In this study, a noninvasive quantitative measure was used to identify short and long term post-concussion syndrome (PCS) both from each other and from healthy control populations. We used Electrovestibulography (EVestG) for detecting neurophysiological PCS consequent to a mild traumatic brain injury (mTBI) in both short-term (N = 8) and long-term (N = 30)  symptomatic individuals. Peripheral, spontaneously evoked vestibuloacoustic signals incorporating - and modulated by - brainstem responses were recorded using EVestG, while individuals were stationary (no movement stimulus). Tested were 38 individuals with PCS in comparison to those of 33 age-and-gender-matched healthy controls. The extracted features were based on the shape of the averaged extracted field potentials (FPs) and their detected firing pattern. Linear discriminant analysis classification, incorporating a leave-one-out routine, resulted in (A) an unbiased 84% classification accuracy for separating healthy controls from a mix of long and short-term symptomatology PCS sufferers and (B) a 79% classification accuracy for separating between long and short-term symptomatology PCS sufferers. Comparatively, short-term symptomatology PCS was generally detected as more distal from controls. Based on the results, the EVestG recording shows promise as an assistive objective tool for detecting and monitoring individuals with PCS after normal recovery periods. Following an mTBI, patients usually experience one or more of the following clinical symptoms: headaches, dizziness, depression, memory loss, confusion, blurred vision and balance problems that may occur with or without loss of consciousness4. The persistence of these symptoms for more than one week is usually referred to as post-concussion syndrome (PCS)5. These symptoms except headache and dizziness are often reported for a few days and up to weeks following the injury. The headache and dizziness symptoms typically occur immediately as well as later in the recovery period. The observable neural dysfunction resulting from a head injury in most cases is temporary3; however, the symptoms can last for days, weeks (short-term PCS (SPCS)), and sometimes much longer with persisting symptoms6 which is referred to as long-term PCS (LPCS)7. Permanent, undiagnosed and untreated symptoms may disable the affected person and; in some cases, it may be fatal8.Mild traumatic brain injury (mTBI), also known as concussion, is defined as a transient alteration of brain function caused by a biomechanical force to the brain after a head injury, which is usually followed by some permanent or transient neurological symptoms and signs. mTBI is more frequent in teenagers, young adults, males and people who are engaged in high impact physical activities 9 which include somatic , cognitive (e.g. poor concentration), mood (e.g. depression), visual  and behavioral (e.g. irritability) problems10. These symptoms can be dominant in the first few hours or days right after the impact but may also persist for weeks, months or even years10. Neuropsychological assessments following a concussion have shown that the cognitive function mostly recover within 1–3 months’ post-injury11. Additionally, some improvements can take place during the first two years but some patients may remain impaired longer than 2 years11.Persistent PCS has been reported in 20–30% of concussed individuals and comprise incomplete recovery12 with (A) the ability to early detect and diagnose the presence of PCS soon after an impact, or (B) the ability to monitor full recovery.The major problems confounding treatment for concussed patients derive from the fact that there is no universally and clinically accepted tool13, Balance Error Scoring System (BESS)14, and computerized cognitive tests such as CogSport15 and ImPACT16 are subjective, and rely on self-reported symptoms. Other objective tools, like most of the brain imaging techniques, i.e. computed tomography (CT) or susceptibility weighted imaging (SWI) - magnetic resonance imaging (MRI)17, cannot reliably diagnose mTBI. In addition, the more reliable ones such as diffuse tensor imagining (DTI) have several limitations . These imaging techniques are more reliable for detecting lesions as the case in SWI-MRI17 or skull fracture by CT13. However, in the majority of patients with PCS the clinical neuroimaging findings are found to be normal19. Such disagreement between the imaging finding and the existence of cognitive impairments in PCS group might be due to a damage in the neural network rather than a focal lesion site likely to be detected by imaging techniques.The most commonly and traditionally applied diagnostic tools for concussion and PCS, such as the Sports Concussion Assessment Tool (SCAT3)20. Thus, having an objective and reliable assessment technique for PCS, such as the one investigated in this study, would be of great interest. There have been some quantitative approaches, such as quantitative EEG (qEEG)23 and robotic-assisted test battery24, investigating PCS and its recovery with some very positive outcomes including, when studying different post-concussion times using qEEG, a reported 77.8–92.3% accuracy in detecting short-term and long-term TBI23. However, there are also some studies that question qEEG’s clinical usefulness25 but recent publications do continue to support its utility27. The search for a universally clinically acceptable, quantitative PCS assessment tool continues.Neuropsychological assessments after mTBI, on the other hand, have a limited ability to accurately detect the presence of PCS mainly due to the fact that the scores can also be affected by several other confounding factors such as intelligence, age, education, depression and malingering28 has shown promise as a diagnostic assist tool for neurological and neuropsychiatric conditions such as Meniere’s29, Depression30 and Parkinson’s Disease31. EVestG signals are recorded in the external ear. The recorded signal is a combination of acoustic and vestibular generated field potentials (FPs)28. The EVestG technology can detect the vestibular response, which is hypothesized to be driven predominantly from the utricle28. The utricle has tonically active (spontaneously firing) hair cells that can be modulated by linear acceleration34. There is support for a vestibular change following mTBI. One of the major symptoms after mTBI is dizziness with about 23–81% of mTBI cases experiencing balance problems and reporting dizziness days after injury35. The prevalence of persistent dizziness after mTBI varies widely from 1.2% at 6 months to 32.5% at 5 years39. EVestG signal analysis can detect the changes in the balance/vestibular system28. Using DTI, the vestibulopathy in mTBI patients was found to have a central axonal injury component40. In addition, vascular compression of the 8th cranial nerve has been shown to lead to dysfunction of the auditory and vestibular systems41. Herein, this study presents staged research to evaluate EVestG as an assistive tool to measure and monitor both short and long-term symptoms of PCS.Electrovestibulography (EVestG)We hypothesize that one of the many reasons that mTBI patients experience balance problems is due to stretching, compressing or twisting of the vestibular nerve leading to a change in the vestibular response, which may also be persistent. Likewise, if the damaged regions of the brain that are bruised, swollen or have bled are connected to the vestibular system, their electrical signals related to balance might have changed. Further, during the metabolic cascade following mTBI, the sensory systems including the peripheral vestibular system will be impacted. Thus, the EVestG signals will likely change following a concussive impact, and that may prove to be indicative of a concussion and its continuing symptomatology. This paper reports on the use of EVestG addressing the above hypotheses.The objectives of this study were to 1) investigate the EVestG detected spontaneous FP changes between concussed individuals with PCS and healthy controls, and 2) investigate whether the features representing those changes have diagnostic classification power between the two groups of PCS and controls.42 followed by an unbiased classification routine to investigate whether the features have a diagnostic classification power.To accomplish the first objective, we recorded EVestG signals from a group of concussed individuals with PCS as well as a control group of age and gender matched healthy individuals. We compared the extracted field potentials (FPs) from the EVestG signals of the two groups. To achieve the second objective, we extracted two characteristic features that had shown sensitivity to PCS in our previous studyth author)43.All patients who presented to neuro-ophthalmology clinic and were diagnosed with PCS, met the inclusion criteria for the study and did not have the exclusion criteria, were referred to the research assistant to be included in this study. All referred patients had diagnosis of PCS, which was made based on the history and the positive examination findings such as convergence insufficiency and abnormal balance, determined by the neuro-ophthalmologist collaborator of the study (4th author), and 5) having normal hearing. The healthy control group’s inclusion criteria were: 1) being over 15 years of age, 2) have no history of head trauma, ear infection/injury, any psychiatric and/or neurological disorder, and 3) having normal hearing.The inclusion criteria for PCS group were: 1) being over 15 years of age, 2) having at least one head trauma with or without loss of consciousness in the last 10 years, 3) having a Glasgow Coma Scale (GCS) >13 within 10 minutes after the head trauma, 4) having continued symptoms and signs of concussion one month after the head trauma at the time of neurological examination  with PCS, out of which 8 were short-term PCS (SPCS- concussion <3 months prior to testing) and 30 patients were long-term PCS (LPCS- concussion >3 months prior to testing). As per International Classification of Headache Disorders-3, post-traumatic headache is called “persistent” if it lasts more than 3 monthsWe also recruited 33 healthy age-and-gender-matched individuals  with no history of concussion as the control group of the study.The duration between the majority of LPCS patient’s mTBI’s and the recording date was between 5 months to 5 years with the exception for two patients remaining symptomatic for more than 10 years prior to the EVestG recording. The aim was to test PCS patients with ongoing symptomology during (1 week to 3 months) and post (>3 months) normal recovery periods to investigate whether we could identify any malingering or psychogenic symptomology with EVestG assessment. Data were recorded at the Neural Diagnostic Laboratory, Riverview Health Center, Winnipeg, Manitoba. Demographic details of the PCS participants can be found in the supplementary Table S1.28. The muscle artifacts were removed from the recorded signals by a high pass filter set to 300 Hz. Nevertheless, if a signal was corrupted by muscle artifacts, poor electrode placement, movement or contact of the electrodes, that signal was removed from the analysis. Approximately 7% of signals were excluded. The NEER algorithm detects a series of FPs to produce an average FP plot like the one shown in Fig. 46  segments of the signals were extracted using a wavelet-based signal processing technique called the Neural Event Extraction Routine (NEER) 46 Fig. . HoweverCharacteristic features of the signals representing the vestibular system and PCS were extracted with the following procedure. Using the FP curve as well as the FP firing pattern, we extracted two types of features as elaborated below.42. However, when we looked at each subject’s AP area individually, we found in some cases there were differences in the descending part of the AP, the ascending part of AP, or on both sides of the AP. The choice of the AP area as our feature can take into account all three differences. Further investigations are needed to see why some have difference in the ascending part while some others in the descending part and some in both parts of the AP.After normalizing the FPs with the absolute value of the action potential (AP) point Fig. , we calc49 that vestibular efferent spontaneous activity is usually seen in the range 10–50 spikes/s. Thus, we also looked for the low frequency (modulated) spontaneous FP interval activity (~ 10 Hz). Since the average measured time gap that NEER algorithm detects between two FPs is ~ 3.3 ms, a 33 FP gap corresponding to about ~100 ms (10 Hz)30 was used  FP gap during the no movement (BGi) phases from the signals of study participants were generated. A significant (p < 0.05) difference was found between both groups as the average distribution of PCS group was shifted towards the right (lower frequencies and longer gaps) as shown in Fig. Beside the FP, the NEER algorithm also provides the time of occurrence of each detected FP. It was shown insed Fig. . TherefoFigure 42, we observed an asymmetry between left and right ear in lateral-impact PCS participants. Indeed, the AP area was always narrower on the coup side, while it was either wider or similar to healthy control response in the contra-coup site. Therefore, we calculated the minimum AP area for left and right ear signals and used this smaller value as a characteristic feature.Thirteen out of the 38 PCS participants had a lateral head impact either from left or right. In our previous pilot studyFigure 50 as the classification routine for separating healthy controls from concussed patients. LDA is a standard approach for supervised classification; it estimates the membership probability of each class as a Gaussian distribution assuming identical covariance matrices for all classes. Due to small size of our dataset and yet keeping the training and testing separate for an unbiased classification and also to avoid the over-fitting problem, a leave-one-out routine was applied for classification, in which one subject’s data was left out for testing and the rest used as training; this routine was repeated until all subjects were used as test once. In each fold, the two features were tested individually using LDA for classifying the two groups and the resultant accuracy was calculated. The same routine was repeated for the combination of the two features and the resultant accuracy calculated.We applied linear discriminant analysis (LDA)Tables The two features used are highly correlated (R = −0.7), however we  used the IH33 feature as well as the AP area feature as both are potentially physiologically meaningful features (see discussion). To give a visual representation of the selected features among the two groups of controls and concussed (SPCS and LPCS), the scatter plot of the features for the two groups of concussed and controls are shown in Fig. The classification accuracy can be improved using other nonlinear classification techniques or a support vector machine (SVM). However, in small sample size studies, LDA is more reliable than other non-linear classification methods because LDA is more robust to the variance. In other words, if a technique shows a reasonable accuracy using the LDA, then we can be confident that the method will perform better using nonlinear classification methods. For comparison, we did run the classification using SVM. The classification accuracy using SVM for separating LPCS and SPCS groups improved to 92% Table  c.f. 79 (c.f. 95The area under the receiver operating characteristic curve (AUC) was calculated Tables –4 as an 52 that can cause balance problems. Poor balance and postural instability have been reported in many studies after mTBI55 and have been correlated with dysfunction in sensory integration57. Some symptoms including vertigo and dizziness can be due to neurovascular compression of the 8th cranial nerve58. Considering this fact and the link between dizziness and the abnormal function of the vestibular apparatus, the representative results of this study support the hypothesis that vestibular activity is perturbed following mTBI. This study has shown that the EVestG evoked averaged FP responses of the two groups of PCS individuals and healthy controls has classification power to separate PCS individuals from controls.Following an mTBI, the most common symptom after headache is dizziness45 and acoustic compound action potentials59 have similar characteristic shape and both are comparable with the extracted vestibular FP of the recorded signals. Many studies discuss the generation of the acoustic compound action potentials  the P1 peak is reduced59. Another study46 showed the P1 peak was recovered one hour after removing the CN; this indicates the P1 peak might be produced  in the 8th nerve, and may not be entirely of CN origin. Brown et al.46 showed that applying pressure to the 8th cranial nerve lead to a decrease in the P1 amplitude; that implies any physical change to the 8th cranial nerve results in an AP shape change.Vestibularals Fig.  and the ent Fig.  of the a3. A physical change of the neuronal cell membrane or the axons leads to indiscriminate flux of ions through ion gates. This abnormal process increases the release of the excitatory neurotransmitters like glutamate, resulting in further ionic flux. Then, in order to maintain an ionic balance, the Na/K ATP-dependent channels become activated, and that increases the glucose metabolism. This mismatch of energy supplies and demand leads to further cell injury and dysfunction3. Linking the process of the development of the AP after stimulation with the generation of N1 and P1 peaks of the acoustic compound action potentials ; that would result in an increase in Ca+2 influx and subsequent proteolysis of the NaCh α-subunit. As a result of the α-subunit degradation, the α-subunit promotes persistent elevation in Ca+2, which helps explain the narrower AP area in long-term PCS. In another study64 plaques composed of amyloid β (Aβ) were found in the damaged axons following a brain trauma in humans. In the same study, the authors described the accumulation of Aβ in the damaged axons as well as in a limited number of neurons of the cortex, hippocampus and cerebellum 3 and 7 days and 6 months after trauma in the TBI mice. As mentioned before, the extracted FP curve was always narrower for PCS individuals compared to healthy controls. We speculate that this change is due to an increase efflux and influx of sodium-potassium ions that might be explained by the accumulation of intra-axonal Ca+2 65. The ability of the Aβ to form neurofibrillary tangles could be the consequence of its ability to increase the Ca+2 influx into neurons65.To explain our results supporting of the long-term symptomatology of our PCS participants, we need to consider the possibility of a more permanent damage that might have occurred in the 846 observation that the generation of N1 and P1 peaks of the acoustic compound action potentials were entirely by the 8th nerve, we believe the changes we observe in the AP area ; this implies more influx of the Na+ and more K+ efflux in the PCS group compared to those of the healthy controls, which in turn implies a difference in the depolarization and repolarization mechanism of both groups.Following Brown’srea Fig.  of the PUsing the two-feature combination, there were seven misclassified PCS patients. It is of interest to note that most (5 out of 7) of the misclassified patients had the head trauma more than 1.5 years prior to our recording. Thus, considering the plasticity factor of the nerves, one may speculate that those patients had more time to recover. However, one of the two patients who had the impact more than 10 years prior to recording, was classified as healthy, while the other patient was classified as PCS. This suggests PCS individuals’ symptoms may persist for a long time and recovery depends on their brain’s plasticity or other confounding influences like anxiety.24, using a robotic-assisted assessment of neurological function, they investigated if PCS following mTBI can be predicted during the initial presentation to an emergency department. However, they only validated their prediction accuracy over a short (3 weeks post-injury) duration.Accurate diagnosis and prognosis of the TBI consequence are essential for patient care and long-term rehabilitation. In a recent study23. However, qEEG has not shown a clear ability to differentiate between SPCS and LPCS23 as both SPCS and LPCS show increased delta and reduced alpha band power27, while PCS within the recovery time and recent to the injury is expected to be different than PCS beyond the recovery time. The authors in27 continue, “EEG/qEEG findings in mTBI have been hypothesized to be related to the known pathophysiology of mTBI, and in some cases have also been corroborated with other investigations such as neuroimaging or histopathology” but also state, “Although the literature indicates the promise of qEEG in making a diagnosis and indicating prognosis of mTBI, further study is needed to corroborate and refine these methods”27. In other study25 it was indicated that qEEG provides, at best, an imperfect assessment of mTBI and reports the high specificity of qEEG evaluations of TBI must be interpreted with care questioning qEEG’s disease specificity. However, these authors also state “The published literature does indicate, however, that it (qEEG) can be an important complement to other assessment procedures”25.In another study, using qEEG, it was claimed that qEEG analysis, independent of other assessments, could predict the severity of the injury with high accuracy in a post-trauma period ranging from months to 8 yearsIn this current study, we showed the use of a quantitative physiological measure of the vestibulo-acoustic response  has promising potential to identify SPCS and LPCS both from each other and from healthy control populations. EVestG is shown to have the potential to monitor the PCS within the non-persisting symptom recovery time and also differentiate it from persistent PCS beyond the normal recovery time. However, according to the literature, the recovery time might be within the first days, weeks, up to 3 months after the injury. Thus, it is possible that some of our SPCS participants were beyond the non-persisting symptom recovery time, and appeared only with persistent symptoms (which may be a reduced form of those in LPCS). That may explain why two out of eight SPCS were classified in the LPCS group Fig. . This isData used in this study was collected through two separate but related projects. One was to investigate the feasibility of the EVestG technology for diagnosis of concussion, and the second was a clinical trial  for treatment and recovery monitoring (using EVestG) of post TBI Syndrome. Both studies were approved by the University of Manitoba Biomedical Research Ethics Board, and all the participants signed an informed consent prior to the experiment. All experimental procedures were performed in accordance with the protocol approved by the Biomedical Research Ethics Board and its regulations.Placing the electrodes: the ear canal wick electrode was placed in each ear canal close to the ear drum . IdentiAfter placing the electrodes, the participant was positioned in an acoustically attenuated (>30 dB) and electromagnetically shielded chamber, and seated in a stationary hydraulic chair, with their head supported by a headrest Fig. . ParticiTM with a sampling rate of 41,666 Hz for compatibility with previous studies.The signals of both ears were recorded using Spike234. In this study, in order to minimize any artifacts caused by body movement which may corrupt the recorded signal and to, at least initially, consider the ability of features based only on the spontaneous activity of the vestibule-acoustic system to discern PCS, the analyzed EVestG recorded signals are only from the stationary phase (BGi) recordings28. The analyzed segment was the average of three BGi recordings.The vestibulo-acoustic system is highly spontaneously activeThe data that support the findings of this study are available from Neural Diagnostics Pty. Ltd. but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of Neural Diagnostics Pty. Ltd.Supplementary Information


NKR-2 are autologous T cells genetically modified to express a chimeric antigen receptor (CAR) comprising a fusion of the natural killer group 2D (NKG2D) receptor with the CD3ζ signalling domain, which associates with the adaptor molecule DNAX-activating protein of 10 kDa (DAP10) to provide co-stimulatory signal upon ligand binding. NKG2D binds eight different ligands expressed on the cell surface of many tumour cells and which are normally absent on non-neoplastic cells. In preclinical studies, NKR-2 demonstrated long-term antitumour activity towards a breadth of tumour indications, with maximum efficacy observed after multiple NKR-2 administrations. Importantly, NKR-2 targeted tumour cells and tumour neovasculature and the local tumour immunosuppressive microenvironment and this mechanism of action of NKR-2 was established in the absence of preconditioning.8, 1×109 and 3×109 NKR-2 per injection. On the identification of the recommended dose in the first segment, based on dose-limiting toxicity occurrences, the study will expand to seven different cohorts examining the seven different tumour types separately. Clinical responses will be determined according to standard Response Evaluation Criteria In Solid Tumors (RECIST) criteria for solid tumours or international working group response criteria in haematological tumours.This open-label phase I study will assess the safety and clinical activity of NKR-2 treatment administered three times, with a 2-week interval between each administration in different tumour types. The study will contain two consecutive segments: a dose escalation phase followed by an expansion phase. The dose escalation study involves two arms, one in solid tumours (five specific indications) and one in haematological tumours (two specific indications) and will include three dose levels in each arm: 3×10Ethical approval has been obtained at all sites. Written informed consent will be taken from all participants. The results of this study will be disseminated through presentation at international scientific conferences and reported in peer-reviewed scientific journals.NCT03018405, EudraCT 2016-003312-12; Pre-result. Standard dose escalation phase and an expansion phase to investigate for the first time the safety and initial clinical activity of NKR-2 -based chimeric antigen receptor (CAR) T cells retrovirally modified) in a multiple injection schedule.Phase I study exploiting the multiple ligand targeting capability of human NKG2D by assessing the safety and initial clinical activity of NKR-2 against a broad range of tumour indications, including haematological and solid tumour types.Based on the unique NKR-2 mode of action, NKR-2 will be administered without any prior preconditioning.The chimeric antigen receptor (CAR) is an artificial construct that endows T cells with predetermined specificity.1 2transplant procedure-like’ approach, that is, a single CAR T cell infusion in preconditioned (lymphodepleted) patients and for which persistence, expansion and survival of the transferred T cells are crucial.The CD19-specific CAR provides the paradigm against which other CAR approaches are currently measured due to the impressive clinical responses seen in early phase clinical trials targeting B cell malignancies. Reports of initial objective clinical responses in 70–90% of patients with advanced chemoresistant B cell leukaemia have fuelled interest in the area with the number of CD19-targeted CAR T cell trials rapidly rising within the last few years.However, single-target therapy may select for and spur escape variants by clonal selection or antigen loss. Several CD19-specific therapies indeed evidenced cases of relapse resulting from CD19 antigen loss in leukaemia cells post-CD19-specific CAR T cell therapy.By contrast, outside of the B cell malignancy setting, the CAR T cell approach has met with much reduced levels of clinical success.+ T cells and, under certain conditions, some CD4+ and γδ+T cell subsetsNKR-2 are autologous T cells genetically modified to express a CAR comprising a fusion of the full-length natural killer group 2D (NKG2D) receptor with the CD3ζ signalling domain. NKG2D is a C-type, lectin-like, type II transmembrane glycoprotein-activating receptor expressed in humans on natural killer (NK), natural killer T, activated CD8In humans, eight NKG2D ligands have been identified, encoded by different loci on two different arms of the chromosome 6 and corresponding to two main families: the major histocompatibility complex (MHC) class I chain-related proteins (MIC) which include MICA and MICB proteins, and the structurally diverse unique long 16-binding proteins (UL16-binding proteins or ULBP) which include ULBPs 1 to 6.35 36The breadth of ligands targeted by NKG2D provides T cells armed with the NKR-2 receptor the potential to challenge an extensive range of tumour indications.33 34Preclinical studies have shown the murine equivalent of NKR-2 (chNKG2D T cells) can mediate potent antitumour activity against both haematological and solid tumours,38Critically, these preclinical data in multiple syngeneic tumour models indicate that antitumour efficacy was not dependent on lymphodepleting preconditioning, while the induction of a tumour-specific adaptive immune response by NKR-2 therapy enabled the long-term protective effects of the approach.49Expressing the NKR-2 CAR into T cells therefore allows the NKR-2 cells to present the broad specificity characteristic of NK cells while exploiting the migration, expansion, long half-life and potential to generate immunological memory characteristic of T cells. In addition to direct recognition of tumour cells, targeting of ligands expressed on the stroma of the TME can also lead to disruption of the essential support mechanisms required for tumour cell survival and growth. Moreover, in contrast to other single or limited target CAR T cell therapies, the fact that NKR-2 targets several ligands from two main families putatively decreases the probability of target loss and associated relapse after treatment. Further, the lack of important in vivo cell expansion and long-term survival of NKR-2, and the induction of tumour-specific adaptive immunity in the absence of patient preconditioning, also bears a safety advantage preventing side effects generally associated with rapid CAR T cell expansionThe THerapeutic Immunotherapy with NKR-2 (THINK) trial is an open-label phase I study which primarily aims to assess the safety and clinical activity of the NKR-2 treatment administered three times every 2 weeks between each administration in different tumour types. The study is split into two segments each with specific study objectives. The first dose escalation segment focuses on determining the recommended dose (RecD) of NKR-2 cells for second segment of THINK. Segment 2 extends the safety study and also investigates initial clinical activity of NKR-2 across multiple tumour indications. In both segments, correlative measures of clinical activity will be examined.+ cells obtained after transduction of autologous T cells with the NKG2D-based CAR, which contains a total viable population of at least 80% CD3+ cells, with at least 50% CD3+ being transduced. NKR-2 will be supplied cryopreserved in bags containing a T cell dose in accordance with the dose level which is to be administered.NKR-2 refers to the viable autologous CD37 total cells) and initial signs of clinical benefit.53NKR-2 has been used in the single infusion dose escalation clinical study CM-CS1 (NCT02203825) which evaluated the safety and clinical activity of NKR-2 administration without prior lymphodepleting conditioning in patients with two distinct haematological malignancies: acute myeloid leukaemia (AML)/higher risk myelodysplastic syndrome (MDS) refractory anaemia with excess blasts, that are not in remission and for which standard therapy options are not available, and relapsed or refractory progressive multiple myeloma (MM) after several lines of standard therapy. The trial has been successfully completed with no observed dose-limiting toxicities (DLTs) at the highest dose infused (3×106 cells to 7.5×106 cells per dose (intravenous single injection). Although a significant antitumour activity was obtained with a single injection of murine NKR-2, 100% overall survival and complete recovery were obtained following repeated three sequential doses of 5×106 cells/mouse without any adverse effect.7 cells, and repeated administration of this dosage to healthy and tumour-bearing mice did not produce any major toxicities.54In preclinical models, the pharmacologically effective dose (PED) of the murine NKR-2 ranged from 1×106 cells/injection per mouse and were identical whatever the tumour-type model . Using the allometry scaling exponent of 0.75, as defined by Food and Drug Administration (FDA) guidelines , human-equivalent PED and MTD were therefore estimated at 0.43–3.2×109 and 4.3×109 cells/injection, respectively. Together these observations of high tolerability in initial single dose phase I clinical trial coupled with preclinical efficacy experimentation strongly support the concept of a dose escalation segment of 3×108 –3×109 NKR-2 per infusion given three times each separated by a 14-day interval.Following these values of PED and MTD, the no-observed effect level was defined at 5×10BoxMen or women ≥18 years old at the time of signing the informed consent form (ICF).Patient with specific cancer indications (see below).Disease must be measurable according to the corresponding guidelines.Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 or 2 based on peripheral neuropathy from prior therapies.Patient with adequate bone marrow reserve, hepatic and renal functions.Left ventricular ejection fraction of >40%.For patients with solid tumour type, the patient must agree to have a tumour biopsy at baseline.Patient with a tumour metastasis in the central nervous system.Patients who have received another cancer therapy within 2 weeks before the planned day for the apheresis.Patients who receive or are planned to receive any other investigational product within the 3 weeks before the planned day for the first NKR-2 administration.Patients who are planned to receive concurrent growth factor, systemic steroid or other immunosuppressive therapy or cytotoxic agent.Patients who underwent major surgery within 4 weeks before the planned day for the first NKR-2 administration.Patients who have active infections necessitating the use of antibiotics/antivirals treatment.Patients with history of autoimmune disease.Patients must have either:A documented metastatic or locally advanced colorectal adenocarcinoma and having received, being intolerant or having refused at least two prior standard cancer therapy regimens as part of their primary treatment regimen or part of their treatment for management of recurrent/persistent disease.A documented recurrent epithelial ovarian cancer or fallopian tube carcinoma and having received or refused at least two prior standard cancer chemotherapy regimens as part of their primary treatment regimen or part of their treatment for management of recurrent/persistent disease.An inoperable locally advanced or metastatic urothelial carcinoma that has progressed after previous platinum-based chemotherapy.A metastatic or locally advanced triple-negative breast cancer and having received at least one prior cancer therapy regimen as part of their treatment for management of recurrent/persistent disease.A metastatic or locally advanced pancreatic ductal adenocarcinoma and having received, being intolerant or having refused at maximum two prior lines of standard cancer chemotherapy regimens.Relapsed or refractory acute myeloid leukaemia  after one prior therapy or patients with myelodysplastic syndrome  or MDS with TP53 mutation who failed from prior treatments.Relapsing or refractory/relapsing multiple myeloma with a minimum of one prior line of systemic therapy.Refractory or relapsing patients with metastatic or locally advanced CRC, urothelial carcinoma, triple-negative breast cancer (TNBC), pancreatic cancer, recurrent epithelial ovarian and fallopian tube carcinoma, AML/MDS and MM, which failed from standard treatments, will be recruited into this protocol .The tumour types targeted in this protocol were selected primarily based on their high expression of the NKG2D ligands .THINK phase I study will contain two consecutive segments: a dose escalation and an expansion segment .dose escalation design will include three dose levels of NKR-2. The assumption is made that the safety profile of the treatment could be different in haematological versus solid tumour types. Therefore, it will be assessed separately in two different arms, one in solid tumours  and one in haematological tumours (AML/MDS or MM). Each arm will use a 3+3 design to determine the RecD of the NKR-2 treatment for the corresponding cohorts in the expansion segment, as based on the occurrence of DLTs . The sample size of the dose escalation part will be between 2 and 18 patients per arm. Six patients will be treated at the MTD (or highest dose level if no DLT was observed).The The RecD will be the MTD unless in case no MTD is determined in the dose escalation segment of the study. In the latter, the RecD will be highest dose evaluated in the dose escalation segment.expansion segment of the study to assess separately the safety profile and initial clinical activity of the NKR-2 treatment in seven cohorts of patients with the distinct tumour types (up to 14 patients per tumour type). The expansion segment in haematological and solid tumours will be initiated as soon as their respective dose escalation arm defines the RecD. During this segment, the RecD can still be adapted according to predefined safety rules in any specific tumour type. The statistical analysis is planned in three steps. A futility analysis will be conducted separately in the first 14 patients with a solid tumour and the first 6 patients with a haematological tumour type. These number of patients have been defined to have a 0.05 probability of finding 0 objective response at this step if the true probability of response were 0.2 (solid) or 0.4 . Subsequently, futility (no response in the first seven patients) and efficacy (three or more responses in 14 patients) will be tested by cohort, based on the Simon’s two-stage optimal design. The assumptions are: type I error rate=0.15, power=0.80 and response probability of poor drug=0.10/beneficial drug=0.30. In total, up to 86 patients are expected to be enrolled in the expansion segment .The RecD will be further evaluated in the 8, 1×109 and 3×109 NKR-2 for each injection  with a schedule of administration of three NKR-2 doses administered with a 2-week interval per patient. No systemic lymphodepleting preconditioning will be performed prior to NKR-2 injections.Three NKR-2 dose levels will be evaluated during this study, that is, 3×10primary endpoint of the dose escalation segment will be the occurrence of DLTs in all patients during the study treatment until 14 days after the last study treatment administration. Secondary endpoints will include the (i) occurrence of adverse events (AEs) and serious adverse events (SAEs) during the study treatment, (ii) occurrence of objective clinical response at D57, M3, M6, M9, M18 and M24 after first NKR-2 administration and according to international guidelines and (iii) progression-free survival (PFS) and overall survival (OS), and duration of response for patients with objective response up to 2 years after first NKR-2 administration.The primary endpoint of the expansion segment will be the objective response rate (ORR) at D57, M3, M6, M9, M18 and M24 after first NKR-2 administration. Secondary endpoints will include the (i) PFS and OS, (ii) duration of response for patients with objective response up to 2 years after first NKR-2 administration and (iii) occurrence of AEs and SAEs and DLTs during the study treatment.The The overall aim of this segment is to understand the safety profile and develop initial observations of clinical activity in a cohort of up to 14 patients that support a deeper assessment of clinical efficacy of NKR2 in larger patient cohorts within a phase II clinical study.Response to treatment will be assessed according to standard Response Evaluation Criteria In Solid Tumors (RECIST) criteria (version 1.1) for solid tumours or international recommendations for response criteria in haematological tumours. Apart from the visits scheduled per protocol, tumour assessment might also be performed at any time if considered as needed per investigator’s decision  and with regard to the RECIST 1.1. response confirmation.For each patient, the overall study duration will be 15 years. The study consists of a screening phase, a treatment administration phase, a treatment follow-up phase and a long-term safety follow-up . The durlong-term safety follow-up that will last for up to 15 years after the day of the first NKR-2 administration to collect safety data for 13 additional years. The surveillance will mimic as much as possible the standard follow-up of the respective tumour type, including monitoring of important safety parameters.As per regulatory requirements, patients who received at least one NKR-2 treatment will need to be followed annually for a period of 15 years. Therefore, patients will also follow a Patients may be withdrawn from the study treatment or from the study at any time at the patients’ discretion or at their investigator’s discretion. The investigator will document on the patient’s record whether the decision to discontinue treatment and/or study participation was made by the patient or the investigator and which reason applies.basis, that is, in case of progression/relapse suspicion. In case of disease progression at M24 or at any time before, the patient will be still asked to attend simplified follow-up procedures, which will include (i) record of survival status, (ii) medication status and (iii) blood sampling for viral copy number (VCN) and replication competent retrovirus (RCR) assessment. Clinical progression at the end of the treatment phase (D57) will not to be considered as a criterion for withdrawal from the study and will allow the continuation of the active follow-up of the patient with or without the initiation of a new anticancer therapy.In case of absence of disease progression at M24, PFS and OS as clinical endpoints will be followed up on a yearly basis or at ad hoc dose escalation phase, but each dose level should enrol at least two different types of cancer. In each arm, patients will be enrolled at the next dose level when all evaluable patients at the same dose level have completed the safety period of 14 days after last NKR-2 injection. Within each dose level, patients will be enrolled as soon as the previous patient has received at least one NKR-2 administration and has completed the 14-day safety period after the first injection.There is no requirement for specific recruitment of each individual tumour indication to corresponding arm within the expansion segment of the study will be performed in three steps, with a statistical analysis at the end of each step . Within each step, all patients can theoretically be enrolled at the same time.Enrolment in the Approximately 15 centres are expected to be initiated for patient enrolment in the expansion segment. Recruitment is anticipated to be completed within approximately 24 months. If this target is not met, then the recruitment period may be extended accordingly.All AEs starting from registration to 28 days after the last study treatment (Visit D57) must be recorded into the Adverse Event form in the patient’s electronic case report form (eCRF), irrespective of intensity or causality with study treatment.To ensure patient safety, every SAE, regardless of suspected causality, occurring after the patient enrolment and until the administration phase concluding visit (at D57) must be reported to the sponsor within 24 hours of learning of its occurrence. Any SAEs experienced outside this period will only be reported to the sponsor if the investigator suspects a causal relationship to the study participation or if fatal SAEs occur after signature of the Informed Consent Form (ICF).DLT refers to a toxicity that is experienced during treatment and within 14 days following any NKR-2 dose, is new and at least possibly related to study treatment. This includes any Grade 3 or higher toxicity or Grade 2 or higher autoimmune toxicity that cannot be controlled to Grade 1 or less within 72 hours with appropriate treatment, with the exception of Fever Grade 3 and immediate hypersensitivity reactions occurring within 2 hours of cell infusion that are reversible to a Grade 2 or less within 24 hours of cell administration with standard therapy.Whenever a patient experiences toxicity that fulfils the criteria for a DLT, the study treatment will be interrupted. The standard time period for collecting and recording DLTs will be 14 days after each study treatment administration. AEs/SAEs that meet the definition of DLTs but that are reported outside of this time window will be reported as AEs/SAEs but not as DLTs.The standard time period for collecting and recording pregnancy will begin at the first receipt of the product and last until the end of the study.In addition to the above-mentioned reporting requirements and in order to fulfil international reporting obligations, SAEs that are related to study participation  will be collected and recorded from the time the patient consents to participate in the study until she/he is discharged.All statistical analyses will be performed using SAS (SAS Institute) and/or StatXact (Cytel Software Corp) softwares. The results will be reported per dose level/tumour-type cohort and overall.total treated population.Physical examination , medical history and baseline tumour characteristics will be tabulated and analysed with appropriate descriptive statistics on the analysis of safety will be performed on the total treated population. All the AEs will be graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE), Version 4.03 and will be coded to the preferred term level using the Medical Dictionary for Regulatory Activities (MedDRA). A summary of AEs by maximum grade and cohort will be generated. The same analysis will be presented for SAEs and events with a suspected relationship to the study treatment. A summary of AEs by maximum grade and cohort per treatment dose will also be performed. SAEs, DLTs and discontinuation due to AEss will be described in detail by patient narratives.The A summary of the concomitant medications, classified by Anatomical Therapeutic Chemical (ATC) term, will be displayed by cohort. Individual listing of concomitant medications per patient and per time point will also be provided. Finally, the total number of study treatments administered to each patient and the length of active follow-up will be summarised.analysis of clinical activity will be performed on the total treated population, that is, all patients who received at least one NKR-2 treatment . Sensitivity analyses related to the clinical activity endpoints will be performed on the according to protocol population which includes all evaluable patients who have complied with all the procedures defined in the protocol up to concluding visit.The The first ORR will be assessed at Visit D57. The occurrence and duration of objective clinical response Complete response (CR), partial response (PR) and stable disease (SD) will be reported. Disease progression, including death, during the study will be described. Estimates of the rates of best clinical response and their 95% CIs will be reported. Kaplan-Meier curves will be estimated for PFS and OS.that is, the clinical activity may be assessed in subpopulations based on the NKG2D ligands tumour expression, types of ligands expressed in tumour, serum levels of soluble NKG2D ligands and so on.In addition, other exploratory analyses may be performed, In the dose escalation segment, safety data will be monitored closely on an ongoing basis. The main analysis (leading to the selection of the RecD) will be conducted separately in each arm once all patients in the last dose level have received the three NKR-2 administrations and have completed the safety period (D43). All data related to the primary endpoints of the study will be analysed. The final analysis will be performed once all patients have attended the concluding visit of the follow-up phase (M24) or have been withdrawn from the study.The expansion segment of the study will proceed in three steps, with a statistical analysis at the end of each step . These sAn interim analysis for futility will be performed on the first 14 patients over all 5 cohorts of patients with solid tumour types and on the first 6 patients over the 2 cohorts of patients with haematological tumour types who have either completed the administration phase (D57) or have withdrawn from the study.The second step consists in cohort futility analyses after the first seven cancer patients in each tumour type have either completed the administration phase (D57) or have withdrawn from the study. At this step, an evaluation of the safety of the NKR-2 treatment in each cohort will also be performed on the first six patients who completed the 6-week treatment period (D43) or had a DLT. This will allow to adapt the RecD in any specific cohort (tumour type) according to both safety rules and futility analysis. Indeed, if, and as soon as, two or more DLTs have been reported within a specific cohort, and the criterion for futility was not reached, the next patients enrolled in this cohort will be treated at the next lower dose level. In contrast, if no DLT has been reported within a specific cohort, and the criterion for futility was not reached at Step 2, the next patients enrolled will be treated with the next higher dose level.Finally, the final evaluation of the objective clinical response rate will be evaluated once all the first 14 cancer patients in each cohort have either completed their administration period (D57) or have withdrawn from study due to disease progression or DLT. If, in any cohort, there are >2 out of 14 patients presenting an ORR, the study will be considered as successful in this tumour type.Peripheral blood samples, bone marrow aspirates from patients with haematological malignancies, as well as tumour biopsies from patients with solid malignancies, will be collected at regular time points during the study see  and usedAs previously mentioned, the use of CAR T cells is limited by potentially severe toxicities, including CRS, neurotoxicity and tumour lysis syndrome (TLS), on-target off-tumour toxicity , clonality and insertional oncogenesis, vector persistence and immunogenicity. While these events may have been seen in relation with other CAR T cell therapies, and are provided for information, there are currently no events considered as expected for NKR-2 therapy (which can be different from other CAR T cell studies based on the unique mode of action) and all serious adverse reactions (SARs) will be considered as suspected unexpected SARs (SUSARs).The management of toxicities related to CAR T cell therapies was described elsewhereet alBriefly, CRS toxicity is currently of highest concern in the field. It is caused by a release of inflammatory cytokines such as interleukin (IL)-6, IL-2, Interferon (IFN)-γ and Tumour Necrosis Factor (TNF)-α, following the recognition of the target antigen, resulting in a systemic inflammatory response similar to sepsis. Therefore, CRS will correlate with both efficacy and toxicity in patients receiving T cell-engaging therapies. Tocilizumab (anti-IL-6) will be used as the first-line agent in the treatment of CRS. If the patient’s condition does not improve or stabilise within 24 hours of the tocilizumab dose, administration of a second dose of tocilizumab and/or a second immunosuppressive agent (such as corticosteroids) will be considered, based on the CRS revised grading system created by Lee TLS resulting in renal insufficiency, rapidly rising uric acid level or evidence of organ dysfunction will be managed with fluids and rasburicase as needed and as determined by the treating physicians.Further, as NKG2D ligands may be upregulated on stress signals, expression of ligands on non-cancer cells can be expected in tissues that are actively infected or inflamed or experienced cell stress of other aetiology. Infections could therefore invoke a significant inflammatory response by NKR-2 by potentially inducing a CRS due to the increased recognition of ligands by NKR-2 cells. Therefore, any inflammation, infection, concurrent or previous treatment with anticancer therapies will be carefully registered and any Grade 2 or higher autoimmune toxicity will be closely followed to detect any evidence of on-target off-tumour toxicity. Evidence of acute infection, as well as fever of 38°C/100 °F or above, will be therefore considered as an exclusion criterion at screening see  or postpSince NKR-2 construct consists entirely of human sequences, it is not expected to result in immunogenicity sometimes observed with other CARs using scFv from murine origin (see section Introduction), and therefore multiple NKR-2 injections are allowed without the anticipated risk of humoural immune responses.Uncontrolled expansion and proliferation of the transformed cells increase the risks for onset of CRS and other CAR-associated toxicities. However, in contrast to more classical CAR T cell therapies, NKR-2 demonstrated neither significant in vivo expansion nor long-term persistence until now,Finally, following FDA recommendation for retroviral and lentiviral vector based gene therapy products, the patient monitoring schedule will also include analysis of the RCR of patient samples up to 15 years after CAR T cell injections.The THINK trial aims primarily to determine the safety and clinical activity of NKR-2 cell therapy, without preconditioning chemotherapy, in patients with advanced cancer. The dose escalation phase of the trial is designed to determine the RecD in haematological and solid tumours, which then is used in the expansion phase to assess clinical responses separately in seven different indications. Due to the multitargeting specificity of NKR-2, the dose escalation and subsequent expansion segments are designed to facilitate a rapid assessment of NKR-2 activity across a range of tumours. This is advantageous since the assessment of safety and any evidence of clinical activity will be suitably evaluated across cancer types using a consistently produced cell product, thereby avoiding issues that arise in multiple single tumour indication trials testing a single T cell product. Interestingly, the number of ligands targeted by NKR-2 (eight ligands from two main families), combined with their stress-induced tissue expression, also limits the potential risk of clonal selection or antigen loss after injection, which has been observed for classical CAR constructs targeting a single antigen. This is expected to translate into limiting the risk for potential relapse.For patients, the lack of preconditioning chemotherapy removes the need for an inpatient stay during the transient period of lymphodepletion translating to a likely improved patient experience of the trial. Additionally, the absence of preconditioning will also reduce the potential toxicity of this treatment and facilitate providing a therapeutic solution to a larger patient population, including, notably, the elderly population.The challenge facing classical CAR T cell therapies in the human patient with advanced cancer  is major and, in the solid tumour situation, requires the cells to traffic to sites of tumour, overcome the highly suppressive TME and initiate direct tumour cell killing to drive a sustained antitumour response.The multiple infusion strategy, absence of preconditioning chemotherapy, diversity of putative mechanisms of action, as well as the diversity of tumour types that can potentially be targeted, therefore stand the NKR-2 CAR T cell approach described in this protocol apart from the current ‘gold standard’ CAR T cell therapy paradigm. In this context, the THINK trial provides a first approach of a global comprehensive clinical programme embedding other phase I clinical protocols that will evaluate the combination of the NKR-2 with specific anticancer treatments and the locoregional administration, among others.The investigator is responsible for ensuring the study is conducted in accordance with procedures and evaluations described in the clinical protocol. Deviations from the protocol shall not be made without prior approval by the sponsor, except to treat a medical emergency and reduce immediate risk to the patient. In such a case, the investigator shall immediately notify the independent ethics committee (IEC) or institutional review board and the sponsor, according to local requirements.If changes to the protocol are deemed necessary, a written amendment should be prepared and signed by the investigator and the sponsor and approved by the IEC and applicable competent authorities prior to being implemented.steering committee will be composed of three members independent of investigators and the sponsor. The role of the steering committee will be to advise on events which may impact the general process of the clinical study. All safety data will be made available to the steering committee in a way that the steering committee can timely advise.The safety data review meetings (or teleconferences) on a regular basis during the study to discuss and evaluate all of the gathered safety data. This safety follow-up will include teleconference at the end of each dose level of the phase I dose escalation arms and at any new relevant safety event onset . In addition, members of the steering committee will be requested to review the clinical study report(s) generated from data collected in this study.Celyad or designee will host specific Clinical data review meetings will be performed at the different study interim analyses for futility by the Celyad clinical trial team and the steering committee in specific. At the time of the interim analyses, the best overall response for each patient will be derived from the overall lesion response assessments recorded in the eCRF. This will be used to calculate the ORR in the respective group(s).For the purpose of compliance with Good Clinical Practises (GCP) and competent authorities guidelines, it may be necessary for the sponsor or a competent authority to conduct a site audit. This may occur at any time from start to after conclusion of the study. Therefore, when an investigator signs the protocol, he agrees to permit drug competent authorities and the sponsor audits, providing direct access to source data/documents.informed consent will be taken from all participants. The patient will be asked to read and review the document. On reviewing the document, the investigator (or delegate) will explain the research study to the patient and answer any questions that may arise. The patients consent should be appropriately recorded by means of the patients personally dated signature prior to any procedures being done specifically for the study. The patients should have the opportunity to discuss the study with their surrogates or think about it prior to agreeing to participate. The patient’s participation is voluntary and the patient may withdraw consent at any time throughout the course of the study.Written Patient confidentiality is strictly held in trust by the participating investigators, their staff, and the sponsor(s) and their agents. This confidentiality is extended to cover testing of biological samples and genetic tests in addition to the clinical information relating to participating patients.The study protocol, documentation, data and all other information generated will be held in strict confidence. No information concerning the study or the data will be released to any unauthorised third party, without prior written approval of the sponsor.study data. Final data set will be stored in a central database maintained by the sponsor. At the conclusion of the study, the sponsor will archive the study data in accordance with internal procedures. All information provided by the sponsor and all data and information generated by the site as part of the study  remain the sole property of the sponsor.The investigator remains accountable for the The results of this study will be disseminated through presentation at international scientific conferences and reported in peer-reviewed scientific journals.insurance policy covering damages arising out of Celyad’s sponsored clinical studies. Financial compensation to investigators and/or institutions will be in accordance with the agreement established between the investigator and/or institutions and the sponsor.The sponsor holds and will maintain an adequate The THINK trial  is currently recruiting participants in Europe and USA.


In evidence-based weight-loss programs weight regain is common after an initial weight reduction. Eating slowly significantly lowers meal energy intake and hunger ratings. Despite this knowledge, obese individuals do not implement this behaviour. We, thus tested the hypothesis of changing eating behaviour with an intra-oral medical device leading to constant weight reduction in overweight and obesity.2) with increased CVRF profile were included in this prospective study. All patients had been treated for obesity during the last 10 years in a single centre and had at least 3 frustrate evidence-based diets. Patients received a novel non-invasive intra-oral medical device to slow eating time. Further advice included not to count calories, to avoid any other form of diet, to take their time with their meals, and to eat whatever they liked.Six obese patients  of their initial weight. At 12 months, all patients had lost >5%, and 67% (4/6) achieved a >10% bodyweight loss. In the course of the study, altered eating patterns were observed. There were no complications with the medical device. Of note, all patients continued to lose weight after the initial intervention period (p<0.001) and none of them had weight regain.With this medical device, overweight and obese patients with a history of previously frustrating attempts to lose weight achieved a significant and sustained weight loss over two years. These results warrant the ongoing prospective randomised controlled trial to prove concept and mechanism of action.Trial registration: German Clinical Trials Register DRKS00011357 Substantial and sustained increase of total energy intake worldwide has led to increased body weight across the global population, with an estimated 1.5 billion overweight individuals  3], res, res3], Consequently, rectifying this imbalance by caloric restriction and increased physical activity form the basis in the management of evidence-based interventions of obesity  9]. How. How9]. Obesity can be regarded as a neurobiological disease with a psychological element. The mechanisms underlying flawed eating behaviour are complex. Several physiologic adaptations after voluntary weight loss favour weight regain. These involve appetite-related hormones , the comWith the acceleration of obesity rates and few effective obesity treatment options, new strategies addressing changes of eating behaviour are urgently needed. Successfully maintaining weight loss requires a permanent change of behaviour.We thus tested that hypothesis that a relatively short period of using an intraoral device to change eating behaviour in overweight and obesity could lead to a constant and long-lasting weight reduction.This was a prospective investigator-initiated, open-label therapy trial to test an innovative oral medical device designed to reduce chewing area . The study was conducted in a single centre in a specialist practice from July 2012 to January 2015. Recruitment of patients was from July 2012 to August 2014 Patients with clinical indication for weight reduction were prospectively recruited to test the intraoral device. Sponsor of this study were the physician and patent holder and the collaborating orthodontist. The physician conducted the study together with assisting personnel. He took no part in analysis or preparation of the manuscript. All study participants gave written informed consent. This study in accordance with the Helsinki convention and was approved by the locals Ethics Committee (FF 1/2015) Landesärztekammer Hessen . The study has been registered in the German Clinical Trials Register on 8.12.2016 and has been assigned the number DRKS00011357. The study was initiated and carried out by a senior general practitioner and his befriended patients to prove the concept. They were not aware of the requirement to register.2 with other risk factors  or BMI over 30.0 kg/m2; at least 2 previous evidence-based weight management programmes with consecutive weight regain in the last years (≥ 1kg / year); will to reduce weight; age 18–75 years; increased cardiovascular risk profile to develop cardiovascular disease by considering age >65 years, male sex, hypertension, diabetes, and smoking for coronary artery disease as single cardiovascular risk factors ; dentalPatients who fulfilled the above-mentioned inclusion and exclusion criteria were sent to the orthodontist (consultant to the project) for baseline testing and preparation of dental impressions using a purpose-designed kit . The devParticipants attended follow-up appointments in the practice at weeks 2, 4, 6, 12, and at months 6, 9, 12, 15, 18, 24, 30 and 36. Weight, compliance, adverse events (AEs), and qualitative feedback were obtained during each consultation. For AEs, a standardised questioning at each contact was performed.The novel intraoral device has been designed to slow the eating process, with the intent of prolonging the chewing process and delaying the swallowing of a single bite in order to improve the function of physiological satiation mechanisms. Additionally, the duration of the single bite in the mouth should be extended to intensify the tasting of the food. Thereby the device aims to promote mindful eating and a long-lasting change in eating behaviour. for the upper and lower jaw . On top 2) calculated. Supine blood pressure was measured in the left arm. Venous blood samples were taken at baseline and after 3 and 6 months, as clinically indicated, to assess plasma glucose, insulin, haemoglobin A1c (HbA1c), total, high-density lipoprotein and low-density lipoprotein cholesterol and triglycerides. These samples were taken after an overnight fast. Concomitant medication was assessed at baseline and changes monitored throughout the study.Weight was measured at baseline and each follow-up (see above), using the same digital scales. Patients’ historical weight was retrieved from the individual records. At baseline and last follow-up, height, weight and waist circumference were quantified and the body mass index  . A questCompliance throughout Phase I of the study period was defined as reported by participants. Good compliance as use of a minimum use of 3 meals/day, 6 days/week; moderate compliance as use of 15 to 18 meals/week; poor compliance as use of 9 to 14 meals/week; less than 9 meals/week was defined as no compliance. These values were averaged to obtain a “mean uses per week” across the Phase I of the study for each participant. Additionally, length of use in weeks and cumulative duration of use in hours were recorded.At each visit, participants were asked to report any adverse events; these were recorded, and where necessary, follow-up action was taken by the medical officer or dentist.In their first week, the last three participants included in the observation volunteered to participate in an additional acute eating experiment with the device. For this, they were asked to eat an identical ad-libidum meal (lunch) on two following days at home without the device (baseline) and on the following day with the device. All volunteers were instructed to eat a light breakfast on the test days. They all reported to have followed the instructions.Appetites were measured with a German translation of the Visual Analog Scale (VAS)  and addi2 test and the Fisher’s exact test. Primary outcomes (weight loss in Phase I and Phase II) were assessed on an intention-to-treat basis. A total of 6 patients will enter this study. The probability is 80 percent that the study will detect a relationship between the independent and the dependent variables at a one-sided 0.05 significance level, if the true change in the dependent variables is 2.484 units per one standard deviation change in the independent variable in a test to find an association if the dependent variable is affected by the treatment. This is based on the assumption that the standard deviation of the dependent variable is 2.Data are expressed as mean ± standard deviation (SD). All continuous variables were assumed not to be normally distributed due to the small number of patients included and compared with the Mann-Whitney U test. Comparison of categorical variables was generated by the Pearson χStatistical significance was assumed if a null hypothesis could be rejected at p≤0.05. All statistical analysis was performed with SPSS 21.0.From among the practice, 12 patients were identified as meeting eligibility criteria and offered to test the novel medical device. Of these, 9 consented to participate. Of these, 1 patient had dental exclusion, 1 lost his device on a longer trip abroad (week 2) and 1 patient felt uncomfortable with the device (week 1). Six patients participated in and completed the study and were used for analysis . None ofAll patients significantly lost weight during the initial Phase I  weeks. The wide range is explained by a self-reported significant weight loss due to a change in eating behaviour in 2 patients, who were than allowed to eat without the device. Four patients were defined as fully compliant, 1 as moderately and 1 as poor compliant according to the practitioners assisting personnel (2 independent judgements). Cumulative use of the device for the individual patient was 20 to 160 (100 ± 57) hours.There were no adverse events with the device reported. All patients reported uncomfortable foreign body sensation with the device that subsided after 1 week (n = 2), 2 weeks (n = 3), or after 4 weeks (n = 1). Further discomfort was reported by 2 patients, related to mild gingivitis that required re-adjustment by the orthodontist (n = 1), intermediate difficulties with biting and chewing some food  and attracting another person’s attention (n = 1) during eating and were reported to be “rare” or “very rare”. Apart from these events related to the device, no other incidences occurred. In general, patients reported that handling of the device for insertion and removal was “very good” or “good” in 4 cases and to be “acceptable” in 2 cases. Three patients would recommend the device “unreservedly” and 3 “more yes” .As shown in Changes in food intake were considerable and similar in all three volunteers between eating without (baseline) or with the device, as shown in The findings of this study support the hypothesis that this medical device is the first non-invasive medical device to reduce weight in overweight and obesity effectively and enduringly. Our data indicate that this weight loss is a result of a sustainable change in eating behaviour. What’s more, the approach of this therapy strongly contrasts with contemporary weight loss strategies in that all patients were instructed to eat whatever they liked.Treatment of obesity includes food consumption because this is the primary source of caloric intake and manipulating eating behaviour thus is a primary target. Commonly, weight loss strategies to fight individual and populations overweight and obesity include behaviour modification, pharmacotherapy, meal replacements, and more drastically, gastric bypass surgery, or gastric balloon. Despite proven short- and medium-term weight reduction of these evidence-based programs, rapid weight gain is a serious health problem  for the All patients with increased cardiovascular risk profile who were included in our study had increases in weight despite previous evidence-based strategies to reduce weight over the last years. Participants all lost weight in the first phase, when they used the device for their meals and reported to have changed both the size of their meals as well as the choice of food consumed during this first acute phase of only a few weeks. To verify this finding, an acute experiment was conducted in a subgroup of patients, where patients showed prolonged eating duration, increased awareness of food composition and feelings of satiety, although they had eaten smaller meals when eating with the device. Satisfaction and compliance with the medical device in our study was acceptable after initial discomfort and no adverse events occurred.Thus, this device is different from other medical intra-oral devices that attempt to prevent an individual from eating but do not change eating behaviour.There is a strict relationship between bite size and food intake  as well The change of eating behaviour translated into sustained weight loss in the consecutive months in the second phase during long-term follow-up, where the patients continuously lost weight without using the device. During long-term follow-up, all of the patients in our study significantly lost weight in a range defined as effective program .Complex neurophysiological changes during cognitive control and unusual eating patterns of individuals have been identified as strong inducers of weight regain. Our approach to help overweight and obese patients to lose weight thus used this knowledge to change their behaviour and allow them to eat what they pleased. Our patients were not simply forced to eat slowly, but given the opportunity to savour  and induThis study has several limitations, such as the small number of 6 patients, the retrospective design of long-term follow-up with limited phenotypic data at inclusion and the long recruitment period as well as the lack of a control arm. We cannot exclude the possibility that patients have been included in this study who previously failed to reduce weight because of cravings caused by the sensory deprivation of monotonous diets.Thus, a more powerful study is now conducted with a large number of patients in a prospective, randomised, controlled multicentre trial. This is to prove the concept and the underlying mechanism. Since the majority of patients will regain weight after conventional dieting, our approach may prove a serious alternative treatment for obesity in obese and overweight patients for whom evidence-based options have failed.S1 File(TIF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(TIF)Click here for additional data file.S1 Protocol(DOCX)Click here for additional data file.