CAR-T Cell Therapy for ALL

A Pilot, Single-Arm, Open-Label Feasibility Study of Autologous Anti-CD19 Chimeric Antigen Receptor T-Cell (CAR-T) Therapy in Pediatric and Young Adult Patients With Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia (r/r B-ALL)

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children and the second most frequent acute leukemia in adults. B-cell ALL constitutes approximately 85% of all ALL diagnoses. In Pakistan, ALL represents the most prevalent haematological malignancy presenting to tertiary centres, with AFBMTC receiving the largest national referral volume for haematological malignancies and transplantation.

First-line combination chemotherapy achieves complete remission (CR) in >95% of paediatric patients; however, 15-20% relapse. Outcomes following first relapse are substantially inferior: second-line salvage chemotherapy achieves CR2 in 30-50% of patients, and long-term event-free survival (EFS) after conventional chemotherapy alone is <10%. Outcomes in adult ALL are even more dismal, with OS at 5 years below 40% even in first CR without allogeneic transplant.

Patients with primary refractory ALL or multiply relapsed ALL have an unmet medical need for novel therapeutic approaches. The classical paradigm of chemotherapy followed by allogeneic haematopoietic stem cell transplantation (allo-HSCT) is limited by donor availability, conditioning-related mortality, and inability to achieve remission before transplant.

1.2 CD19 as a Therapeutic Target CD19 is a B-lineage surface glycoprotein expressed on the majority of B-cell ALL blasts, retained through disease evolution, and absent on haematopoietic stem cells, non-haematopoietic tissues, and most normal tissues. CD19 expression is confirmed in >95% of B-ALL cases, making it the optimal target for immunotherapy. The only significant on-target off-tumour consequence is B-cell aplasia and hypogammaglobulinaemia, which are clinically manageable with immunoglobulin replacement.

1.3 CAR-T Cell Therapy: Scientific and Clinical Rationale Chimeric antigen receptor T-cell (CAR-T) therapy involves ex vivo genetic modification of autologous T cells to express a synthetic receptor comprising: (1) an extracellular antigen-binding domain (anti-CD19 scFv), (2) a hinge and transmembrane domain, (3) an intracellular costimulatory domain (4-1BB/CD137), and (4) a CD3ζ signalling domain. Upon infusion, CAR-T cells recognise CD19+ target cells in an MHC-independent manner, inducing direct cytotoxicity, cytokine release, and in vivo proliferation.

The 4-1BB costimulatory domain (used in this protocol) confers superior persistence and memory formation compared to CD28-based constructs, as demonstrated in seminal studies from the University of Pennsylvania and subsequently confirmed in the ELIANA global Phase II trial of tisagenlecleucel (Maude et al., NEJM 2018), which demonstrated an overall remission rate of 81% and 12-month EFS of 50% in heavily pre-treated paediatric and young adult r/r B-ALL.

1.4 Institutional Rationale and LMIC Context AFBMTC is Pakistan's first and largest haematology and cellular therapy centre, established in 2001 under NUMS, with a cumulative experience of >2,000 allogeneic, autologous, and haploidentical HSCTs. AFBMTC possesses critical institutional infrastructure for CAR-T implementation including GMP-equivalent cleanroom facilities, validated cryopreservation and leukapheresis capabilities, and an established mesenchymal stromal cell (MSC) GMP program with over a decade of experience - directly translatable to CAR-T manufacturing quality standards.

Access to commercially approved CAR-T products (tisagenlecleucel, axicabtagene ciloleucel) is prohibitively expensive and effectively unavailable in Pakistan. AFBMTC's model of on-site CAR-T manufacturing using an internationally validated lentiviral vector platform (BIOCCUS, China) and automated closed-system bioreactor technology (CELLBRI) - analogous to the NexCAR19 program (ImmunoACT/IIT Bombay, India) in the LMIC context - provides a feasible, cost-recoverable, and sustainable approach to making this therapy accessible. This pilot study represents Pakistan's first academic CAR-T trial and aims to demonstrate manufacturing feasibility, safety, and early efficacy to support future regulatory approval and programme scale-up.

1.5 Investigational Product Description

The investigational product is autologous anti-CD19 CAR-T cells generated from patient-derived peripheral blood T cells using:

• Lentiviral vector: BIOCCUS-supplied, replication-deficient third-generation lentiviral vector encoding the anti-CD19 CAR transgene (CD19 scFv-CD8 hinge/TM-4-1BB-CD3ζ)
• Ex vivo manufacturing: CELLBRI automated closed-system bioreactor platform at AFBMTC GMP-equivalent cell therapy laboratory
• CAR construct: Single-chain variable fragment (scFv) targeting human CD19, fused to CD8α hinge and transmembrane domain, 4-1BB (CD137) costimulatory domain, and CD3ζ activation domain
• Cell source: Non-mobilised leukapheresis product from the patient
• Administration: Single intravenous infusion following lymphodepleting chemotherapy

Inclusion Criteria:

• Inclusion Criteria

All of the following criteria must be met for enrolment:

• 1. Age ≥5 years and ≤50 years at the time of consent

• 2. Morphologically or immunophenotypically confirmed B-cell ALL (CD19+) meeting one or more of the following relapsed/refractory criteria:

  • a. Second or subsequent bone marrow relapse (BM ≥2nd relapse)
  • b. Any BM relapse following prior allogeneic HSCT, provided ≥6 months have elapsed from SCT to planned CAR-T infusion
  • c. Primary refractory disease: failure to achieve CR after ≥2 cycles of standard induction chemotherapy; or chemorefractory: failure to achieve CR after ≥1 cycle of standard salvage chemotherapy for relapsed ALL
  • d. Philadelphia chromosome-positive (Ph+) ALL: intolerant to or failed ≥2 lines of TKI therapy, or TKI contraindicated
  • e. Ineligibility for allo-HSCT due to: comorbid disease, lack of suitable donor, contraindication to conditioning, or patient refusal after documented discussion with a BMT physician not part of the study team

• 3. CD19 expression on tumour cells confirmed by multi-parameter flow cytometry within 3 months of enrolment (≥20% CD19-positive blasts required)

• 4. Bone marrow blast burden ≥5% by morphological assessment at screening

• 5. Adequate organ function at screening:

  • a. Renal: Age-adjusted serum creatinine within normal limits per CTCAE paediatric reference tables, or GFR ≥30 mL/min/1.73m² (MDRD/CKD-EPI)
  • b. Hepatic: ALT/AST ≤5× ULN; Total bilirubin <2.0 mg/dL
  • c. Cardiac: LVEF ≥45% or LVSF ≥28% on echocardiogram (performed within 28 days of screening)
  • d. Pulmonary: ≤Grade 1 dyspnoea; SpO₂ ≥91% on room air

• 6. ECOG performance status ≤2 (age ≥16 years); Lansky performance scale ≥50 (age <16 years)

• 7. Life expectancy >12 weeks in the opinion of the investigator

• 8. Adequate haematological status to tolerate leukapheresis (ALC ≥100/µL and CD3+ count ≥100/µL at time of apheresis, or acceptable stored product available)

• 9. Patients who have undergone prior allo-HSCT must have: (a) no active acute GVHD (Grade ≥2) or extensive chronic GVHD; (b) no systemic immunosuppression for GVHD within 4 weeks prior to CAR-T infusion

• 10. Written informed consent from patient (and parent/guardian if age <18 years); assent from patients aged 7-17 years

• 11. Willingness and ability to comply with study procedures, visit schedule, and long-term follow-up requirements including the 15-year gene therapy safety surveillance

• 12. Negative pregnancy test (serum or urine β-hCG) within 48 hours of CAR-T infusion for females of childbearing potential (defined as post-menarche and not surgically sterilised) 4.3 Exclusion Criteria

Patients meeting ANY of the following criteria will be excluded:

• 1. Isolated extra-medullary (CNS-only or testicular-only) disease relapse without bone marrow involvement

• 2. Active CNS involvement by ALL, defined as CNS-3 status per NCCN criteria (CSF blasts on cytospin, cranial nerve palsy, or brain parenchymal disease) at time of screening. Patients with prior CNS disease that has been effectively treated and cleared are eligible

• 3. Burkitt's lymphoma/leukemia (mature B-ALL with sIg positive, FAB L3 morphology and/or MYC translocation)

• 4. T-cell ALL or ambiguous lineage leukemia

• 5. Known congenital bone marrow failure syndromes: Fanconi anaemia, Shwachman-Diamond syndrome, Kostmann syndrome, Diamond-Blackfan anaemia, or any other inherited aplastic anaemia. (Down syndrome patients are NOT excluded)

• 6. Prior treatment with any CAR-T or adoptive T-cell product

• 7. Prior anti-CD19 therapy of any kind (including blinatumomab) within 4 weeks of screening; or confirmed CD19-negative (antigen-loss) relapse on anti-CD19-based therapy. [Note: prior blinatumomab ≥4 weeks before screening is not exclusionary if CD19 positivity is confirmed at re-screening]

• 8. Prior gene therapy with a viral vector (non-CAR gene therapy); or prior receipt of a gene-edited cellular product

• 9. Active uncontrolled infection at screening (bacterial, fungal, viral or parasitic). Patients with controlled or treated infection may be enrolled at investigator discretion

• 10. Active Hepatitis B (HBsAg positive, or anti-HBc positive with detectable HBV DNA); active Hepatitis C (anti-HCV positive with detectable HCV RNA); or HIV positive (confirmed within 8 weeks of screening)

• 11. Active Grade 2-4 acute GVHD or active moderate/severe chronic GVHD

• 12. Prior malignancy other than B-ALL in the last 3 years (except carcinoma in situ of cervix or skin treated with curative intent, with no evidence of active disease)

• 13. Investigational medicinal product (IMP) exposure within 30 days prior to screening, or within 5 half-lives, whichever is longer

• 14. Pregnant or breastfeeding women

• 15. Uncontrolled psychiatric condition or severe cognitive impairment that would preclude informed consent or compliance with protocol procedures

• 16. Any medical condition that, in the opinion of the investigator, would place the patient at unacceptable risk from the study procedures

• 17. Prohibited concomitant medications at time of CAR-T infusion (detailed in Section 6.5):

  • Systemic corticosteroids >physiologic replacement (>12 mg/m²/day hydrocortisone equivalent) within 72 hours prior to CAR-T infusion
  • Anti-T-cell antibody therapy (ATG, alemtuzumab) within 8 weeks prior to CAR-T infusion
  • Systemic GVHD immunosuppression within 4 weeks prior to CAR-T infusion
  • Donor lymphocyte infusion within 6 weeks prior to CAR-T infusion

Exclusion Criteria:

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