How to increase lymphocyte count before harvesting Chimeric Antigen Receptor T (CAR-T) cells?

Boosting Lymphocyte Count Before CAR-T Cell Harvesting

The primary strategy to optimize lymphocyte collection for CAR-T manufacturing is to perform leukapheresis earlier in the disease course (ideally at first relapse before salvage therapy) and ensure adequate recovery from prior cytotoxic chemotherapy, with an absolute lymphocyte count ≥0.2 × 10⁹/L recommended before proceeding with collection. 1, 2

Timing of Leukapheresis

Early lymphocyte collection is superior to delayed collection:

• Perform leukapheresis at first relapse, before salvage treatment, rather than waiting until second relapse or beyond. This approach yields T cells with increased percentages of naïve phenotypes, improved in vitro functionality, and lower exhaustion profiles compared to cells collected after multiple lines of therapy 2

• Allow sufficient time for recovery from prior cytotoxic chemotherapy and immunosuppression before leukapheresis 1

• A minimum washout period of 3 days from steroids is required before leukapheresis (though physiological replacement doses of hydrocortisone, topical, and inhalational steroids are permitted) 1

Pre-Leukapheresis Requirements

Minimum lymphocyte thresholds must be met:

• Absolute lymphocyte count (ALC) should be ≥0.2 × 10⁹/L before proceeding with leukapheresis 1

• For pediatric patients, a CD3+ count of 0.2 × 10⁹/L is the minimum recommended threshold 1

• Low lymphocyte counts indicate insufficient hematological recovery and may predict manufacturing failure 1

Additional pre-leukapheresis parameters:

• Hemoglobin >80 g/L is recommended to establish a good interface during leukapheresis 1

• Platelet count >30 × 10⁹/L (transfuse as required, particularly for central line insertion) 1

• Performance status: ECOG <2 or Karnofsky >60% 1

Factors Associated with Poor Lymphocyte Collection

Patient and disease characteristics that predict low collection efficiency:

• Advancing age (every 10-year increase in age is associated with lower collection efficiency, OR = 1.51) 3

• Disease type matters: Patients with acute lymphoblastic leukemia have significantly lower collection efficiency compared to non-Hodgkin lymphoma (OR = 0.20) or chronic lymphocytic leukemia (OR = 0.24) 3

• Higher pre-apheresis platelet counts are paradoxically associated with lower lymphocyte collection efficiency (every 10 × 10³/μL increase, OR = 1.07) 3

Growth Factor Support

G-CSF (pegfilgrastim) can be considered but requires careful timing:

• Pegfilgrastim is FDA-approved to decrease infection incidence in patients receiving myelosuppressive chemotherapy 4

• Do not administer pegfilgrastim between 14 days before and 24 hours after cytotoxic chemotherapy 4

• The recommended dose is 6 mg subcutaneously once per chemotherapy cycle for patients ≥45 kg 4

• For pediatric patients <45 kg, weight-based dosing is required (ranging from 0.1 mg/kg for patients <10 kg to 4 mg for patients 31-44 kg) 4

Bridging Chemotherapy Considerations

If bridging chemotherapy is required between leukapheresis and CAR-T infusion:

• The goal is to maintain disease control while CAR-T cells are manufactured (2-4 week period), not to act as primary treatment 1

• Select bridging regimens carefully to minimize toxicities that might disqualify patients from proceeding to CAR-T infusion 1

• Monitor for tumor lysis syndrome and provide antimicrobial prophylaxis during this period 1

Common Pitfalls to Avoid

Critical errors that compromise lymphocyte collection:

• Avoid corticosteroids before leukapheresis as they are lymphocytotoxic and will reduce collection yield 1

• Do not proceed with leukapheresis if the patient has not adequately recovered from prior cytotoxic therapy 1

• Do not delay leukapheresis until late in the disease course after multiple lines of therapy, as this results in T cells with exhausted phenotypes and reduced functionality 2

• Ensure adequate marrow recovery from prior chemotherapy before attempting collection 1

Manufacturing Success Despite Low Collection Efficiency

Reassuring data on manufacturing outcomes:

• Even with low collection efficiency (<40% in 45% of collections), 100% manufacturing success was achieved in one study of adult patients with B-cell malignancies 3

• This suggests that while optimizing collection is important, even suboptimal collections can often yield sufficient cells for CAR-T manufacturing 3