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Author contributions | INFLAMMATIONS | Authors SST, BMS, JJH, SM, JRL, and CJ designed the S-LiTE randomized trial. CJ, SBKJ, JRL, CRJ, and RMS contributed to the execution of the trial. RMS performed the statistical analyses. RMS, CRJ, SBKJ, MBB, MR, SST, and CA contributed to the methodology. LG performed sampling, analysis, and interpretation of inflammations marker hsCRP. JEBJ provided resources, analysis, and interpretation of DXA-scans. RMS, CRJ, SBKJ, and AFB performed visualizations. RMS, CRJ, SBKJ, and SST drafted the study. SST acquired funding. All authors read and approved the final manuscript. | PMC9960425 | |
Funding | Nordisk | The trial was supported by two Novo Nordisk Foundation grants (NNF15SA0018346 and Excellence Grant NNF16OC0019968) and Helsefonden, all in Denmark. Cambridge Weight Plan supplied diet-replacement products. Novo Nordisk, Denmark, supplied Saxenda and placebo pens. Supporting partners did not influence or participate in trial execution, data collection or analysis, interpretation or writing, or communication of the study results. | PMC9960425 | |
Availability of data and materials | The study protocol and statistical analysis plan have been published [ | PMC9960425 | ||
Declarations | PMC9960425 | |||
Ethics approval and consent to participate | ICH | The trial was approved by the Committee of Health Research Ethics (H-16027082) and the Danish Medicines Agency (EudraCT: 2015-005585-32) and was conducted according to the Declaration of Helsinki and ICH Good Clinical Practice guidelines. Participants provided written informed consent before inclusion. | PMC9960425 | |
Consent for publication | Not applicable. | PMC9960425 | ||
Competing interests | RMS: Family member holds Novo Nordisk stocks. MR: Currently employed at Novo Nordisk. JJH: Advisory boards: Novo Nordisk. SM: Advisory boards: AstraZeneca; Boehringer Ingelheim; Eli Lilly; Merck Sharp & Dohme; Novo Nordisk; Sanofi Aventis. Lecture fees: AstraZeneca; Boehringer Ingelheim; Merck Sharp & Dohme; Novo Nordisk; Sanofi Aventis. Grant Recipient: Novo Nordisk, Boehringer-Ingelheim. SST: Grant and lecture fee recipient, Novo Nordisk. JRL, CJ, CRJ, SBKJ, MBB, AFB, LG, JBJ, CA, BMS have no disclosures. | PMC9960425 | ||
References | PMC9960425 | |||
Subject terms | toxicity, toxicities, germ cell tumors | DISEASE, EVENT, SOLID TUMORS, CYTOKINE RELEASE SYNDROME, GERM CELL TUMORS, IMMUNE EFFECTOR CELL-ASSOCIATED NEUROTOXICITY SYNDROME, SEQUELAE | The oncofetal antigen Claudin 6 (CLDN6) is highly and specifically expressed in many solid tumors, and could be a promising treatment target. We report dose escalation results from the ongoing phase 1/2 BNT211-01 trial evaluating the safety and feasibility of chimeric antigen receptor (CAR) T cells targeting the CLDN6 with or without a CAR-T cell-amplifying RNA vaccine (CARVac) at two dose levels (DLs) in relapsed/refractory CLDN6-positive solid tumors. The primary endpoints were safety and tolerability, maximum tolerated dose and recommended phase 2 dose (RP2D). Secondary endpoints included objective response rate (ORR) and disease control rate. We observed manageable toxicity, with 10 out of 22 patients (46%) experiencing cytokine release syndrome including one grade 3 event and 1 out of 22 (5%) with grade 1 immune effector cell-associated neurotoxicity syndrome. Dose-limiting toxicities occurred in two patients at the higher DL, resolving without sequelae. CAR-T cell engraftment was robust, and the addition of CARVac was well tolerated. The unconfirmed ORR in 21 evaluable patients was 33% (7 of 21), including one complete response. The disease control rate was 67% (14 of 21), with stable disease in seven patients. Patients with germ cell tumors treated at the higher DL exhibited the highest response rate (ORR 57% (4 of 7)). The maximum tolerated dose and RP2D were not established as the trial has been amended to utilize an automated manufacturing process. A repeat of the dose escalation is ongoing and will identify a RP2D for pivotal trials. ClinicalTrials.gov Identifier: In the ongoing phase 1/2 BNT211-01 trial, CLDN6-specific chimeric antigen receptor (CAR)-T cells given with or without CARVac, a CAR-T cell-amplifying RNA vaccine, were well-tolerated and exhibited encouraging clinical activity in patients with relapsed or refractory CLDN6-positive solid tumors, with the highest response rate in patients with germ cell tumors. | PMC10667102 |
Main | tumor, toxicity, cancer, contributeWe, tumors | MULTIPLE MYELOMA, TUMOR, B-CELL NEOPLASM, CANCER, SOLID TUMORS, TUMORS | Chimeric antigen receptor (CAR)-engineered T cell therapies have shown remarkable results in patients with relapsed/refractory (r/r) B-cell neoplasms and multiple myelomaA key obstacle for CAR-T cell approaches in solid tumors is the lack of highly cancer-specific cell-surface targets for efficient tumor eradication without on-target/off-tumor toxicity. Other common challenges are poor expansion and persistence, to which lack of antigenic stimulation due to inaccessibility of the target antigen in the periphery and an immunosuppressive tumor microenvironment may contributeWe have previously identified the primitive tight junction protein claudin 6 (CLDN6) as a potentially ideal immunotherapy targetWe have designed a highly sensitive, strictly CLDN6-specific second generation CAR with fusion of a CLDN6-specific single-chain variable fragment, the CD8α hinge and transmembrane region and 4-1BB and CD3ζ signaling moieties, which mediates rapid rejection of CLDN6-positive tumors by engineered T cells in xenograft and syngeneic mouse modelsBased on this work, we initiated a phase 1/2 first-in-human clinical trial to evaluate CLDN6 CAR-T cells alone and in combination with CARVac in patients with relapsed or refractory (r/r) CLDN6-positive solid tumors in a dose escalation part followed by cancer type-specific dose expansion cohorts. The manufacturing process of the CAR-T product is being developed in parallel to its clinical testing to implement a mature process with favorable product characteristics to facilitate a transition from manual to automated manufacturing before starting potentially pivotal trials.Here we present a non-prespecified interim analysis of 22 patients treated at two dose levels in the phase 1 dose escalation part of this trial, comprising the complete set of data obtained with the manual version of the manufacturing process. These data provide insights into the feasibility, safety and antitumor activity of the first-in-human concept of targeting the CLDN6 with a CAR-T therapy and combining with a CAR-amplifying vaccine. | PMC10667102 |
Results | PMC10667102 | |||
Study design and execution, patient selection and treatment | Tumors, tumor | TUMOR, SPINAL METASTASES, DISEASE, SOLID TUMORS, TUMORS, SECONDARY, ONCOLOGY | The study design comprises a dose escalation part and a dose expansion part with indication-specific cohorts including GCT, EOC and other indications to start when the recommended phase 2 dose (RP2D) obtained with the dedicated phase 2 manufacturing process is defined.The primary endpoints for the dose escalation part are to characterize the safety and tolerability of CLDN6 CAR-T cells ± CARVac and to identify the maximum tolerated dose (MTD)/RP2D. Evaluation of the antitumor activity of CLDN6 CAR-T cells ± CARVac per Response Evaluation Criteria In Solid Tumors (RECIST) 1.1 and characterization of soluble immune factors (cytokines) induced by treatment are the secondary endpoints. An MTD/RP2D was not established, as dose escalation is being repeated with CAR-T cells manufactured with an automated manufacturing process.The dose expansion part of the study enrolled adults diagnosed with advanced metastatic solid tumors of any type lacking further systemic treatment options, Eastern Cooperative Oncology Group (ECOG) 0–1 and measurable disease per RECIST 1.1. Patients with new or growing brain or spinal metastases during screening were excluded. Patients for the study were recruited from a CLDN6 prescreening. Additional eligibility criteria are noted in the Of 180 patients prescreened for CLDN6 expression with a semiquantitative immunohistochemistry assay between September 2020 and November 2021, 54 (30%) met the inclusion criteria for tumor CLDN6 positivity defined as ≥50% of tumor cells displaying an intermediate (2+) or strong (3+) membrane staining intensity (Fig. | PMC10667102 |
Safety | DLTs, impaired bone marrow function, toxicity, treatment-emergent adverse, toxicities, CRS, RNA-LPX cancer vaccine platformTransient, cytopenia, headache, deaths, pancytopenia, Cancer | ADVERSE EVENT, DISEASE PROGRESSION, ADVERSE EVENT, CYTOPENIA, IMMUNE EFFECTOR CELL-ASSOCIATED NEUROTOXICITY SYNDROME, PANCYTOPENIA, CANCER | All 22 patients treated within the study were included in the safety analysis. The data cutoff date was 6 October 2022, with a median follow-up of five months (range: 29–416 days, Extended Data Table Nineteen of 22 patients had treatment-emergent adverse events (TEAEs) greater than or equal to grade (G) 3 (Extended Data Table TEAEsAEs graded for severity using National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v.5.0, with CRS graded according to American Society for Transplantation and Cellular Therapy consensus gradingTEAE is defined as any adverse event (AE) with an onset date on or after the first administration of CLDN6 CAR-T (if the AE was absent before the first administration of CLDN6 CAR-T) or that worsened after the first administration of CLDN6 CAR-T (if the AE was present before the first administration of CLDN6 CAR-T). AEs with an onset date more than 90 days after the last administration of any investigational medicinal product (IMP) were considered as treatment-emergent if assessed as related to any IMP by the investigator.CRS was seen in ten patients (46%), eight of whom were treated at DL2 (Extended Data Table One patient treated at DL1 plus CARVac had symptoms (headache and decreased level of alertness) classified as G1 immune effector cell-associated neurotoxicity syndrome (ICANS) that occurred in conjunction with G2 CRS and resolved spontaneously within 24 h (Extended Data Table Dose-limiting toxicities (DLTs) emerged in two patients treated at DL2, one with monotherapy and one in combination with CARVac (Extended Data Table The other DLT was prolonged G4 pancytopenia observed in a heavily pretreated GCT patient reported 21 days post-ACT. The patient was nonresponsive to granulocyte colony-stimulating factor, received an autologous peripheral blood stem cell support on day 32, upon which the patient’s bone marrow function recovered within 14 days. The patient was redosed with CAR-T cells 41 weeks after the first CAR-T cell administration. This DLT experience led to the decision to consider GCT patients who had recently undergone HDCT as at risk for treatment-related persistent cytopenia. Consequently, we introduced protocol amendments to make the availability of autologous peripheral blood stem cells a prerequisite for treatment of patients with HDCT within the last 12 months or with impaired bone marrow function with the full dose of LD. Alternatively, we allowed dose-reduced LD.No further DLTs were observed after enrollment of three additional patients into each of these two DL2 cohorts. The Safety Review Committee (SRC) determined that the MTD (primary endpoint) was not reached. The recommended phase 2 dose of CLDN6 CAR-T cells was not further pursued as enrollment to the planned DL3 cohorts was canceled per the study protocol amendment to facilitate a repeat of the CAR-T dose escalation component with CAR-T cells manufactured with an automated process, including potential testing of higher dose levels. All eight deaths in the study were classified as disease progression and not attributed to CAR-T cell toxicity.The safety profile of CARVac was in line with previous reports related to our RNA-LPX cancer vaccine platformTransient release of IFNγ and IFNγ inducible protein 10, peaking 3–6 h post intravenous CARVac administration, was observed in serum cytokine measurements (Extended Data Fig. In summary, the safety profile of both dose levels of CLDN6 CAR-T cells as monotherapy or in combination with CARVac was manageable (Table | PMC10667102 |
Efficacy | DISEASE, SECONDARY | Twenty-one of the 22 patients were treated per protocol and qualified for efficacy analysis for the secondary endpoints ORR, disease control rate (DCR) and duration of response (DOR). Partial responses (PRs) in six patients and one complete response (CR) resulted in an unconfirmed ORR of 33% (Fig. | PMC10667102 | |
Pharmacokinetics | Characterization of the pharmacokinetics of CLDN6 CAR-T cells was an exploratory endpoint. For lymphodepleted patients at DL1 and DL2, CAR-T cell peak expansion (CARVac was administered to 11 patients who had received CLDN6 CAR-T cells after LD and two patients who received CAR-T cells without prior LD (Extended Data Fig. Given the limited sample size, the heterogeneity of patients and unequal initial engraftment within the subgroups at DL2, the impact of CARVac on persistence of CAR-T cells cannot be statistically evaluated with the data available at this initial cutoff. However, we noted a transient increase in CAR-T cells immediately following CARVac administration in some patients (Extended Data Fig. Further analysis with a larger patient cohort treated with CARVac at an earlier time point is planned to provide a more conclusive understanding of the impact of CARVac on CAR-T cell dynamics. | PMC10667102 | ||
Discussion | heterogenous, cancers, tumor, fever, impaired bone marrow function, toxicity, cancer, non-tumor, TEAEs, CRS, cytopenia, HLH, hematotoxicity | CANCERS, TUMOR, CANCER, HEMATOLOGICAL MALIGNANCIES, DISEASE, SOLID TUMORS, RECRUITMENT, CYTOPENIA, BONE MARROW SUPPRESSION, SEQUELAE | Extending the success of CAR-T cells to solid tumors presents significant hurdles, primarily due to the scarcity of distinct, tumor-specific targets, complicating the development of efficacious CAR-T therapies for these cancers. Notwithstanding these obstacles, several early-phase clinical trials are underway targeting molecules including mesothelin, Muc16, Folate receptor and carcinoembryonic antigenThe effectiveness and safety of CAR-T cell therapy largely hinges on on-target specificity. Targeting antigens such as her2neu, GD2 and CLDN18.2 that are also expressed by non-tumor tissue could lead to on-target, off-tumor toxicity where the antigen in healthy tissue is accessible to CAR-T cellsTo our knowledge, this is the first report on the clinical performance of CAR-T cells directed against CLDN6, an oncofetal antigen with no detectable CLDN6 expression in healthy adult human tissuesThe addition of the RNA-LPX-based CARVac at doses determined in previous trials did not alter the safety profile of CLDN6 CAR-T cells substantially.The TEAEs we observed (for example, CRS and one case each of HLH (G4) and ICANS (G1)) have been reported for CAR-T cell products used in hematological malignancies, and are most likely associated with this treatment modality. CRS was mostly G1–2, well manageable and resolved without sequelae.Other TEAEs ≥G3 were hematological ones, for example, cytopenia. GCT patients with impaired bone marrow function due to recent prior intensive myelosuppressive treatment were found to be especially at risk for treatment-related prolonged bone marrow suppression. Our observations indicate that omitting LD altogether may not be an option due to lack of CAR-T expansion without LD. To mitigate risks of enhanced hematotoxicity associated with prior treatments typical for the cancer types in this study, we continue to assess approaches such as dose-reduced LD protocols supported by CARVac-mediated CAR-T cell expansion. All reported safety-relevant findings (including asymptomatic transaminase/lipase elevations) had resolved by the time of this interim analysis.Overall, the study supports CLDN6 as a safe target for cell therapy in solid tumors and does not raise concerns for either combining CAR-T cells with CARVac or for exploring higher doses of CLDN6 CAR-T cells. Given that CARVac dosing improved engraftment for patients treated at DL1 dosed with CARVac four days post-ACT but not for those at DL2 dosed following peak expansion (23–51 days post-ACT), future cohorts will be treated at the earlier time point regardless of the dosing level. With this trial being continued, the safety data set will be extended, allowing more detailed characterization of the preliminary observations we made in the first 22 patients.This interim analysis showed encouraging clinical activity of CLDN6 CAR-T cells with and without CARVac in a heterogenous set of patients. Enrolled patients were heavily pretreated, had active disease with r/r solid tumors, and the baseline CT scans were performed on average almost six weeks before ACT, altogether being conditions that may underestimate the potential clinical activity of CLDN6 CAR-T cells.In the higher DL of 1 × 10More than half of the patients suffered from GCT. Subgroup analysis of those GCT patients treated with 1 × 10The remaining nine enrolled patients were too heterogenous in terms of tumor types for indication-specific assessment of CAR-T cell activity. Of the three patients with EOC treated at the higher DL, two experienced an objective response warranting further exploration of this tumor type.An important finding was the robust engraftment and expansion of CLDN6 CAR-T cells after a single administration, which has been a limiting factor for a number of clinical trials of CAR-T cells in solid tumorsCARVac administration was well tolerated among trial participants, signifying its potential for combination therapy with CAR-T cells. Upon receiving CARVac, patients typically experienced a brief period of fever. Transient interferon-gamma release was observed post-CARVac administration indicating proper innate immune stimulation. In addition to these indicators of immune stimulation, we observed temporary increases in the frequency of CAR-T cells in some patients who received CARVac. While these preliminary findings indicate that CARVac may aid in boosting the expansion of CAR-T cells, potentially enhancing their therapeutic potency, the overall impact of CARVac on CAR-T cell pharmacokinetics is difficult to quantify based on the highly heterogeneous and small-sized set of patients analyzed. Given the importance of long-term persistence and robust expansion of CAR-T cells and motivated by these promising early findings, we continue to investigate the effects of higher doses of CARVac and assess the impact of repeated vaccine administration on prolonging persistence in this ongoing trial.As this study is a single arm trial with no comparator or control group, selection bias cannot be ruled out. The study is further limited by the small size, a relatively short follow-up duration with patients still on trial at the time of data cutoff, and by the all-comer nature of the trial, which resulted in recruitment of individual patients with distinct tumor indications, meaning that the suitability of these indications for treatment with CLDN6-directed CAR-T cells remains unclear. Treatment of more patients with standardized CARVac treatment algorithms are required to draw clear conclusions about the potential role of CARVac in supporting CAR-T cell engraftment and persistence.In conclusion, CLDN6 CAR-T cells resulted in robust engraftment, with a manageable safety profile and strong signals of clinical activity in patients with CLDN6-positive solid tumors with high unmet medical need. The encouraging response rates in these high medical need populations led us to develop an automated manufacturing process to scale up manufacturing, and we have initiated a repeat of dose escalation with this IMP, introducing further dose levels for both CAR-T cells and CARVac. Following identification of a recommended phase 2 dose, the trial will proceed to open expansion cohorts in select indications. | PMC10667102 |
Methods | PMC10667102 | |||
Trial design, patients and treatment | tumor, prostate cancer, cancer, human immunodeficiency virus/hepatitis C, non-melanoma skin cancer, non-metastatic carcinoma, primary cancer, autoimmune disease, Cancer | TUMOR, VIRUS, PROSTATE CANCER, SPINAL METASTASES, CANCER, DISEASE, RECRUITMENT, PROSTATE, SOLID TUMOR, REMISSION, CERVICAL CARCINOMA IN SITU, SUPERFICIAL BLADDER CANCER, PRIMARY CANCER, AUTOIMMUNE DISEASE, CANCER | This first-in-human, open label phase 1/2 trial is being conducted at six sites in Germany (University Medical Center Hamburg-Eppendorf, University Hospital of Cologne, University Hospital Regensburg, Hannover Medical School, University Hospital Erlangen and the University Medical Center Mainz) and one site in the Netherlands (Netherlands Cancer Institute, Amsterdam) to assess dose levels of CLDN6 CAR-T cells with or without CARVac using a 3 + 3 dose escalation approach. We recruited adults with a diagnosis of metastatic or unresectable solid tumor of any indication with no further treatment options, intermediate or high CLDN6 membrane staining intensity in at least 50% of tumor cells as assessed by semiquantitative immunohistochemistry, and measurable disease per RECIST 1.1. Patients were screened for adequate coagulation, hepatic, renal and hematologic function. GCT patients without initial measurable disease per RECIST 1.1 were eligible for this trial if they had disease evaluable by cancer antigen-125, Alpha-fetoprotein (AFP) or human chorionic gonadotropin (hCG, as applicable), which was introduced by protocol amendment (note that all patients recruited to this trial had measurable disease). Men were not allowed to father a child or donate sperm, and women of child-bearing potential had to agree not to become pregnant. Patients previously treated with another CAR-T cell therapy, who had received vaccination with live virus vaccines within six weeks before the start of LD, who had an active autoimmune disease or human immunodeficiency virus/hepatitis C positivity, or who were receiving concurrent systemic steroid therapy >10 mg were not eligible. Evidence of new or growing brain or spinal metastases during screening was a further exclusion criteria. A history of another primary cancer within the two years before enrollment also precluded entry into the trial, except for the following: non-melanoma skin cancer, cervical carcinoma in situ, superficial bladder cancer, prostate cancer with currently undetectable prostate specific antigen, or other non-metastatic carcinoma that has been in complete remission without treatment for more than two years. The full list of inclusion/exclusion criteria can be found in the trial protocol, available online at 10.1038/s41591-023-02612-0.As the decision was taken to repeat dose escalation using CAR-T cells manufactured using an automated process, a protocol amendment was introduced to cease recruitment to a planned third dose level using the manually produced CAR-T cells. This analysis of the 22 patients dosed between December 2020 and February 2022 with two DLs of CLDN6 CAR-T cells manufactured from autologous leukapheresis material by a manual process was therefore introduced by protocol amendment. All but two patients underwent LD (500 mg mThe dose for CARVac was selected based on prior clinical trial experience with the RNA-LPX vaccine platform in several hundred patients (for example, For further details, please refer to the trial protocol and statistical analysis plan in the | PMC10667102 |
Study oversight | The study protocol was approved by institutional review boards of the authorities in Germany (Paul-Ehrlich-Institute) and the Netherlands (Central Committee on Research involving Human Subjects). The study was conducted in accordance with the Declaration of Helsinki and International Conference on Harmonization guidelines for Good Clinical Practice. Written informed consent was obtained from all patients. Initial drafts of the manuscript were prepared by a subset of the authors and all authors contributed to the final manuscript. All authors made the decision to submit the manuscript for publication. | PMC10667102 | ||
Endpoints and assessments | death, tumor, CRS, encephalopathy, PD | SECONDARY, RECURRENCE, ENCEPHALOPATHY, TUMOR | The primary endpoints of the study were safety and tolerability of CLDN6 CAR-T cells with or without CARVac and identification of the MTD/recommended phase 2 dose. AEs and serious (S)AEs were collected from signing the informed consent form until 90 days after the last administration of the study drug. Key secondary endpoints included the ORR, the DCR and the DOR per RECIST 1.1. DOR was calculated as the time from first occurrence of CR or PR to the first occurrence of PD (recurrence or death from any cause, whichever occurs first). If patients were redosed with CAR-T cells, the time from first PR/CR to first PD was counted. Exploratory endpoints included overall survival and PFS, CLDN6 expression in tumor tissue, serum cytokines and CAR-T cell persistence.Safety and efficacy parameters were reviewed weekly by the SRC. The DLT period was 28 days. Relatedness of AEs was captured for bridging therapy, LD, CAR-T cells and CARVac. CRS was graded according to American Society for Transplantation and Cellular Therapy guidelines, and ICANS was assessed by immune effector cell encephalopathy scoreCLDN6 CAR-T cell frequency in peripheral blood measured by quantitative polymerase chain reaction (qPCR) was used to calculate CAR-T cell counts, AUC and maximal concentration ( | PMC10667102 |
Statistical analysis | tumor | TUMOR | This interim analysis was conducted with a data cutoff date of 6 October 2022 and followed the statistical analysis plan. The primary efficacy analysis is defined as the ORR, but PFS, overall survival (OS) and DOR were included as secondary/explorative endpoints. The efficacy analyses were conducted with the modified intent to treat population (that is, any patients who had been treated with a conformant dose of the CAR-T product and who had a baseline and at least one on-treatment/posttreatment tumor assessment). The safety analysis set comprises all patients treated with a CAR-T cell product, while patients in the efficacy analysis set were treated with a conformant CLDN6 CAR-T product (≥1 × 10The rates of binary endpoints such as efficacy variables (ORR and DCR) were calculated by cohort along with the corresponding two-sided 95% confidence intervals using the Clopper–Pearson method.Time-to-event endpoints (DOR and PFS) were analyzed using Kaplan–Meier methodology and censored in accordance with the Food and Drug Administration guidance The median event/survival time in days (including two-sided 95% confidence limits according to Brookmeyer and CrowleyCorrelative analyses are descriptive and exploratory. Data were analyzed and depicted using R programming (v.4.1.2) and GraphPad Prism 9. | PMC10667102 |
CAR-T cell manufacturing | The CLDN6-CAR was constructed by linking the signal peptide sequence of an immunoglobulin heavy chain variable region (GenBank no. | PMC10667102 | ||
CAR-T cell immunophenotyping | CD45RA-BV510 | Standard staining and flow cytometry techniques were used to perform immunophenotyping of surface markers on BNT211 CAR-T cell products at the manufacturing site. Cells were stained with CD3-FITC, CD4-APC and CD8-PE (BD Multitest catalog no. 342417; 1:11 dilution) in the bulk drug product, and CD3-PerCP, CD4-FITC, CD8-PE (BD, Tritest, catalog no. 342414; 1:6.25 dilution) and murine F(ab’)2-fragment-AF647 (anti-CAR antibody) (Jackson ImmunoResearch catalog no. 115-605-006; 1:100 dilution) in the drug substance. An AF647 conjugated murine isotype control (Jackson ImmunoResearch catalog no. 005-600-003; 1:143 dilution) was used as a control. The frequencies of naïve-like T cells, central memory T cells, effector memory T cells and effector memory re-expressing CD45RA T cells were defined by CD3-PerCP (BD catalog no. 345766; 1: 5 dilution), CD4-BV421(BD catalog no. 565997; 1:50 dilution), CD8-BB515 (BD catalog no. 564526; 1:20 dilution), CD197-PE (BD catalog no. 560765; 1:5 dilution) and CD45RA-BV510 (BD catalog no. 563031; 1:25 dilution) in the bulk drug product. All antibodies for analysis were purchased from BD Biosciences and Jackson ImmunoResearch. Samples were analyzed on a FACSVerse flow cytometer (BD Biosciences) using the gating strategies shown in Supplementary Fig. | PMC10667102 | |
CLDN6 CARVac manufacturing | RNA, liposomes and RNA-LPX were manufactured under good manufacturing practice conditions. The CLDN6 CARVac RNA is a highly purified, non-nucleoside-modified, uridine-containing RNA.The single-stranded, 5′-capped mRNA was produced by in vitro transcription from the corresponding DNA template encoding CLDN6. The general structure of the CLDN6 RNA consists of 5′-cap, 5′-and 3′-untranslated regions, CLDN6 coding sequence and poly(A)-tail. In addition to the coding sequence, the RNA comprises sequence elements to enhance RNA stability and translational efficiency of the encoded antigen that have been optimized during development. The RNA backbone is optimized for translation of the coding sequence in human dendritic cellsLiposomes with net cationic charges were used to complex the RNAs to form RNA-LPX. The cationic liposomes were manufactured using an adopted proprietary protocol based on the ethanol injection technique from the cationic synthetic lipid (R)-N,N,N trimethyl-2-3-dioleyloxy-1-propanaminium chloride (R-DOTMA) (Merck and Cie) and the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine phospholipid (DOPE) (Corden Pharma)The injectable RNA-LPX drug products were prepared in a dedicated pharmacy by incubating the individual concentrated RNA drug products with an isotonic NaCl solution (0.9%) (Fresenius Kabi) and cationic liposomes according to a proprietary protocol | PMC10667102 | ||
Clinical assessments | tumor, death, toxicities, encephalopathy, PD, Laboratory anomalies | RECURRENCE, DISEASE, TUMOR, ENCEPHALOPATHY | Laboratory anomalies were screened by hematology testing, coagulation assay, serum chemistries, cytokine levels and urinalysis. Physical exams including 12-lead electrocardiograms and vital signs were performed daily during hospitalization periods for assessment of toxicities, including assessment of the immune effector cell encephalopathy score. Treatment responses were monitored by disease marker evaluation, measurement of serum cytokines, and assessment of tumor responses according to RECIST 1.1 (including time from first objective response to first occurrence of objective progression, recurrence, or death from any cause) using unidimensional measurement such as a CT scan or magnetic resonance imaging during pretreatment screening (average 5.9 weeks before ACT), at six weeks post-CLDN6 CAR-T cell infusion, then every six weeks for 50 weeks and then every 12 weeks thereafter. All patients had at least one tumor assessment post treatment, except for a treated patient who died before the first CT scan and was classified as PD. | PMC10667102 |
Histology | SE, tumor, Embryonal rabbit kidney tissue | TUMOR | Histological and immunohistochemical analyses of all cases were performed in the central histology lab at BioNTech SE on representative tissue slides of formalin-fixed, paraffin-embedded neoplastic tissues. In brief, slides were stained with hematoxylin and eosin on a Leica ST5020 multistainer and manually stained using a monoclonal mouse anti-human CLDN6 antibody (CLAUDENTIFY6, BioNTech Diagnostics) and a normal control reagent. Embryonal rabbit kidney tissue served as a positive control for each immunohistochemical staining. All samples were analyzed by board-certified pathologists regarding tumor content (hematoxylin and eosin) and CLND6 expression in neoplastic cells. CLDN6 staining positivity was classified as negative (0), weak intensity (1+), medium intensity (2+) and strong intensity (3+). Only membranous staining was considered. | PMC10667102 |
Spearman rank correlation analysis | tumor | TUMOR | The Spearman’s rank correlation coefficient was calculated between the age of the archival tumor tissue and the percentage of tumor cells with 2+/3+ positive CLDN6 staining at prescreening for both EOC and GCT samples.The figure was created with the SAS 9.4 Graph Template Language, using the SAS Enterprise Guide v.8. The Spearman coefficients and | PMC10667102 |
Monitoring of CAR-T engraftment by PCR | Woodchuck Hepatitis | CAR-T cell engraftment was assessed in peripheral blood by a duplex qPCR amplification assay targeting the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), a sequence part of the vector encoding the CLDN6-CAR and an intronic sequence from FOXP2, which serves as a reference for normalization of cell numbers. Genomic DNA (gDNA) was isolated from cell pellets, and DNA concentration was measured and corrected to a target concentration of isolated gDNA of 125 ng µl | PMC10667102 | |
Cytokine multiplex assay | REGRESSION | Quantification of cytokines (for example, IL-6, IFNɣ) in serum was performed using the Meso Scale Discovery (MSD) 10-plex assay using the Proinflammatory Panel 1 Multiplex Test Kit. IFNɣ inducible protein 10 was measured using the Chemokine Panel 1 Kit (MSD). Assays were performed per the manufacturer’s protocol with a seven-point standard curve generated using a fourfold dilution series in triplicate. The standards were used as described in the package insert. All samples were evaluated in duplicate at standard 1:2 dilution and maximal 1:8 dilution; calculated percentage coefficients of variation for the duplicate measures were less than 20%. Data were acquired on a Quickplex SQ120 Imager instrument (MSD) and analyzed using an MSD Discovery Workbench and a four-parameter logistic model. Reported values included those within the standard curve range and those calculated by the logistic regression analysis. | PMC10667102 | |
AFP quantification | Serum AFP levels were quantified locally using either the Siemens Atellica IM AFP Assay or the Roche Elecsys AFP immunoassay and a Cobas e801 analytical unit. Assays were performed as per the manufacturer’s protocol. | PMC10667102 | ||
Reporting summary | Further information on research design is available in the | PMC10667102 | ||
Online content | Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41591-023-02612-0. | PMC10667102 | ||
Supplementary information |
Supplementary Figs. 1–4, clinical trial protocol and statistical analysis plan.Reporting SummaryPrevious lines of treatment for patients in the trial. | PMC10667102 | ||
Extended data | PMC10667102 | |||
Transient cytokine release upon CARVac administration. | Interferon-gamma and interferon-gamma inducible protein 10 serum levels were measured in a lymphodepleted patient prior to and following ACT of CAR-T cells (DL1, solid green line). CARVac (25 µg) dosing started 4 days post-ACT and was administered on d51, d72, and d93 (50 µg), denoted by green dashed lines. Cytokine measurements were taken prior to CARVac dosing, then 6 hr, 24 hr, and then weekly after each CARVac dose.
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GCT patient with complete response and long-term CAR-T cell persistence after a single CAR-T cell dose. | The patient was initially diagnosed with a mixed GCT, had been heavily pretreated (5 lines of chemotherapy in total) including cisplatinum-based chemotherapy, HDCT/ASCT,gemcitabine/oxaliplatin/paclitaxel, multiple surgeries and radiotherapy. After the 3
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CLDN6 CAR-T cell frequency over time in all treated patients. | DSRCT, cancer of unknown primary, CUP, serous carcinoma, Cancers | DESMOPLASTIC SMALL ROUND CELL TUMOR, FALLOPIAN TUBE, GERM CELL TUMOR, SEROUS CARCINOMA, ENDOMETRIAL CARCINOMA, EPITHELIAL OVARIAN CANCER, CANCERS, GASTRIC ADENOCARCINOMA | CAR-T cell engraftment and kinetics was recorded in all treated patients (n = 22) from day of ACT until end of FU or data cut-off. Assessments ongoing at the time of data cut-off are marked by arrowhead. Patients are grouped by cohorts at indicated DL (solid line) including the two patients treated with ‘<DL1’ or with ‘50% LD’. Redosing of patients occurred at same DL in combination with CARVac either with (*, n = 4) or without (**, n = 1) LD. Crossover at DL2 (n = 2) is indicated by circle at start of additional CARVac treatment. CAR-T cells were detected in peripheral blood using quantitative PCR on DNA isolated from PBMCs. CAR-T frequency was calculated as CAR copy number divided by µg DNA of the sample. Cancers of ‘other’ origin than GCT and EOC were each a single case of DSRCT, GC, serous carcinoma of the fallopian tube, endometrial carcinoma and CUP. ACT, adoptive cell transfer; CUP, cancer of unknown primary; DL, dose level; DSRCT, desmoplastic small round cell tumor; EOC, epithelial ovarian cancer; FU, follow-up; GC, gastric adenocarcinoma; GCT, germ cell tumor; LD, lymphodepleting chemotherapy.
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CAR-T cell peak numbers in patients treated with CLDN6 CAR-T cells +/- CARVac. | Peak numbers were reached on average within 18 days (range 17–24) for patient treated at DL1 (circles) and 15.6 days (range 8–24) for patients treated at DL2 (squares). 3 patients treated at DL1 received CARVac 4 days after ACT, 7 patients treated at DL2 received CARVac on day ≥ 23 post-ACT. Numbers are presented as CAR copies per µg genomic DNA. Shown are patients treated with LD, peak CAR-T number for one patient treated with a 50% reduced course of LD is shown as a non-filled square. Box plots show median and upper and lower quartiles, with whiskers indicating 1.5x the interquartile range. Individual data points are overlaid.
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Extended data | is available for this paper at 10.1038/s41591-023-02612-0. | PMC10667102 | ||
Supplementary information | The online version contains supplementary material available at 10.1038/s41591-023-02612-0. | PMC10667102 | ||
Acknowledgements | P. | BNT211-01 was sponsored and funded by BioNTech. We thank the patients and their families for making this trial possible. We thank P. Young, K. Vemuri and J. Hinson for statistical support; P. Oehm for support with pharmacokinetic/pharmacodynamic analyses; S. Wöll, A. Feldner and S. Brachtendorf for histological analyses; K. Star and S. Wirsching for clinical operations support; I. Bigalke and P. Schröder for IMP manufacturing; M. PinoClaveria and S. Engel for CAR-T characterization; O. Lysenko for medical monitoring support; G. Rauser for project management support; and K. Reinhard for her role in the development of the IMPs. We also thank the Safety Review Committee and the clinical trial teams. | PMC10667102 | |
Author contributions | U.Ş., Ö.T., A.F. and B.R. conceptualized the project. B.R., L.P., U.Ş., Ö.T., Y.H., T.H., A.M.S. and K.K. were responsible for the methodology. A.M., J.B.H., C.K., W.A., E.W.-D., P.B., D.H., B.F., S.K., C.B., A.D., F.L., N.K., F.M., E.S., P.H. and P.K. carried out the investigation. Visualization was created by B.R., T.H. and Q.K.-F. C.S.-E. was responsible for project administration. B.R., L.P., U.Ş. and Ö.T. supervized the project. All authors gave feedback to a storyboard drafted by U.Ş., Ö.T., A.F. and B.R. from which a draft was developed that was reviewed and edited by all authors. | PMC10667102 | ||
Peer review | PMC10667102 | |||
Data availability | This trial is currently ongoing. Upon completion of this clinical trial, summary-level results will be made public and shared in line with clinical data-sharing guidelines. Requests for access to aggregated data (the tables produced in line with the statistical analysis plan) will be reviewed and approved by the Safety Review Committee on the basis of scientific merit. The datasets generated and/or analyzed during the current study are not publicly available due to proprietary considerations beyond the data that were made available here. All data provided are anonymized to respect the privacy of patients who have participated in the trial, in line with applicable laws and regulations. Data requests pertaining to the manuscript may be made to the corresponding author (U.Ş.; ugur.sahin@biontech.de). Requests will be processed within 16 weeks. The CAR-T (WO2020/161186) and CARVac (W2016/180778) technology is patented. | PMC10667102 | ||
Competing interests | SE | Y.H., T.H., Q.K.-F., A.M.S., C.S.-E., A.F., C.F., K.K., L.P., B.R., Ö.T. and U.Ş. are employees at BioNTech SE. E.W. previously worked as consultants to BioNTech SE. U.Ş. and Ö.T. are management board members of BioNTech SE (Mainz, Germany). U.Ş., Ö.T. and B.R. are inventors on patents and patent applications, which cover parts of this article. The remaining authors declare nocompeting interests. | PMC10667102 | |
References | PMC10667102 | |||
Introduction | Technology has enabled students and teachers to have interactive and engaging learning experiences, and providing teachers with more advanced teaching strategies boosts the educational environment. Animation is one of the most effective tools for creating engaging learning experiences for students, as it can bring topics to life and make them easier to understand. Additionally, animation can help keep students motivated and interested in the learning process, which can help them retain information better. Based on Korakakis et al. [Korakakis et al. [Boxing is a combat sport that depends on physical factors, technical skills, tactical insight, and mental strength. For beginners, the mental aspect is the most important to increase physical, technical, and tactical effectiveness. Cognitive awareness is key to developing psychomotor skills, spatial awareness, and motivation to learn. A correct understanding of boxing skills contributes to improving educational and training processes and reaching optimal motor skills [Therefore, the current study aims to answer the question: what is the effectiveness of using the reciprocal teaching style when combined with 3DHT on students’ performance level of basic boxing skills?From this main question, the following sub-questions emerge: is there a statistically significant difference between the pre- and post-measurements of the experimental group regarding fundamental boxing skills; what is the significance of the differences between the experimental and control groups on the post-measurement of fundamental boxing skills; and what is the effect size of the reciprocal style combined with 3DHT? | PMC10204982 | ||
Theoretical framework | PMC10204982 | |||
The constructivist cognitive development theory | Constructivist cognitive development is the dominant paradigm of educational development, based on Piaget’s cognitive development and Vygotsky’s sociocultural theory. This theory emphasizes that students construct their own knowledge, not that teachers transmit it to them. Through constructivism, teachers become guides and counselors for students, presenting problems and requesting them to identify and analyze details, data, and relationships, thus constructing a cognitive structure. Teachers organize learning environments, provide tools, manage and evaluate learning, and provide back-up information to their students [According to Merriam et al. [Oxford [Initiation of new knowledge.The process of acquiring new knowledge begins with learning as a whole, then paying attention to details.Developing knowledge by building tentative hypotheses, discussing them with colleagues in order to obtain answers, and then developing and modifying that knowledge based on collaborative responses.Putting knowledge into practice and gaining experience.Considering the development of knowledge.According to Oxford [ | PMC10204982 | ||
Sociocultural constructivist theory | Lev Vygotsky developed this theory, which emphasizes that knowledge exists in a social context [According to Vygotsky and Cole [Berk and Winsler [Students’ ZPD is explored through quizzes or discussions to determine what they already know about the learning topic.Various abilities of students are grouped into teams to increase their learning potential. Make sure that every member of the group participates actively.Vygotsky’s scaffolding can be counterproductive when the teacher provides students with too much assistance, making them passive rather than engaged.Students are encouraged to think aloud to determine their current abilities, thereby determine their ZPD and to ensure active learning. | PMC10204982 | ||
Cognitive load theory (CLT) | Working memory ability is a crucial determinant of academic achievement, according to the CLT. As working memory has a finite capacity, it often becomes overwhelmed while processing complicated instructional content. Working memory has to be able to withstand the demands of learning in order to learn well. Therefore, instructional methods should be carefully structured to exert the right amount of cognitive burden on working memory in order to assure learning success [Sweller [ | PMC10204982 | ||
The spectrum of teaching styles in physical education theory | Muska Mosston developed the teaching styles spectrum in physical education in 1966 to ensure all students understood the fundamental concepts, abilities, and ideas of physical education. Spectrum is based on the premise that teaching behavior can be explained as a chain of decisions. There is always a previous decision behind every deliberate act of teaching. There are three types of decisions in each style: pre-impact, impact, and post-impact. A pre-impact set of decisions includes decisions regarding the lesson’s objectives, the teaching method, the subject matter, the classroom environment, the location of the lesson, organizational arrangements, grading policies, and time (e.g., start and end times). The impact decision set includes options for putting pre-impact decisions into practice and, if necessary, modifying them. Post-impact decision categories include gathering data on learners’ performance, evaluating students’ performance against standards, giving feedback to students, and evaluating teaching approaches. There are many different teaching styles on the spectrum, from teacher-led instruction to more student-centered instruction. Physical education teachers are able to implement PE programs in dynamic settings by utilizing a number of teaching styles [ | PMC10204982 | ||
Reciprocal style | The reciprocal style is one of the direct styles in spectrum theory. In this style, learners are organized in pairs, each with a specific role to play. As the doer, one learner performs the task. As observer, learner provides constant and immediate feedback to the doer using a criteria sheet designed by the teacher. At the end of the practice, the observer and the doer switch roles [By giving feedback to a peer, learners are able to do better because the observer provides feedback more frequently, which results in a higher number of correct responses.As learners receive and give feedback to a peer, their socialization skills expand.Observer analyse movements by monitoring the doer’s performance, comparing it against criteria, and drawing conclusions about the accuracy of the performance.In the Reciprocal Style, the teacher’s (T) job is to decide on all subject matter, criteria, and logistical issues (pre-impact decisions), as well as to watch over students and provide them individual comments about their roles (post-impact statements). Throughout the learning process, learner (L) works in a partnership relationship. The doer (L | PMC10204982 | ||
Instructional design models | Instructional design (ID) is a framework for integrating technology into education. It focuses on learners rather than teachers, making the program more applicable and meaningful to them. Koper [ | PMC10204982 | ||
Materials and methods | PMC10204982 | |||
Ethics statement | The Scientific Research Committee at the Faculty of Physical Education, Port-Said University (Approval No. 2022-7-1), approved the study. Informed consent was obtained from the participants in writing, including voluntary participation, withdrawal rights, objectives, importance, procedures, and confidentiality. At the end of the form, participants can select Agree or Disagree to indicate their agreement to participate in the study. | PMC10204982 | ||
Design | A quasi-experimental design was used by creating two experimental and control groups. For the experimental group, the reciprocal method is combined with 3DHT to teach some basic boxing skills. In contrast, the control group is taught a program based on a teacher command style. Pretest-posttest designs were made for the two groups. The conceptual framework was illustrated in ( | PMC10204982 | ||
Study population and sample | The sample consisted of 40 boxing beginners aged 12 to 14 who were enrolled in the 2022/2023 training season at Port Fouad Sports Club in Port Said, Egypt. The participants were randomly divided into two experimental and control groups. There were homogenized based on age, height, weight, general intelligence, physical fitness, and skill level. | PMC10204982 | ||
Data collection tools and equipment | PMC10204982 | |||
The general IQ test | The study used an IQ test developed by Saleh [ | PMC10204982 | ||
An educational program combining reciprocal style and 3DHT | A proposed educational program combining reciprocal style with 3DHT was designed using the ADDIE model, in stages as follows: | PMC10204982 | ||
The pre-measurements process | At Port Fouad Sports Club, four boxing coaches assisted in taking pre-measurements from the experimental and control groups between 16-17/7/2022, in order to verify the homogeneity and the equivalence of the groups with respect to age, height, weight, IQ, special physical abilities, and the degree of skillful performance of the fundamental boxing skills. The homogeneity of the pre-measurements for each group is depicted in | PMC10204982 | ||
Descriptive statistics of the control group and Experimental group ( | PMC10204982 | |||
The basic experiment process | A boxing coach from the Port-Fouad club delivered three sessions per week to each group starting on 23/07/2022. The teaching period for both groups ended on 24/08/2022. The coach was accredited by the Egyptian Boxing Federation, and has over 10 years’ experience in boxing training. Additionally, he has experience as an international boxer. The author explained to him the reciprocal style and how 3DHT could be integrated into the basic experiment. Furthermore, he explained how the same procedures could be applied to both groups. In order to ensure a proper teaching environment and to monitor progress, the author visited the coach regularly. | PMC10204982 | ||
The post-measurements process | Post-measurements were applied immediately after the two groups completed the basic experiment on 25/08/2022. Under the same conditions and timing, arbitrators applied the skill performance evaluation checklists [ | PMC10204982 | ||
Statistical analysis | IBM SPSS Statistics for Windows (2017; version 25; IBM Corp, Armonk, NY, USA) was used for the following statistical analysis: Mean ( | PMC10204982 | ||
Results | The Kolmogorov-Smirnov test was used to verify the normality of the post-measurements for the control and experimental groups as shown in | PMC10204982 | ||
A normality test for post-measurements. | PMC10204982 | |||
Statistical comparison between pre and posttest for experimental group with paired samples t-test ( | *Reject the null hypothesis | PMC10204982 | ||
Statistical comparison between control and experimental groups`posttest using independent samples t-test ( | *Reject the null hypothesisIn order to investigate the statistical differences between the two groups, | PMC10204982 | ||
Statistical comparison between the control and experimental groups’ overall boxing skill degree in the posttest ( | *Reject the null hypothesisThe Cohen (d) value was 1.07, which was greater than 0.8. It confirmed the previous finding of With the use of the G*Power 3.1 tool, a Power Post-hoc analysis was conducted by Cohen’s (d) = 1.07 at a significance level of .05 for independent samples t-test (two groups), while sample sizes for each group were | PMC10204982 | ||
Discussion | mistakes | The results of the first question indicated that the experimental group performed fundamental boxing skills better in the post-measurements than in the pre-measurements. Considering that the participants were beginners to boxing, these results demonstrated the effectiveness of the proposed education program that used the reciprocal style by integrating 3DHT. According to the second question, the experimental group performed better in four straight punches than the control group during the post-measurements. These results were due to the reciprocal relationship between the players in the experimental group. This relationship included executing the required tasks using worksheets that allowed the observer player to provide feedback to the performer player. It also included exchanging roles between them, which positively influenced learning straight punches. These results are in agreement with those reported by Moreover, the experimental group’s superiority in post-measurements was attributed to using 3DHT, which created a 3D illusion of the educated skill, attracting students’ attention and allowing them to visualize and monitor how the skill was performed. Additionally, 3DHT enhances spatial capacity, an important factor in education, as the learner should absorb spatial information from sensory memory. This is consistent with previous studies such as Prado Ortega et al. [The well-designed learning program incorporating 3DHT assisted in placing an appropriate cognitive load in working memory, constructing schemas, and transferring and retaining information in long-term memory, which is consistent with cognitive load theory [Regarding the lack of statistically significant differences between the experimental and control groups in a boxing stance and footwork skills performance degree in the post-measurements, we attribute this to the fact that these skills are first acquired and trained in the first and second educational modules, followed by repetition and correction throughout the entire educational program. This allowed learners to master these skills regardless of the teaching strategy used on them. This is consistent with previous studies such as [The application of 3D hologram technology to teach boxing fundamentals offers an advanced virtual learning environment and provides knowledge that can be applied to real-world scenarios. The visual effect of the 3D hologram captured students’ attention and held their interest, leading to greater motivation among students. The students realized their pre-test mistakes after watching the 3D hologram animation, and in the post-test, the experimental group demonstrated confidence in their ability to perform boxing skills accurately. In this study, participants indicated that they were interested in this kind of learning method compared to conventional teaching methods. Students enjoyed this type of technology and were asked to use it in other courses as well. Several faculty members also expressed excitement about 3D hologram technology when they saw it. Students showed that this technology is beneficial for various reasons, one of which is that it increases their attention span when studying.Based on the large effect size, the post-hoc power analysis further confirms that the effect size was not due to chance, demonstrating that the difference between the two groups was statistically significant. Therefore, the findings can be generalized to the entire population. | PMC10204982 | |
Conclusions | The purpose of this study was to investigate the effectiveness of 3DHT combined with a reciprocal teaching style for learning some fundamental boxing skills. The experimental group performed fundamental boxing skills better in the post-measurements than in the pre-measurements. The experimental group also achieved higher skill levels than the control group, which relied only on the teacher’s command style. Due to this, it is imperative to utilize hologram technology as a pedagogical resource to enhance the learning process and combine it with teaching strategies that support active learning. Further, the power post-hoc analysis confirmed that the large effect size was not caused by chance, showing that the difference between the two groups was statistically significant. Thus, the findings are generalizable to the entire population. | PMC10204982 | ||
Limitations | A WiFi 3D Holographic Projector Hologram is available at electronic stores and is considered one of most accessible and affordable devices. In order to give the illusion that the 3D virtual objects are actually embedded in real life, it uses specialized software to create 3D movies with black backgrounds. The learner should avoid extending their hands too close to the projector in an attempt to grab the 3D virtual objects. Additionally, it does not provide sound, but external speakers can play 3D movies simultaneously through a computer or smart phone. | PMC10204982 | ||
Implications | Holograms allow students to interact with 3D virtual objects of motor skills, which helps to improve their understanding of how the body moves and how to perform movements correctly. Additionally, holograms can be used to help players visualize how to combine different elements of a skill and how to link those elements into a smooth and effective performance. This helps to improve player’s spatial awareness and coordination, which makes it easier for them to learn and practice the skill. Therefore, more studies should be conducted to investigate how to integrate hologram technology into education in conjunction with active learning strategies. | PMC10204982 | ||
Supporting information | PMC10204982 | |||
An example of an educational module used by the experimental group. | (PDF)Click here for additional data file. | PMC10204982 | ||
The Skill performance evaluation checklists by arbitrators [ | (PDF)Click here for additional data file.My sincere thanks go out to the officials of Port Fouad Sports Club in Port Said, the club’s boxing head coach (Captain Nader Al-Sahrawi), and all the players who participated in the study. | PMC10204982 | ||
References | PMC10204982 | |||
Subject terms | MISFORTUNE | Evidence-based interventions to favor more harmonious interactions in difficult relationships remain scarce. This study examined whether compassion training may have beneficial effects in an ongoing tense relationship with a disliked person, by reducing schadenfreude toward them and increasing felt interpersonal closeness. 108 participants were assigned to one of three 5-week trainings in a longitudinal randomized controlled study: compassion training, reappraisal training (emotion regulation control condition), or Italian language training (neutral active control condition). The disliked person was not targeted during the trainings to test potential transfer effects. Misfortune scenarios and a measure of interpersonal closeness were used to test whether schadenfreude and closeness feelings toward a disliked person changed from pre- to post-training, across different experimental and control groups. Only compassion and reappraisal trainees reported a decrease of schadenfreude feelings toward the disliked person compared to their pre-training ratings, no changes were observed in the Italian language training. Importantly, feelings of closeness toward the disliked person increased in the compassion training group compared to the other two groups. This increase of closeness feelings could be a central mechanism for improving social interactions. These transfer effects open new perspectives concerning emotion regulation interventions in conflict resolution. | PMC10603062 | |
Introduction | Social relationships are crucial for humans. So far, studies investigating ways to enhance relationships have mainly focused on romantic couplesTo date, theoretical as well as empirical work increasingly emphasizes that emotions are central to all meaningful interactions as they can influence behaviors and thus social relationshipsIn light of the relevant implications of emotions for social interactions, emotion-based interventions have been developed to promote better relationships. Previous studies tested the effects of emotion regulation strategies, such as cognitive reappraisal training (i.e., reinterpreting a situation that triggered an emotion in order to modify the emotional impact) in conflicts (for instance in couple conflicts, see Ben-Naim et al.In line with this body of research, a theoretical review has pointed out another emotion regulation strategyConsistent with this idea, cultivating compassion has been suggested to benefit social relationshipsPromising evidence comes from a study, which is, to the best of our knowledge, the only study that tested the effect of compassion meditation toward a negative target, a transgressorTherefore, it would be important to test whether compassion training can also have beneficial transfer effects in an actual, ongoing, and tense relationship with a disliked person. In other words: can compassion training change emotions and attitudes when they are directed to a disliked person? Among the emotions that arise from dislike, researchers have identified schadenfreude | PMC10603062 | ||
The present study | groupOur, decrease schadenfreude feelings | Consequently, the purpose of this randomized controlled trial (RCT) study was to test the efficacy of a compassion training (intending to cultivate benevolent wishes toward a benefactor, oneself, and all living beings) in a difficult interpersonal context such as the relationship with a disliked person. To improve the ecological validity of our research, participants were instructed to select a person they disliked, i.e., a person with whom they had personally interacted and with whom they may have had a conflict (as opposed to selecting an imagined or unknown disliked person). We aimed to determine whether compassion training changed schadenfreude and feelings of closeness toward this disliked person even if the disliked person was not targeted during the training (transfer effect). Schadenfreude feelings were measured using misfortune scenarios involving the disliked person, a typical paradigm in schadenfreude research. Closeness feelings were assessed with the Inclusion of the Other in the Self ScaleThe current study tested the efficacy of compassion training against a reappraisal training and a neutral active control group (i.e., learning Italian) to overcome one of the main limitations in meditation research, which is the lack of RCT with an active control groupOur hypotheses were that both compassion training and reappraisal training compared with an active control training (i.e., learning a non-native language), would decrease schadenfreude feelings toward a disliked person. In addition, we hypothesized that both compassion and reappraisal training compared to the control condition would lead to an increase in interpersonal closeness toward the disliked person. Finally, because this is the first study of its kind to compare compassion training and reappraisal training in tense ongoing relationships, we explored the differences between the two emotion regulation interventions without a directed hypothesis. | PMC10603062 | |
Method | PMC10603062 | |||
Participants | Volunteers were recruited through advertisements in Geneva as well as in surrounding areas. Participants with previous meditation experience, psychology students, and Italian speakers were excluded. A power analysis was conducted with G*Power, based on an effect size (0.76) reported for a comparison between a control group (waitlist) and a reappraisal training group in a study with similar measures and designParticipants with previous meditation experience, psychology students, and Italian speakers were excluded. “Other” category includes reasons such as some individuals participated in a similar study and knew the study aims or some participants withdrew at this stage.At the end, a total of 108 participants (60 females, 48 males; | PMC10603062 | ||
Trainings | The three trainings were delivered by experienced instructors and followed the same structure: they started with an introductory session of 1 h that was followed by two group sessions of 2.5 h of duration each. The three sessions took place over a 5-week period and were complemented with 20 min of guided audio recordings for daily listening during the training. All interventions were equal in time and procedure and took place in rooms of the University of Geneva in order to maximize the similarity between them. Furthermore, the instructors were not involved in the design, analysis, or writing of the study. To control for training adherence, participants were asked to log into a platform daily to specify how many minutes they listened to the audio recordings, as well as to indicate the amount of informal practices during the intervention period (i.e., application of the learned techniques in everyday life). Researchers encouraged participants to engage in daily practice of their respective trainings routines to maintain a consistent practice (see | PMC10603062 | ||
Compassion training | Compassion training consisted of a well-established procedure based on meditation sessions and guided audio instructions led by a compassion-based instructor having over 20 years of experience in teaching meditation | PMC10603062 | ||
Reappraisal training | The reappraisal training sessions were led by a psychologist and researcher in affective sciences with 2 years of experience in teaching courses on emotion regulation. The reappraisal training was designed based on previous studies using reappraisal interventions | PMC10603062 | ||
Italian training | The content of the control condition was selected based on another study that compared meditation training to foreign language training as an active control group | PMC10603062 | ||
Measures | PMC10603062 | |||
Schadenfreude feelings | First, participants were asked to identify a familiar disliked person, namely someone with whom they may have experienced a conflict in person and a neutral person, namely someone they barely know (see full description of the instructions in | PMC10603062 | ||
Closeness feelings | aggression, prosocial behaviors | The Inclusion of the Other in the Self Scale (IOSS; Aron et al.Other measures were included in the design to either check for potential differences (compassion traits, emotion regulation skills, and prosociality) between conditions after the randomization or were used for exploratory purposes on the effects of the two emotion regulation interventions (negative attitudes, prosocial behaviors, and aggression behaviors). Due to their exploratory nature and because no specific hypotheses were formulated for these measures, details on these measures are provided in the | PMC10603062 | |
Procedure | Individuals interested in the study received a Qualtrics link to an online demographic questionnaire via email. Participants who met inclusion criteria were then asked to identify a disliked person and a neutral person. Then, participants were invited to a first session in the laboratory for pre-training measures one week before the beginning of the trainings. For each laboratory session, a maximum of 8 participants were invited. Participants assessed their emotions (schadenfreude and compassion) after reading misfortune scenarios involving the disliked person and identical scenarios involving the neutral person. Then, participants evaluated their feelings of closeness toward the disliked person as well as the neutral person. At the end of the laboratory session, each participant was individually informed by the experimenter (orally) about the training they were randomly assigned to. Participants were then enrolled to their respective training (compassion, reappraisal, or Italian training) for 5 weeks. For post-training measures, participants were invited again to the laboratory and were required to not mention the training they had been following to the other participants as the sessions were grouped. They completed the same measures that were used for the pre-training, namely the emotions felt during the misfortune scenarios, and the feelings of closeness. At the end of the experiment, participants were asked to rate their motivation and interest for the training. | PMC10603062 | ||
Statistical analysis | Five one-way ANOVAs were used to examine whether the groups differed in their motivation and interest in the assigned training, in training attendance, formal practice, and informal practice. Differences between groups were then controlled and measures of interest (schadenfreude, and closeness) and the manipulation check (compassion) were analyzed with repeated-measures ANCOVAs. Our hypotheses were tested by planned contrasts. All data were analyzed using Statistica version 14.0.0 and the R version 3.5.1 and the packages “psych”, “car”, & “MBESS”. | PMC10603062 | ||
Ethics declarations | This research was approved by the ethics committee of Ethic Committee of the University of Geneva in October 2017 (ethical committee approval No. PSE.20170803.40) and performed in accordance with the Declaration of Helsinki. Written informed consent was obtained after the procedures had been fully explained to each participant. | PMC10603062 | ||
Results | PMC10603062 | |||
Training | To test whether groups differed in their training adherence, two one-way ANOVAs with condition (compassion, reappraisal, Italian training) as a between-subjects factor, and motivation and interest ratings (separately), as dependent variables were conducted. These analyses did not reveal any difference in terms of motivation or interest for the assigned training (all | PMC10603062 | ||
Compassion decreased feelings of schadenfreude toward the disliked person | Two 2*2*3 repeated-measures ANCOVAs and planned contrasts were run to analyze the effects of the trainings on compassion feelings (manipulation check) and schadenfreude feelings in response to the misfortune scenarios. These latter analyses comprised a between-subjects factor condition (compassion, reappraisal, Italian training), two covariates (audio recording practice and informal practice), and two within-subjects factors: time (pre-training, post-training) and target person (disliked person, neutral person).For compassion feelings, the repeated-measures ANCOVA indicated a main effect of the target person, suggesting that participants felt less compassion toward the disliked person than the neutral person, We then ran similar analysis with schadenfreude feelings as a dependent variable. The repeated-measures ANCOVA yielded a main effect of target person, Self-reports of schadenfreude feelings at pre- and post-training toward the disliked person as a function of condition (compassion, reappraisal, Italian). Errors bars indicate ± 1 | PMC10603062 | ||
Discussion | decreasing schadenfreude feelings | The aim of this study was to test whether compassion training can change attitudes and feelings toward a disliked person. Furthermore, we tested here if the beneficial effects of compassion training were transfer effects as the training did not target or mention the disliked person. Our manipulation check confirmed that only compassion trainees expressed an increase of compassion feelings in response to a misfortune of a disliked other after 5 weeks of intervention. In addition, participants in both emotion regulation interventions—compassion and reappraisal trainings—reported less schadenfreude feelings toward this disliked person at post-training compared to their pre-training ratings. Finally, only compassion trainees showed an increase of closeness feelings toward the disliked person at post-training. The findings reported here suggest that compassion training is a beneficial strategy to enhance difficult social relationships and, thanks to its transfer effects, could be considered an indirect conflict resolution intervention.Here we show for the first time that both emotion regulation interventions (compassion training and reappraisal training) were efficient in decreasing schadenfreude feelings. Future research may study whether the two interventions decrease schadenfreude through distinct mechanisms. Roseman and SteeleImportantly, the increase found in closeness feelings toward the disliked person extends past research in which contemplative interventions were found to promote social connectednessThus, a possible implication of this finding is that increased feelings of closeness may overcome consequences of nonvoluntary interactions and promote constructive dynamics in social relations with disliked others. A next step that future research should address is to use a longitudinal design aimed at testing whether increases in closeness translate into sustained changes in relationships with the disliked person, and result in more constructive interactions.Conversely, reappraisal participants in the current study did not experience a significant increase in closeness feelings toward the disliked other, differing from previous findings showing that the frequent use of reappraisal techniques leads to closer relationshipsIn addition to these potential contributions, the current study also has limitations that could be addressed in future research. This research was limited by the use of self-reports. As schadenfreude may be affected by social desirability due to its attributed immoral nature, scholars have suggested to use implicit measures of schadenfreude such as the use of facial electromyographyDespite these limitations, the current study shed light on the role of emotion regulation interventions in tense interpersonal relationships. The present findings indicate that reappraisal training and compassion training are efficient interventions to decrease schadenfreude for a disliked person. Additional results suggest that compassion training is a way to promote healthier relationships by enhancing compassion feelings for a disliked person. Furthermore, compassion training acted as “social glue” as it increased closeness feelings toward a disliked person. Importantly, effects of compassion training were found toward the disliked person although the disliked person was not targeted during the training. This generalization is key as it may help to overcome one of the major obstacles faced by peacebuilding scholars and practitioners: the lack of motivation to change attitudes or emotions felt toward foes or outgroup members | PMC10603062 | |
Supplementary Information | The online version contains supplementary material available at 10.1038/s41598-023-45363-1. | PMC10603062 | ||
Acknowledgements | BRAIN | We thank Alexandra Zaharia, Mirka Del Prà, and Jean-Philippe Jacques for their contribution as instructors for this study. In addition, we would like to specially thank Floriane Moulin and Vanessa Gottofrey with the data collection. Finally, we thank Ben Meuleman, Simon Schaerlaeken, and the Brain and Behavior Lab technical staff for their help in the study. Trainings of this study were conducted at Uni Mail (Geneva) and the experimental part was conducted at the Brain and Behavior Laboratory. | PMC10603062 |
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