A Phase I/IIa open label single ascending dose study to assess safety and tolerability of regulatory T cells to promote discontinuation of tacrolimus monotherapy in liver transplant recipients - LiveTreg

The primary objective is to determine that autologous polyclonal ex-vivo expanded regulatory T cells (Treg02) therapy in liver transplant recipients receiving tacrolimus monotherapy is safe, well tolerated, and modulates the immunologic response to enable permanent withdrawal of immunosuppression. This objective addresses a critical clinical need to minimize long-term immunosuppressive therapy-related complications while maintaining graft function in liver transplantation.

The secondary objectives include:

• Investigating preliminary efficacy of Treg02 on graft survival and function

• Investigating the effect of Treg02 on quality-of-life and immune cell subsets in peripheral blood

The sponsor did not provide information regarding the total number of participants enrolled in this clinical trial. The study population consists of **adult patients** aged 18 years and older of both genders who have undergone **single liver transplantation** for **end-stage liver disease**. Participants are selected based on specific post-transplant criteria, including a transplantation timeframe of more than 24 months but less than 60 months prior to enrollment, and must demonstrate stable **liver function** with defined laboratory parameters including direct **bilirubin** below 17.1 µmol/l, **albumin** above 3 g/l, and **ALT** below 62 IU/l. Eligible participants must be maintained on stable **tacrolimus monotherapy** for at least 6 months with target trough levels of 5 ng/ml without complications. The trial excludes patients with evidence of **hepatic autoimmune disease** such as **primary biliary cholangitis**, **primary sclerosing cholangitis**, or **autoimmune hepatitis**, as well as those with marked abnormalities in liver biopsy findings. Additionally, participants must meet minimum hematological requirements including **hemoglobin** of at least 7.0 g/dl, **platelets** of at least 80x10^9/l, and total **leukocyte count** of at least 3.0x10^9/l. A negative **SARS-CoV-2** test is required according to current recommendations at the time of enrollment.

This is a Phase I/IIa, open-label, single ascending dose clinical trial designed to evaluate the safety and tolerability of autologous polyclonal ex-vivo expanded **regulatory T cells** (Treg02) in **liver transplant recipients** currently maintained on **tacrolimus** monotherapy. The primary objective is to determine whether Treg02 therapy is safe, well tolerated, and capable of modulating the immunologic response to enable permanent withdrawal of immunosuppression. The trial employs a non-randomized, non-blinded design with sequential dose escalation to assess the safety profile of the investigational **advanced therapy medicinal product** (ATMP). Participants will receive Treg02 as a **solution for injection** administered via **intravenous injection**, while concomitant medications include tacrolimus formulations administered orally, as well as auxiliary medications such as **paracetamol** and dimetindene for supportive care.

The trial is expected to commence recruitment in October 2024 and conclude in October 2027, representing an overall study duration of approximately three years. Eligible participants include adult patients aged 18 years or older who have undergone single liver transplantation for **end-stage liver disease**, are between 24 and 60 months post-transplant, and have maintained stable liver function on tacrolimus monotherapy for at least six months with target trough levels of 5 ng/ml. Key inclusion criteria require stable liver function parameters including direct **bilirubin** below 17.1 µmol/l, **albumin** above 3 g/l, and **ALT** below 62 IU/l, as well as acceptable histopathological findings with inflammation grade below 4 and fibrosis stage below 3. Participants must also demonstrate adequate hematological parameters including **hemoglobin** at or above 7.0 g/dl, **platelets** at or above 80×10⁹/l, and total **leukocyte count** at or above 3.0×10⁹/l. Exclusion of hepatic autoimmune diseases such as **primary biliary cholangitis**, **primary sclerosing cholangitis**, and **autoimmune hepatitis** is required, along with a negative SARS-CoV-2 test at screening.

The study visit schedule begins with a screening visit to assess eligibility criteria, including clinical laboratory assessments, liver function tests, and histopathological evaluation of liver biopsy material when indicated. Following enrollment, participants will undergo Treg02 infusion and enter a structured follow-up period extending to 14 months post-treatment, during which immunosuppression tapering will be attempted. Follow-up visits will include clinical assessments, monitoring for **acute rejection** episodes using **biopsy-proven acute rejection** (BPAR) as the principal measure, histopathological grading according to **Banff criteria**, surveillance for opportunistic infections including **CMV**, **EBV**, **HBV**, and **HCV** reactivation, and evaluation for neoplasia development. Serial biomarker assessments will be conducted to measure functional and molecular indices reflecting immune response, treatment safety, and efficacy. Health-related quality of life will be assessed using the SF-12 questionnaire. The end-of-study visit will occur at the completion of the 14-month follow-up period, with final assessment of immunosuppression levels, graft function, and overall safety outcomes. The expected duration of individual participant involvement is approximately 14 months following Treg02 administration.

Primary safety endpoints include assessment of acute toxicity associated with Treg02 infusion, specifically evaluating pulmonary complications, immunological reactions resulting in **anaphylactic reactions**, immediate cardiovascular compromise, acute organ failure, and biochemical perturbations related to cellular apoptosis. Over-suppression of the immune system will be monitored through surveillance for major and opportunistic infections, early development of neoplasia, and laboratory anomalies unrelated to transplanted liver function. The primary clinical endpoint is the incidence of acute and chronic rejection and prevalence of **adverse events**, with BPAR within 14 months following Treg transfer serving as the principal efficacy measure. Secondary endpoints include time to acute rejection episode, severity of rejection based on treatment response and histological scoring, level of total immunosuppression at final visit, incidence of subclinical acute rejection, prevention of **chronic graft dysfunction** assessed by clinical and histopathological criteria, and reduced incidence of adverse events following immunosuppression tapering including cardiovascular complications and drug toxicity.

Conditions that may lead to early termination from the study include development of biopsy-proven acute or chronic rejection requiring intensification of immunosuppression, occurrence of serious adverse events related to Treg02 administration or immunosuppression withdrawal, development of severe infections or malignancy, significant deterioration in liver function parameters, withdrawal of informed consent, protocol violations, or investigator determination that continued participation is not in the participant's best interest. Throughout the trial, participants will be closely monitored for signs of graft rejection, infectious complications, and other safety concerns to ensure timely intervention when necessary.

The experimental treatment consists of **Treg02**, an **advanced therapeutic medicinal product** classified as a **cell therapy** product. Treg02 contains **autologous**, **ex-vivo expanded** **polyclonal regulatory T cells** characterized as **CD4+CD25+FoxP3+**. The product is formulated as a **solution for injection** administered via **intravenous injection**. The study follows a **single ascending dose** design to evaluate the safety and tolerability of Treg02 in **liver transplant recipients** receiving **tacrolimus monotherapy**.

**Tacrolimus** serves as the background **immunosuppressive therapy** in this trial and is available in multiple pharmaceutical formulations. **Advagraf 1 mg prolonged-release hard capsules** contain tacrolimus as the active substance and are administered via **oral use**. **Envarsus 1 mg prolonged-release tablets** represent an alternative prolonged-release formulation of tacrolimus for oral administration. **Prograf 0.5 mg hard capsules** provide another oral formulation of tacrolimus. All tacrolimus-containing products are utilized as part of the existing **monotherapy regimen** in the study population, with the objective of enabling permanent **withdrawal of immunosuppression** following Treg02 administration.

**Paracetamol-ratiopharm 500 mg tablets** are included in the study as an **auxiliary medication**. This product contains **paracetamol** as the active substance and is formulated as **tablets** for **oral use**. Paracetamol may be used for symptomatic management of minor adverse events such as fever or discomfort.

**Histakut Dimetindenmaleat 1 mg/ml solution for injection** serves as an additional auxiliary medication in the trial. This product contains **dimetindene maleate** and is administered via **intravenous use**. The medication may be used for management of potential **allergic reactions** or **hypersensitivity** events during the study procedures.

Efficacy will be assessed through multiple parameters focusing on prevention of graft rejection and immune system modulation following Treg02 therapy. The principal measure of immunomodulation is biopsy-proven acute rejection within 14 months following Treg transfer. Histopathological grading of biopsy material will be assessed using the established Banff criteria. The incidence of acute and chronic rejection and the prevalence of adverse events will be evaluated as primary clinical endpoints.

Secondary efficacy indices include assessment of time to acute rejection episode, severity of acute rejection episodes based on response to treatment and histological scoring, and the level of total immunosuppression at the final trial visit. The incidence of patients treated for subclinical acute rejection based on histopathological findings will be documented. Prevention of chronic graft dysfunction will be evaluated through clinically impaired levels of liver enzymes and histopathological measures according to Banff staging criteria. The reduced incidence of adverse events and rejections following tapering of immunosuppression will be monitored.

A validated biomarker panel will measure functional and molecular indices reflecting the immune response as well as treatment safety and efficacy of Tregs, as detailed in the clinical trial protocol. Health-related quality of life will be assessed using the SF-12 questionnaire.