Traumatic Brain Injury (TBI) is an alteration of brain function caused by an external force. Long-term mortality in TBI is substantial, TBI survivors can develop chronic progressive disabilities and have a life expectancy shortened by 6 years. Treatment consists in supportive therapy directed at prevention of second insults, but no neuroprotective therapy is available. Given the multifaceted nature of TBI, mesenchymal stromal cells (MSCs) are an ideal candidate: they release multiple soluble factors shown to ameliorate the injury microenvironment through immunomodulatory, protective, reparative and regenerative processes. Preclinical data across a range of different TBI models and injury severities show that human MSCs improve outcome through pleiotropic mechanisms of protection and repair. Thus, data indicate MSCs as strong therapeutic candidate and support a clinical study in TBI.

Aim: the study is designed to assess the safety and the efficacy of the MSCs, intravenously administered in severe TBI patients within 48h from injury. The study will be conducted in a stepwise manner. Step 1 will enroll 36 patients (randomized 1:1:1 in arms 80 x 10^6 MSCs vs 160 x 10^6 MSCs vs placebo) to define safety, and will allow to select the most promising dose. Step 2 will enroll 30 patients (1:1 in arms MSCs selected dose vs placebo) to define the MSC activity based on the quantification of the plasmatic levels of the neurofilament light (NFL) at 14 days, as biomarker of neuronal damage.

Secondary objectives are aimed to assess:

1. brain injury evolution and white matter damage by longitudinal neuroimaging (at 4 days and 14 days post-TBI and at 6 months)
2. brain immunomodulatory changes by temporal profiling of circulating biomarkers of brain damage and neuroinflammation (daily for 3 days after TBI, at day 7 and 14, and at 1, 6 and 12 months)
3. clinical outcome by a structured clinical and neuropsychological assessment at both 6 and 12 months

Methods: a multicenter, double blind, randomized, placebo-controlled, adaptive phase II dose finding study.

Duration of the study: 36 months (24 of enrolment and 12 of follow up).

Funding: Fondazione Regionale per la ricerca Biomedica, FRRB (Call "Unmet medical needs", proposal number 3440227) and Italian Ministry of health (Ministero della Salute, Bando di Ricerca Finalizzata 2021; proposal number RF-2021-12372642).

Study Description: Multicenter, double blind, randomized, placebo-controlled, adaptive phase II dose finding study meant to define if MSCs, administered at dosage of 80 or 160 x 10^6 cells within 48h from TBI, are safe in patients with severe TBI, and to define if MSCs, administered at the dosage found to be safe and more promising, decrease the plasmatic neurofilament light (NFL) biomarker of brain damage at 14 days. Patients will be recruited at the Neurointensive Care Unit at Fondazione IRCCS San Gerardo dei Tintori, Monza, at ASST Ospedale Papa Giovanni XXIII Bergamo and at Fondazione IRCCS Ca Granda Ospedale Maggiore Policlinico of Milano. Collection of multiple clinical, neuroimaging and biological parameters will describe TBI evolution.

Preclinical studies: Studies in rodent TBI models have shown that administration of MSCs produces functional improvement with amelioration of sensorimotor and cognitive deficit, reduction of contusion volume and neuronal loss, modulation of the inflammatory response with decreased inflammation, stimulation of beneficial endogenous mechanisms (angiogenesis, neurogenesis, synaptic plasticity). These results support the notion that MSCs can reprogram the microenvironment mitigating the progression of brain damage and fostering recovery of function. Only few clinical studies have assessed the safety, feasibility and efficacy of MSC therapy. No adverse events (AEs) or severe adverse events (SAEs) were reported. No AEs were associated to the intravenous route, which is the one the investigators propose for the study.

Target Sample Size:The total number of evaluable patients to be analyzed will be 66 (27 on the control arm, 12 in the experimental arm stopping at first step and 27 in the experimental arm reaching the second step). In details, the total number of evaluable patients to be analyzed will be 24 (12 patients in each experimental arm) for the safety interim analysis and 54 for the final efficacy analysis (27 patients in both the control arm and in the experimental arm reaching the second step).

Expecting a 13% of deaths of TBI patients in ICU, the number of patients to be randomized is about 78 (32 in the control arm, 14 in the experimental arm stopping at first step and 32 in the experimental arm reaching the second step). In case of the trial closure after the first step, the number of patients to randomize will be about 42 (14 in each arm).

Statistical design and sample size:The statistical study design is conceived in 2 steps.

Step 1: An interim safety analysis will be performed to evaluate separately in each of the two experimental dosage groups, at one-sided type I error rate of 10%, whether more than 30% of patients experience at least one serious adverse drug reaction (SADR) within 14 days from treatment, and at the same time at a power of 80% whether 5% or less of the patients do not experience any SADR. Adopting the Fleming design with A'Hern's approach, 12 evaluable patients will be analyzed in each group (36 patients overall). The maximum number of patients experiencing at least one SADR to observe in each experimental group is 1 out of 12, since this result is associated with an upper limit of the 80% exact confidence interval of 28.8%.

The experimental treatment will be considered as safe for this step only if none or one patient experiment a SADR.

According to the above role:

• if both experimental treatments are considered not safe, the study will be stopped for safety issues and the step 2 of the study will not be performed
• if one experimental treatment is considered safe and the other not safe, only the safe arm will proceed with the step 2 of the study
• if both the experimental dosages are safe, then the more active schedule will be selected for the step 2 The more active schedule will be defined in terms of proportion of patients who reaches a NFL increase at 14 days equal or lower than baseline level by 5 folds (defined as "responder patients").

This cut-off has been defined based on longitudinal quantification kindly provided by BIO-AX-TBI collaborators, showing a median increase of 12.8-fold at 14 days compared to baseline, with first quartile equal to 5-fold increase (see paragraph 5.4 for details). In case of an equal number of responder patients in the two experimental arms, the cut-off defining the response will be increased by 1 unit iteratively since a difference between the two experimental arms will be observed. This assessment will only be performed on the MSC treated arms; the control group will not be considered. According to Simon, Wittes and Ellemberg randomized phase II design, the study has more than 80% probability of correctly select the more active schedule when the proportion of responder patients is 15% higher in the first of the two schedules of treatment.

Step 2: the primary endpoint will be the NFL at 14 days. The final efficacy analysis will be performed by increasing the sample size to 27 evaluable patients in the experimental and control arms. The study is designed to detect an effect size of 0.59 on logarithmic scale of the experimental to control arm, setting a one-sided type I error rate of 10% and a power of 80%.

Data will be analysed by the study biostatistician using the software SAS 9.4 release. A full Statistical Analysis Plan will be written prior to statistical analysis.

Deceased or lost to follow-up patients and patients with missing values will contribute to the analysis of the secondary outcomes only for the time during which data are available.

All patients who have received the treatment will be included in the safety analysis. Each patient will be analyzed in the arm actually received.

The assessment of safety will be mainly based on adverse reactions (ARs) and the frequency and nature of the serious adverse events (SAEs) and will be conducted on the safety analysis set.

Expected results: the investigators expect to prove the safety and efficacy of MSC intravenous administration in acute and severe TBI patients. The investigators will provide a detailed longitudinal description of the effects of MSCs on brain advanced neuroimaging trajectories, on axonal damage and neuro-immunomodulatory changes by blood biomarker analysis and on clinical outcome.

MSC preparation: The preparation of MSCs will be performed as detailed in the IMP brochure according to Good Manufacturing Procedures (GMP) guidelines at Laboratorio Stefano Verri, ASST MONZA. Laboratorio Stefano Verri has standard operating procedures in place for storage, release, thawing, preparing and administering the MSCs.

MSCs are stored in vials containing 40 X 10^6 MSCs each in 4.5 ml of storage solution (2.5 ml albumin 20%, 1.1 ml normal saline solution, 0.45 ml citrate-dextrose solution (ACD), 0.45 ml Dimethyl sulfoxide (DMSO)). Vials containing 4.5 ml of storage solution only will be prepared and stored in the same manner. The MSC injections and the placebo injections will have identical appearance and consistency. Vials are stored in the vapor phase of liquid nitrogen until the time of administration.

The study treatment will be prepared by qualified personnel under a class A laminar flow hood in proximity to the ICU.

IMP will be administered acutely (within 48h after injury) within 15 minutes from the preparation through a central venous line.

Random allocation procedure: Patients will be randomly assigned to one of the three treatment groups (allocation ratio 1:1:1 for the first step and 1:1 for the second step). The study biostatistician will prepare the sequence of treatments according to a randomized permuted blocks procedure. The randomization schedule will be generated using SAS 9.4 release. The randomized allocation of treatment will be centralized.

Upon recruitment of a patient meeting the inclusion criteria, the local investigator will follow a randomization list (provided by the factory member's personnel) (detailed in the Manual of Procedures) to assign the treatment to the patient in a blinded fashion manner.

Blinding: The subjects, the research coordinators and investigators, and the personnel involved in outcomes assessments and monitoring compliance will all be blinded. Treatment assignment will be known only to the Sponsor Data Management Team and the Cell Factory of each center. An unblinded physician will be designated to monitor safety results.

Patient withdrawal: Patients may withdraw (or be withdrawn) from the study at any time. Withdrawals within 14 days will be replaced.

Reasons for patient withdrawal include:

• Adverse event that in the judgement of the Investigator requires study withdrawal
• Patient wishes to withdraw from the study
• Patient is lost to follow-up Benefit/Risk assessment: The use of MSC therapy for TBI patients is experimental and may not result in any direct benefit to the patient. Nevertheless, proposers strongly believe that the benefits awaited from this MSC-based therapeutic strategy is an amelioration of the evolution of TBI pathology with a consequent better outcome with the reduction of the long-term disabilities. MSCs are a particularly promising cell therapy because of their availability, immunologic properties and track record of safety and efficacy.

The risks associated with the intravenous administration procedure include bleeding, swelling and minor pain on injection. These are self-limiting and do not pose any long-term problem.

During the first few hours from the MSC intravenous infusion: headache, fever, rush and allergic reaction may appear. The risk of allergic reaction is mitigated by the use of human platelet lysate instead of the fetal calf serum during the manufacturing process. This also allows to avoid the possible transmission of still unknown zoonosis.

There is also a remote infectious risk that can be treated with appropriate antibiotics. The infection may conduct pain, discomfort and tissue damage.

A possible risk of intravenous MSC infusion is represented by thromboembolic events. MSC infusion could increase pulmonary vascular resistance in the presence of injured pulmonary microcirculation, potentially by aggregating or clumping in capillaries or small arterioles. Subsequent potential risks are acute right heart failure, impaired cardiac output and hemodynamic instability. Additionally, infusion of MSCs could potentially worsen ventilation-perfusion mismatch and result in further impairment of oxygenation or carbon dioxide excretion.

Collection of Data: All data obtained during this clinical trial will be captured electronically in a project specific programmed Electronic Data Capture (EDC) application.

For each subject, all data and trial-related visits will be recorded in the eCRF (electronic Case Report Form). If a subject withdraws from the trial, the reason must be stated in the eCRF.

Informed consent: It is the responsibility of the investigator/delegate to obtain informed consent according to ICH-GCP and Declaration of Helsinki guidelines and local regulations from each individual subject participating in this study and/or his/her legally designated representative/proxy. If the subject is not able to provide personal consent at the time the consent is obtained, then he/she must provide or withdraw this consent as soon as possible once his/her clinical condition has improved to the extent that providing personal consent is possible, unless local regulations state otherwise.

The informed consent form (ICF) will be provided in the country's local language(s).

Regulatory and Ethical Compliance: The trial will be performed in accordance with the Declaration of Helsinki and the Good Clinical Practice. The trial obtained the Regulatory Agency (AIFA) approval. The protocol and trial conduct will comply with the Medicines for Human Use (Clinical Trials) Regulations 2004 and any relevant amendments. Development Safety Update Reports will be submitted to the IEC and Regulatory Agency.

Inclusion Criteria:

• Age: 18-70 years
• Clinical frailty index (CFI) < 5
• Evidence of TBI confirmed by abnormalities consistent with trauma on CT scan upon admission (Marshall's CT Classification >1)
• Feasibility of study drug (MSC/placebo) administration within 48 hours from TBI
• GCS ≤ 8 at recruitment and at least one pupil reactive to light
• ICP monitoring already inserted or planned for clinical indications
• Weight < 100 Kg and > 40 kg

Exclusion Criteria:

• Motor GCS > 5 at recruitment

• High likelihood (> 85%) of death in the first 48 h calculated by IMPACT calculator on early admission data

• Bilateral mydriasis

• Opening ICP > 40 mmHg

• Known history of prior brain injury, psychiatric disorder, neurological impairment and/or deficit

• Brain penetrating injury

• Spinal cord injury

• Previous epilepsy requiring anti-convulsant therapy

• Severe organ failure (including PaO2/FiO2<200 and shock)

• Recent serious infectious process

• Cancer

• Immunosuppression

• Human immunodeficiency virus

• Positive urine pregnancy test or nursing

• Known risk/history of coagulopathy and thromboembolism

• Pre-existing and severe:

  • lung disease (such as asthma, chronic obstructive pulmonary disease),
  • heart dysfunction (as heart failure and reduced cardiac output),
  • liver insufficiency (as cirrhosis)
  • kidney insufficiency
  • and other organ severe abnormalities

• Known hypersensitivity to excipients used in the formulation (Dimethyl sulfoxide (DMSO), Citrate-dextrose solution (ACD))

• Participation in a concurrent interventional study