Car-T cells: expanding the universe of immunotherapy in children with malignant diseases

1

What are chimeric antigen receptor–T cells?

Chimeric antigen receptor (CAR)-T cells are a powerful form of adoptive cell therapy. In their native state, T cells do not express antigens targeted to bind to malignant cells. In CAR-T therapy, T cells are harvested from a patient and then genetically engineered using a viral vector (most commonly a lentiviral or retroviral vector) to produce proteins on their surface that can directly bind to the malignant cells. Binding of CAR-T cells with malignant cells leads to activation and proliferation of CAR-T cells, which results in the killing of malignant cells.

2

Which antigens can be targeted by CAR-T cells in pediatric malignancies?

Several potential antigens have been studied in pediatric malignancies. For B-cell acute lymphoblastic leukemia (B-ALL), CD19 and CD22 have been studied; for T-cell acute lymphoblastic leukemia, it has been CD7 and C5; for acute myelogenous leukemia, CD33 and CD123; for non-Hodgkin lymphoma, CD19; and for neuroblastoma, GD2. The most well-studied CAR to date targets CD19.

3

Describe the history of CAR-T cell development.

In the early 1990s, immunologists Zelig Eshhar and Gideon Gross developed the first CAR-T cells. The original construct for the first Food and Drug Administration (FDA)–approved product, however, tisagenlecleucel (Kymriah), was developed by Dario Campana at St. Jude and then significantly modified by Carl June at University of Pennsylvania for clinical application in patients. The product was then tested in phase I trials of adult and pediatric patients at the University of Pennsylvania and Children’s Hospital of Philadelphia (CHOP), respectively. The first pediatric patient treated with CD19 CAR was Emily Whitehead at CHOP, who had relapsed/refractory B-ALL. She achieved remission after the therapy and remains in remission with evidence of circulating CAR-T cells over 9 years later. The technology developed at University of Pennsylvania was later transferred to Novartis, who conducted a global registration trial (the ELIANA trial) of tisagenlecleucel. On August 30, 2017, the FDA approved tisagenlecleucel, which was marketed as Kymriah.

4

What are the indications for tisagenlecleucel administration in pediatric patients?

The FDA-approved label for tisagenlecleucel includes children and young adults who are up to 25 years of age and have B-ALL that is either (1) refractory or (2) in second or greater relapse. The precise definition of refractory ALL is not clearly defined by the FDA.

5

What are the steps for manufacturing CAR-T cells?

Once a patient is deemed eligible for CAR-T cells, the medical team has to take several steps for collection, manufacture, and infusion of CAR-T cells.

• Step 1: Cell collection

  • A.

    Insurance approval: The medical team has to prepare a letter of medical necessity for insurance authorization. The current cost of tisagenlecleucel is $475,000; medical centers must follow FDA-approved indications to infuse tisagenlecleucel; otherwise, they risk not receiving reimbursement.

  • B.

    Washout period: Medications that could affect T-cell function should be discontinued for a period of time before collection. The specific washout period is dependent on the specific medication (e.g., 7 days for intrathecal methotrexate vs. 2–4 weeks for PEG-asparaginase).

  • C.

    Cell counts: It is recommended that patients have an absolute lymphocyte count (ALC) of over 500/ μL and a CD3 of more than 150 to 200/μL to yield the best T-cell collection. It is preferred that patients do not have a substantial number of circulating blasts, but the exact cutoff is not firmly established.

  • D.

    Apheresis process: The majority of children will require placement of an apheresis catheter. The apheresis procedure usually takes 3 to 4 hours because 3 to 5 blood volumes have to circulate through the machine.

  • E.

    After apheresis, the optimal total nucleated cell count needed for successful CAR-T manufacturing is 1x10 ⁹ cells/kg. To manufacture tisagenlecleucel, the cells are frozen and then shipped to Novartis.

• Step 2: Manufacturing

  • A.

    Cell culture: Cryopreserved cells are thawed at Novartis’ facility and put in the cell culture medium. Cells are genetically modified during cell culture process, which takes approximately 2 weeks. Before release, the product undergoes viability and cytokine level testing. Products that meet FDA-release criteria are cryopreserved and shipped back to the home center. Products that are outside of FDA-specification (generally because of viability < 80%) can be administered under an expanded access protocol.

  • B.

    Manufacturing failure: Up to 7% of products will fail to manufacture. The causes of manufacturing failure are not fully described. We speculate that it could be related to past chemotherapy exposures, such as doxorubicin, cyclophosphamide, and clofarabine, which can alter the energy metabolism of T cells, rendering them with poor proliferative capacity.

6

What are the steps for bridging chemotherapy and lymphodepletion before CAR-T cell infusion?

• Bridging Chemotherapy : The time from leukapheresis to CAR-T infusion is generally 4 to 6 weeks. During that period, most patients will require chemotherapy to decrease or maintain their leukemia burden. It is generally recommended to give the least toxic chemotherapy that will allow for disease control. In addition, it is important to consider avoiding certain agents. For example, inotuzumab ozogamicin is generally not preferred during the bridging period because it can lead to prolonged B-cell aplasia and concerns have been raised about the effectiveness of CD19 CAR in the absence of any CD19 ⁺ antigen load.

• Lymphodepletion chemotherapy: The goal of lymphodepletion is to provide an in-vivo environment that will result in the expansion, function, and persistence of CAR-T cells. The preferred regimen includes cyclophosphamide (500 mg/m ² × 2 days) and fludarabine (30mg/m ² × 4 days). It is hypothesized that these agents result in the depletion of T cells, B cells, and natural killer (NK) cells and the eradication of immunosuppressive T-regulatory cells and cytokines. CAR-T cells can be infused 2 to 14 days after finishing lymphodepletion.

7

What are the steps for infusion of CAR-T cells?

• A.

  Disease assessment: Some centers perform a bone marrow aspiration/biopsy before lymphodepletion; others perform the assessment after lymphodepletion but before CAR infusion.

• B.

  Infection and organ function: Patients should not have any active infections before CAR-T infusion. This includes documenting a negative influenza test during influenza season. The presence of active infection can result in more severe side effects such as severe cytokine release syndrome. Patients should also have adequate organ function before infusion.

• C.

  Setting for infusion: CAR-T cells can be infused in the inpatient or outpatient setting. If performed outpatient, it is important that the patient stays within close proximity of the treating center.

• D.

  Infusion: A single dose of tisagenlecleucel generally ranges from 0.2 to 5.0 × 10 ⁶ CAR-T cells/kg for patients up to 50 kg and 0.1 to 2.5 × 10 ⁸ CAR-T cells for patients over 50 kg. Typically, the actual volume of CAR-T cells is small (< 50 mL); thus the infusion can be completed in a few minutes. Infusion-associated reactions are rare, but premedication with acetaminophen and diphenhydramine is advised (corticosteroids need to be avoided).