCR regions and a p2a self-cleaving peptide linker to facilitate production of separate alpha and beta polypeptide chains of the TCR. The TCR variable fragments were inserted into the expression vector using a single-step Golden Gate reaction and transformed into NEB5-alpha-competent Escherichia coli (NEB). Colonies were screened for the transgene by antibiotic resistance, and an endotoxin-free plasmid was prepared using NucleoBond Extra Maxi EF kit (Macherey-Nagel) for transfection.

TCR-NFAT reporter assay

The cloned TCR expression vector and a nano-luciferase-based NFAT reporter vector (pDONR, with 4× NFAT-response elements) were delivered into Jurkat Δ76 cells (obtained from TRON gGmbH, authenticated using the Multiplexion STR profiling and compared to normal Jurkat cells, regularly tested for mycoplasma contamination and tested negative at all time points) using electroporation (Neon Transfection system, ThermoFisher Scientific). In brief, 2 × 106 cells were used per electroporation with Neon 100-μl tips (8 μg TCR expression vector with 5 μg NFAT reporter vector). Cells were prepared according to the manufacturer’s protocol; electroporated with 1,325 V, 10 ms, 3 pulses; and transferred to antibiotic-free RPM1 1640 medium containing 10% FCS. Patient-autologous PBMCs or REP cells were used as APCs as indicated and thawed 24 h before co-culture in X-VIVO 15 medium (Lonza) containing 50 U/ml benzonase (Sigma-Aldrich), rested for 6–8 h before seeding into 96-well white-opaque tissue culture-treated plates (Falcon) at 1.5 × 105 cells per well, and loaded with peptides at a final concentration of 10 μg/ml in a total volume of 150 μl for 16 h. A pool of human IDH1(R132H) peptides (p122–136, p124–138, p126–140) was used. MOG (p35–55) at equal concentrations and PBS + 10% DMSO (vehicle) at equal volume were used as negative controls. Forty-eight hours after electroporation, Jurkat Δ76 cells were collected and co-cultured with peptide-loaded PBMCs for 6 h at a 1:1 ratio. Human T cell TransAct beads (Miltenyi) were used as positive control. Nano-luciferase induction, indicating TCR activation, was assayed using the Nano-Glo Luciferase assay system (Promega) according to the manufacturer’s protocol and signal was detected on a PHERAstar FS plate reader (BMG Labtech).

In vitro HLA affinity analyses

Peptides were synthesized by Genscript and dissolved in DMSO followed by dilution in assay buffer. The final DMSO concentration was 10%. Peptides did not contain cysteines so no reducing agent was added. As positive controls, peptides CLIP (PVSKMRMATPLLMQA), KLAT (HA306–318, YKYVKQNTLKLAT) or PADRE (AKFVAAWTLKAAA) were used. Peptides were titrated in assay buffer (10,000, 1,000, 100, 10, 1, 0.1, 0.01, and 0.001 nM) and recombinant MHC II of different alleles and paralogues was added. After at least 24 h of refolding, solutions were transferred to optiplates AlphaScreen acceptors and donor beads were added. Raw data was imported into Microsoft Excel and deconvoluted. For some peptides the highest concentrations led to a reduction in signal (hooking effect). These datapoints were deleted. Data were imported into GraphPad Prism software version 9.0.0 and analysed by sigmoid curve fitting. All experiments were done in duplicate with good correlation.


For statistical analyses of primary endpoints, two patient analysis populations were defined. The safety population included all enrolled patients who had at least received one dose of IDH1-vac. This was the analysis dataset for evaluating patient characteristics, study administration, efficacy (overall response rate, i.e. stable disease), and safety endpoints (safety dataset, SDS). The immunogenicity population (immunogenicity dataset, IDS) included all patients who could be evaluated for immunogenicity assessment. A patient was defined as evaluable if they had completed the study up to and including V07, had received at least four vaccinations through V07 and had all intended blood samples collected for immune monitoring through V07; or had received at least 6 of 8 vaccinations, and baseline plus at least two further blood samples had been collected for immune monitoring through V12. Non-evaluable patients were replaced for assessment of immunogenicity, except for patients who left the study early owing to RLT. For the primary endpoints (RLT and immune response), summary tables, percentages and exact 95% CIs according to Clopper–Pearson were generated.

All secondary variables were analysed using explorative and mainly descriptive methods using GraphPad Prism software version 9.0.0. For PLA, Pearson correlation coefficient was calculated. For contingency analyses, Fisher’s exact test was performed. For multiple comparisons, a Kruskal–Wallis test (KWT) by ranks was performed and multiplicity adjusted P values (Dunn’s test) are presented. All statistical tests were two-tailed to a significance level of 5%. For detailed description of exploratory analyses, see Supplementary Table 8. For analysis of selected secondary variables, a molecular dataset was defined. The molecular dataset included all patients whose astrocytomas could retrospectively be defined molecularly according to copy number variation load (CNV-L), methylation class, and CDKN2A/B status.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

Source link

Leave a Reply

Your email address will not be published.