Phase I Trial of CD19-CAR_LENTI_ALLO, Fludarabine Phosphate, and Cyclophosphamide in Pediatric and Young Adult Relapsed/Refractory B-cell Acute Lymphoblastic Leukemia

The primary objective of this Phase I clinical trial is to evaluate the **safety** and establish the recommended dose of donor-derived, CD19-directed second-generation CAR T cells (CD19-CAR_Lenti_ALLO) in pediatric patients and young adults with relapsed/refractory B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL). This condition may occur either after allogeneic hematopoietic stem cell transplantation (alloHSCT) or before alloHSCT in cases of extremely refractory disease with the availability of a familiar HLA-fully matched donor. The clinical relevance of this objective lies in determining a safe and effective dose of CD19-CAR T cells, which could potentially improve treatment outcomes for this patient population.

Secondary objectives include: - Incidence of grade II-IV acute and chronic graft-versus-host disease (GvHD). - Determination of the rate of bone marrow complete remission with minimal residual disease negativity at day +28. - Improvement of 1-year event-free and overall survival rates compared to historical controls. - Cumulative incidence of both molecular and morphological relapse. - Toxicity of treatment with CD19-CAR_Lenti_ALLO compared to historical controls. - Incidence of cytokine release syndrome (CRS) and neurotoxicity. - Assessment of CD19-CAR_Lenti_ALLO expansion and persistence. - Duration of B-cell aplasia. - Evaluation of disease response in patients with previous CD19-directed immunotherapy.

The clinical trial involves a study population comprising **pediatric patients and young adults** aged between 1 and 35 years, diagnosed with **relapsed/refractory B-cell Acute Lymphoblastic Leukemia**. The trial includes both male and female participants, and the population is considered vulnerable due to the nature of the disease. The sponsor has not provided information regarding the total number of participants. Participants were selected based on specific eligibility criteria, including a diagnosis of CD19 expressing B-cell Acute Lymphoblastic Leukemia with relapse after allogeneic hematopoietic stem cell transplantation or refractory disease. Lifestyle considerations such as diet and physical activity are not specified, but participants must meet clinical performance standards, with a Karnofsky score of at least 60% for those over 16 years and a Lansky scale score of at least 60% for those under 16 years. Additionally, women of childbearing potential are required to have a negative pregnancy test and agree to use contraception, as must male participants of reproductive capacity, for a specified duration following the infusion of the CD19-CAR_Lenti_ALLO drug product.

The clinical trial is a **Phase I** study designed to evaluate the safety and establish the recommended dose of donor-derived, CD19-directed second-generation CAR T cells (**CD19-CAR_Lenti_ALLO**) in pediatric patients and young adults with relapsed/refractory B-cell Acute Lymphoblastic Leukemia (B-ALL). The trial employs a **randomized, double-blind, controlled** design to ensure the reliability and validity of the results. The estimated duration of the trial is from January 2024 to July 2027, with participant involvement expected to last approximately 12 months from the time of infusion.

Participants will undergo a series of study visits, beginning with an inclusion (screening) visit to confirm eligibility based on specific criteria, such as age, disease status, and previous treatments. Following successful screening, participants will receive the investigational product, **CD19-CAR_Lenti_ALLO**, administered intravenously. The primary endpoint is to evaluate the safety of the infusion by assessing dose-limiting toxicity at two dose levels, depending on the donor HLA-matching. Secondary endpoints include estimating the rate and severity of acute and/or chronic Graft-versus-Host Disease (GvHD) and evaluating the proportion of patients achieving complete remission (CR) or CR with incomplete blood count recovery (CRi) and minimal residual disease (MRD) negativity by day 28.

Follow-up visits will be scheduled to monitor the participants' response to treatment and any adverse events. These visits will include assessments of clinical performance status, laboratory tests, and imaging studies as necessary. The end-of-study visit will occur at the conclusion of the 12-month follow-up period, or earlier if the participant experiences significant adverse events or disease progression that necessitates withdrawal from the study. Conditions that may lead to early termination include severe adverse reactions, non-compliance with study procedures, or withdrawal of consent by the participant or their legal guardian.

The experimental medication in this clinical trial is **CD19-CAR_Lenti_ALLO**, a cell suspension for injection. This investigational product is a donor-derived, CD19-directed second-generation CAR T cell therapy, specifically designed for pediatric patients and young adults with relapsed or refractory B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL). The pharmaceutical form is a cell suspension, and it is administered intravenously. The dosing schedule and frequency of administration are determined based on the study protocol, with the primary objective being to evaluate safety and establish the recommended dose.

In addition to the experimental treatment, the study utilizes **Fludarabine Phosphate** as a non-experimental, auxiliary treatment. Fludarabine is provided in the form of a powder for solution for injection or infusion, with a concentration of 50 mg. It is administered as a solution for injection or infusion, and the dosing regimen is aligned with standard preconditioning protocols for CAR T cell therapy. The pharmaceutical form and administration route are consistent with its use as a chemotherapeutic agent to enhance the efficacy of the CAR T cell therapy.

Another auxiliary treatment used in the study is **Cyclophosphamide**, which is provided as a solution for injection with a concentration of 1 g. Similar to Fludarabine, Cyclophosphamide is administered as a solution for injection or infusion. It serves as part of the preconditioning regimen to facilitate the engraftment and activity of the CAR T cells. The dosing schedule is determined according to established protocols for preconditioning in hematological malignancies.

Participant compliance with the treatment regimen is monitored throughout the study to ensure adherence to the dosing schedules and to assess any potential adverse effects. The administration of both auxiliary treatments, Fludarabine and Cyclophosphamide, is conducted under controlled conditions to support the primary investigational therapy, CD19-CAR_Lenti_ALLO, in achieving its therapeutic objectives.

The efficacy of the clinical trial will be assessed through several key endpoints. The primary endpoint focuses on evaluating the safety of the infusion of **CD19-CAR_Lenti_ALLO** by assessing two dose levels for each cohort of patients, based on the donor HLA-matching. The dose-limiting toxicity (DLT) of the cellular product will be established, with specific dose levels defined for fully matched, familial or unrelated donors, and haploidentical donors.

Secondary endpoints include estimating the rate and severity of acute and/or chronic graft-versus-host disease (GvHD), evaluating the proportion of patients achieving complete remission (CR) or CR with incomplete blood count recovery (CRi), and assessing minimal residual disease (MRD) negativity by flow-cytometry or qPCR at day 28, defined as a value less than 1x10^-4. Additionally, the probability of obtaining CR with MRD negativity will be stratified according to the disease burden at the time of enrollment.