CAR19TIF（CD19 CAR-T）治疗白血病：I/II 期临床试验

Inclusion Criteria:

1. Age 18-70 (inclusive),gender unrestricted.
2. Patient with r/r CD19+ B-ALL, as per guidelines (NCCN, 2019)

   * morphologically confirmed with ≥ 5% leukaemic blasts in the bonemarrow;
   * or presenting a quantifiable MRD load of 1x10\^-3 , assessed by multiparameter flow cytometry and/or quantitative polymerase chain reaction, at the end of the last induction treatment.
   * who has exhausted alternative treatment options.

   Relapsed disease is defined as:
   * second or subsequent bone marrow relapse or,
   * any bone marrow relapse after allogenic hematopoiesis stem cell transplant (allo-HSCT).

   Refractory disease is defined by not achieving an initial complete response (CR) after 2 cycles of a standard chemotherapy regimen (primary refractory). Subjects who were refractory to subsequent chemotherapy regimens after an initial remission were considered chemorefractory.
3. Toxicities due to prior therapy must be stable and recovered to ≤ Grade 1 (except for hematological toxicities and clinically non-significant toxicities such as alopecia).
4. Eastern Cooperative Oncology Group (ECOG) performance status ≤ 2.
5. Adequate renal, hepatic, pulmonary and cardiac function defined as:

   * Serum creatinine≤1.5 upper limit of normal (ULN) or creatinine clearance (as estimated by Cockcroft Gault) ≥ 60 mL/min.
   * Serum alanine aminotransferase / aspartate aminotransferase (ALT/AST) ≤ 3×ULN; Total bilirubin ≤ 1.5×ULN.
   * Cardiac ejection fraction ≥ 50%, no evidence of pericardial effusion as determined by an echocardiogram (ECHO), and no clinically significant electrocardiogram (ECG) findings.
   * Coagulation Function: International Normalized Ratio (INR) ≤ 1.5 ×ULN, and Activated Partial Thromboplastin Time (APTT) ≤ 1.5×ULN .• Baseline oxygen saturation \>91% on room air.
6. Subjects of both genders who are willing to practice birth control from the time of consent through 6 months after the completion of conditioning chemotherapy. Females of childbearing potential must have a negative serum or urine pregnancy test (females who have undergone surgical sterilization or who have been postmenopausal for at least 2 years are not considered to be of childbearing potential).
7. Voluntarily participate in this clinical trial and sign an informed consent form.

Exclusion Criteria:

1. Expected survival time \< 3 months per Principal Investigator's opinion.
2. Previous or concurrent cancer within 3 years prior to treatment start EXCEPT for curatively treated cervical cancer in situ, non-melanoma skin cancer, superficial bladder tumors \[Ta (non-invasive tumor), Tis (carcinoma in situ) and T1 (tumor invades lamina propria)\].
3. Prior CD19 targeted therapy
4. Prior CAR-T therapy or other genetically modified T cell therapy.
5. Active central nervous system (CNS) leukaemia (CNS-3).
6. B-ALL with clinically suspected extra-medullary involvement.
7. Burkitt cell (L3 ALL) or mixed lineage acute leukaemia.
8. Clinically active significant CNS dysfunction

   * History or presence of CNS disorder such as seizure disorder, cerebrovascular ischemia/hemorrhage, dementia, cerebellar disease, or any autoimmune disease with CNS involvement.
   * Known history of irreversible severe neurological toxicity related to previous antileukaemic treatment leading to organic central nervous system lesions.
   * Radioimmunotherapy, radiotherapy, within 8 weeks (except prophylaxis of CNS involvement) before Inclusion.
9. Use of previous anti-leukemic therapy within 5 half-lives prior to CAR19TIF cells administration; participation in non-interventional registries or epidemiological studies is allowed.
10. History of severe, immediate hypersensitivity reaction attributed to lymphodepletion drugs or any component of CAR19TIF cells.
11. Presence or suspicion of fungal, bacterial, viral, or other infection that is uncontrolled or requiring intravenous (IV) antimicrobials for management.
12. Uncontrolled or active infectious diseases, such as human immunodeficiency virus (HIV) infection, acute or chronic active hepatitis B or C, epstein-barr virus (EBV), and cytomegalovirus (CMV) infection.
13. Subjects with cardiac atrial or cardiac ventricular lymphoma involvement.
14. History of myocardial infarction, cardiac angioplasty or stenting, unstable angina, or other clinically significant cardiac disease within 12 months of enrollment.
15. Expected or possible requirement for urgent therapy within 6 weeks due to ongoing or impending oncologic emergency (eg, tumor mass effect, tumor lysis syndrome).
16. Primary immunodeficiency.
17. History of autoimmune disease (e.g. Crohn's, rheumatoid arthritis, systemic lupus) resulting in end organ injury or requiring systemic immunosuppression/systemic disease modifying agents within the last 2 years.
18. History of symptomatic deep vein thrombosis or pulmonary embolism requiring systemic anticoagulation within 6 months of enrollment.
19. Any medical condition likely to interfere with assessment of safety or efficacy of study treatment.
20. Vaccine ≤ 6 weeks prior to planned start of conditioning regimen.
21. In the investigator's judgment, the subject is unlikely to complete all protocol-required study visits or procedures, including follow-up visits, or comply with the study requirements for participation.

主要终点：Phase 1: Incidence of Adverse Events (AEs) · AE is defined as any adverse medical event from the date of randomization to 12 months after CAR T cells infusion. Among them, cytokine release syndrome(CRS) and immune cell-associated neurotoxicity syndrome (ICANS) were graded according to American Society for Transplantation and Cellular Therapy (ASTCT) criteria. Other AEs were graded according to common terminology criteria for adverse events (CTCAE) v5.0. · Up to 12 months since the initiation of CAR-T cell therapy.；Phase 1:Incidence of Incidence of Dose-Limiting Toxicities (DLTs) · DLT is defined as any AE related to the investigational drug that occurs within 28 days after administration of the CAR19TIF cells and meets any one of the criteria listed in the DLT criteria: * Grade 3 CRS that does not resolve to grade 2 or lower within 2 weeks; * Grade 3 ICANS lasting for ≥ 7 days; * Any Grade ≥ 4 CRS or ICANS; * Any other Grade ≥ 4 and Grade 3 AEs related to the CAR19TIF cells that lasts for ≥ 14 days, except hematology toxicity. · [Time Frame: Up to 28 days since the initiation of CAR-T cell therapy]；Phase 1:RP2D · The recommended dose for phase 2 was determined through phase 1 study. · 12 months；Phase 2:Objective response rate (ORR) · Objective response definition: a molecular response (MRD \< 10\^-4 post treatment) assessed by multiparameter flow cytometry and/or qPCR, or a morphologic complete response (CR), or a CR with incomplete blood count recovery (CRi). ORR is defined as the proportion of patients who have achieved objective response assessed by investigators. · 24 months；Phase 2:Overall Survival (OS) · OS is defined as the time from CAR-T cells infusion to the date of death. Subjects who have not died by the analysis data cutoff date will be censored at the last contact date. · 24 months；Phase 2: Progression Free Survival (PFS) · PFS is defined as the time from the CAR-T cells infusion date to the date of disease progression assessed by investigators, or death any cause. Participants not meeting the criteria for progression by the analysis data cutoff date were censored at the last evaluable disease assessment date. · 24 months
次要终点：Phase 1 and phase 2: Level of CAR-positive T cells circulating in blood over time；Phase 1 and phase 2: Level of CD19+ cells in peripheral blood

• CAR19TIF cells · EXPERIMENTAL · Patients with r/r B-ALL A conditioning chemotherapy regimen of fludarabine and cyclophosphamide will be administered followed by investigational treatment, CAR19TIF cells.

In this single-center, single-arm, prospective, Phase 1/2 study, the safety and efficacy of autologous BCOR and ZC3H12 genes knock-out CD19-targeting chimeric antigen receptor (CAR) T-cell therapy will be evaluated in patients with refractory/relapsed (r/r) B-cell acute lymphoblastic leukaemia (B-ALL). In phase 1, 3 eligible patients will be enrolled and receive BCOR and ZC3H12 genes knock-out CD19 CAR T cell therapy at a initial dose of 5×10\^5 cells/kg. Based on the results, . Subsequently an additional 3-15 patients will be enrolled in a "3+3" dose-escalation/decline design to adjust the dose of BCOR and ZC3H12 genes knock-out CD19 CAR T cells to achieve optimal safety and efficacy. The recommended Phase 2 dose (RP2D) will then be established. 10 to 12 subjects will be enrolled and receive BCOR and ZC3H12 genes knock-out CD19 CAR T cell infusion at dose of RP2D.