Peri-CAR T-Cell Management

Screening

Once identified as a candidate for chimeric antigen receptor (CAR) T-cell therapy, patients must undergo screening for eligibility. Prediagnostic evaluations are required to assess patient candidacy to undergo CAR treatment, fulfill regulatory criteria for cell product collection, and satisfy insurance standards for reimbursement.

Patient's disease burden should be reevaluated per usual practice prior to receiving CAR T-cell therapy. Target antigen expression should be assessed by tissue biopsy prior to initiation of collection or treatment. This is particularly important if patients have received prior therapies sharing the same antigen specificity as the CAR T-cell, such as blinatumomab prior to CD19-targeted CARs, which may predispose to escape mutations, leading to loss of antigen expression.

Interestingly, there has been evidence of CAR T-cell activity in antigen-negative disease; for example, in the phase II study of CD19-targeted axicabtagene ciloleucel (Yescarta), eight patients with CD19-negative disease were treated with no suggestion of reduced efficacy. As a result, there is no absolute requirement for CD19 expression prior to treatment with axicabtagene. However, based on the mechanism of action of CAR T-cells, it is unlikely that CAR T-cells can recognize truly CD19 negative tumors, and “CD19-negative” responders likely express low levels of CD19 below the detectable level of the diagnostic assay. Therefore, the decision to treat an “antigen-negative” tumor should be made cautiously and only after discussion with pathology and CAR T-cell experts.

Location of disease is also important, as the successful use of cellular therapy is predicated on the ability of infused cells to traffic to the disease site. Regarding CD19 CAR T-cells, it has been shown that cells will penetrate the central nervous system (CNS) and mediate CNS lymphoma regression. However, all large trials of currently available, commercially developed CAR products have excluded active CNS disease (e.g., CNS 3 in B-cell acute lymphoblastic leukemia [ALL] or radiographic evidence of disease for B-cell non-Hodgkin's lymphoma [NHL]). Patients previously and successfully treated for CNS disease have generally not been excluded on these trials. However, it is currently not well characterized what role CNS disease has on the occurrence or severity of postinfusion neurologic toxicity. Physicians should consider precollection and/or pretreatment diagnostics for CNS disease such as brain MRI or lumbar puncture as clinically appropriate. Future clinical trials and/or next-generation CAR T-cell designs may target patients with active CNS lesions.

Patient screening also involves a detailed clinical history including recent travel, immunizations, transfusion history, and infectious disease exposure. Basic screening laboratory testing including infectious disease markers (IDMs) should be obtained prior to collection. Additional diagnostics including echocardiogram and 12-lead electrocardiogram ( Table 4.1 ) should be obtained when clinically indicated. Organ function assessment should confirm patient suitability to withstand potential adverse events following treatment including cytokine release syndrome (CRS) or other target organ toxicity. A pregnancy test for females of reproductive potential should be confirmed negative prior to collection and treatment.

Regulatory standards for IDMs are set by federal agencies such as the Food and Drug Administration, accrediting organizations such as the Foundation for the Accreditation of Cell Therapy (FACT), and the American Association of Blood Banks (AABB), as well as state and local agencies. The timing of IDM testing varies in these standards between 7 to 30 days prior to collection. Clinicians should check with national, local, and institutional regulatory agencies to determine additional testing requirements.

Critical to successful use of CAR T-cell therapy is the patient's ability to withstand any potential postinfusion toxicity such as CRS and/or end organ toxicity. To this end, organ function criteria to qualify for treatment can be extrapolated from the relevant clinical trials of commercially approved CAR T-cell products, which are described in Table 4.2 . Of note, all trials required robust performance status (ECOG of ≤1 or Karnofsky/Lansky ≥60%). Additional studies in populations that would not have met eligibility criteria for the registration trials are ongoing.

Informed Consent

Informed consent is a required element for institutions in compliance with FACT Immune Effector Cell (IEC) guidelines. Due to the complexity and potential toxicity of CAR T-cell therapy, we recommend the development of institution-specific consent procedures for all patients treated outside of a clinical trial. Informed consent should explain the rationale for the use of CAR T-cells in terms the patient can understand including clinical benefit of therapy, risk of toxicities, and alternative treatment options. Long-term toxicity (e.g., long-term B-cell aplasia with CD19-specific CAR T-cells) should also be described, including theoretical adverse events such as insertional oncogenesis, when appropriate.

Consent should be documented per institutional guidelines. Any additional investigational protocols or biospecimen banking will require separate consents, as will central line placement and leukapheresis. Practical requirements, such as the need for a caregiver and need to reside in proximity to the treatment facility, should also be highlighted.

Collection

Most commonly, T-cells for CAR modification are harvested from leukapheresis of peripheral blood mononuclear cells. Leukapheresis involves removal of whole blood, separation of peripheral blood mononuclear cells, and reinfusion of unselected components. Compared with peripheral blood stem cell collection, which typically takes 4–6 hours per day over several days, collection of mononuclear cells for CAR T-cell manufacture is quicker, usually requiring a single 2–3 hour collection.

Standards for the necessary personnel, physical space, and organization of apheresis facilities are provided by regulatory agencies including FACT. In addition, for the initial development of commercial CAR T-cell products, manufacturers have required a standardization and assessment process of apheresis facilities, which is required prior to selection as a treatment site. These protocols may change as CAR T-cell commercial products are brought to a wider group of facilities.

Leukapheresis is generally well tolerated, and the rate of serious adverse events is extremely low. Commonly reported low-grade adverse effects include fatigue, nausea, dizziness, cold sensation, and tingling in the fingers and mouth. More serious adverse events include pre-syncopal and syncopal reactions, as well as citrate-related hypocalcemia, which are rare, reported at 0.37% in one series. ACE inhibitors should be held for 24 hours prior to collection to reduce the risk of bradykinin-mediated reactions. Most pediatric and adult patients will require a dedicated apheresis catheter. At our center, the decision as to whether a large bore, nontunneled catheter is placed is made in discussion with transfusion medicine physicians, nurses, and as per institutional guidelines developed for IEC collection.

Since the desired cells are lymphocytes, no myeloid growth factors, mobilization factors, or chemotherapy is given to enhance collection. In fact, patients require a washout period without any active treatment to optimize lymphocyte counts. The tisagenlecleucel trials advised 2 weeks without cytotoxic chemotherapy prior to collection ( Table 4.3 ). Pegylated asparaginase was held at least 4 weeks prior to collection, and therapeutic doses of steroid were held for at least 72 hours prior to collection. Since peripheral lymphocyte count is predictive of CD3+ T-cell harvest and ultimate CAR T-cell dose generated, the axicabtagene ciloleucel trial in NHL required a minimum absolute lymphocyte count (ALC) of 100 cells/μL, whereas the tisagenlecleucel trial suggested a minimum ALC of 500 cells/μL and/or a minimum CD3+ cell count of 150 cells/μL. Thus, acceptable cutoffs vary by trials and products.

For patients receiving CAR T-cells after allogeneic stem cell transplantation, immunosuppressive drugs (e.g., calcineurin inhibitors) that suppress T-cell expansion and function must be held for at least 2 weeks prior to leukapheresis. Additionally, CAR T-cell candidates who have had a prior allogeneic transplant cannot have active graft-versus-host disease or be in the early posttransplant period where immunosuppression is required or lymphopenia is expected. Clinical trials typically required a period of at least 3 months between transplant and CAR therapy, but the period required will depend on immune reconstitution, graft-versus-host disease, and ability to wean from immunosuppression.

Numerous product-specific factors can affect the quality of leukapheresis. For example, the amount of circulating T-cells is predictive of yield, whereas other cells in the collection product, particularly myeloid cells, can inhibit subsequent T-cell growth. Furthermore, the identity and composition of the T-cells themselves can affect quality. In pediatric ALL and NHL, patients with a higher proportion of “early lineage” T-cell phenotypes such as T central memory and T stem cell memory cells had better in vitro CAR expansion. This mirrors data that suggest effector differentiation after T-cell manufacturing and expansion leads to impaired proliferation and efficacy.

Manufacturing

Following apheresis, T-cells must be selected, genetically modified, and expanded to produce CAR T-cells. The manufacturing process for both academic medical centers and commercial manufacturers is reviewed in depth by Wang and Riviere, Levine et al., and Roberts et al. and will be discussed only briefly here. The initial step for CAR T-cell manufacturing includes the isolation, activation, and expansion of the target T-cell population. Several methodologies are utilized, including the commercial expansion platforms such as Life Sciences/Invitrogen Dynabeads and Miltenyi TransAct beads, which stimulate T-cells via CD3 and CD28. Other platforms such as soluble CD3 antibodies are also utilized, and the optimal method is still an area of active research.

Viral and nonviral methods are next used to genetically modify the targeted T-cell population for CAR expression. Most commonly, gamma retroviral and/or lentiviral vectors are utilized to introduce the CAR vector into the target T-cell population, as this method allows for stable integration of the CAR into the T-cell genome. The transduction and expansion process takes place in a bioreactor culture system to avoid contamination and adhere to good manufacturing practice (GMP) requirements.

Prior to release, cell products must meet release criteria including (1) dose threshold with predetermined minimum viability, (2) identity criteria such as percentage of cells expressing the CAR construct, and (3) safety criteria such as Gram stain, endotoxin, and mycoplasma levels. Assays to determine potency may also be performed at this time. A major commercial innovation has been the development of shipping and logistics infrastructure necessary to produce and track cell products. Failure to fulfill predefined release criteria is a major obstacle to the utilization of this therapy, as those products may be rendered unusable (e.g., manufacture failure), though release criteria standards and the use of “out-of-specification” products are under investigation.