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I. Yakoub-Agha et al.
order to prevent or rapidly treat complications that might arise while awaiting the arrival of the CAR T-cell product.
Lymphodepleting conditioning
The use of lymphodepleting (LD) conditioning prior to the CAR T-cell infusion creates a ‘favorable’ environment for CAR T-cell expansion and survival in vivo, probably by eliminating regulatory T cells.27 In addition, it can lead to the upregulation of tumor immunogenicity and improve disease control.28 Furthermore, there are data demonstrat- ing that LD conditioning works to promote homeostatic proliferation of adoptively transferred T cells via increases in the pro-survival/proliferation cytokines, interleukin (IL)-7 and IL-15, and in conjunction with a lack of compe- tition with wildtype T cells.29-31
Many drugs have been used for LD conditioning includ- ing cyclophosphamide, fludarabine, pentostatin and ben- damustine as well as total body irradiation.32 In a clinical trial involving 30 patients with B-ALL at the Fred Hutchinson Cancer Research Center, fludarabine and cyclophosphamide was associated with superior CAR T- cell persistence and better disease-free survival when com- pared to single-agent cyclophosphamide or cyclophos- phamide in combination with etoposide.33,34 Fludarabine- cyclophosphamide is the most widely used LD condition- ing regimen.35,36
LD conditioning is usually administered on a 3-to-5 day schedule prior to the infusion of the CAR T cells. If the center does not have established policies and infrastruc- ture to allow for safe outpatient-based administration, hospitalization is recommended during this period to ensure close monitoring and optimal hydration.
Items to consider before starting LD conditioning are shown in Table 5A.
Laboratory tests to review before starting LD condition- ing are shown in Table 5B.
If there is a long delay (in general, more than 3 weeks) between completing LD conditioning and the subsequent CAR T-cell infusion, and the white blood cell count is >1.0x109/L, then consideration should be given to re-treat- ing the patient with LD chemotherapy prior to adminis- tration of the CAR T cells.
Product receipt and thawing
The currently licensed CAR T-cell products are delivered frozen and must be maintained at very low temperatures during shipping, receipt and temporary storage until they are thawed immediately prior to use. Hospitals have adopt- ed different approaches to product receipt, taking into account local organizational and regulatory issues. The unit receiving the CAR T-cell products will need to have suitable storage containers and facilities for genetically manipulated material; depending on national legislation, a storage site may need regulatory approval as gene therapy medicinal products are also genetically modified organisms.21 As the manufacturing companies use differently sized cryostorage cassettes, custom-made cryo racks, at least one for each company, must be obtained. A storage site with secured access and an adequate number of trained staff licensed to work with biohazards and liquid nitrogen are required, both at the hospital pharmacy and at the cell processing facility.
The designated receiving laboratory will receive advance notice from the manufacturer and the product will be delivered in a sealed liquid nitrogen dewar (vacuum flask). Upon receipt, the seals of the dewar are inspected for breaches; seals are broken, if applicable; the temperature log is read out; and the product is inspected for bag integri- ty and identity according to the label; the bag in its cassette is subsequently transferred to a liquid nitrogen storage con- tainer until it is brought to the bedside. The company-spe- cific product receipt documentation must be completed; personnel authorized to handle products are provided with specific and detailed training from the relevant manufac- turer. When the ward is ready to receive the product, the cassette is transferred to a laboratory dewar and this is transported to the ward.
In some countries, the use of water baths, carefully cali- brated to 35-37°C, remains acceptable; use of an automat- ed thawing device is preferable. Representative examples of such devices are the SaharaTM (Sarstedt) and PlasmathermTM (Barkey) devices. While the thawing of CAR T cells is, in principle, the same as for cryopreserved hematopoietic progenitor cells collected by apheresis, the much smaller volumes of CAR T-cell products only require very short thawing times. We recommend that thawing times be established locally with similarly-sized mock products, ideally with mononuclear cell suspensions in protein-saline-dimethylsulfoxide freezing buffer and test- ing of post-thaw viability, but at a minimum, with protein- saline-dimethylsulfoxide buffer without cells and observa- tion of the time until the buffer assumes the slushy consis- tency of a ready-to-spike cryo product. If thawing is con- ducted in a water bath, the spike ports that protrude out of the water must be carefully massaged to ensure that they thaw in synchrony with the rest of the product. The spike ports of the thawed product are uncapped, disinfected and aseptically spiked with the transfusion set, the air trap is filled completely with the cell suspension (no falling drops, as this shears cells) and air is evacuated from the infusion line. The individual responsible for the thawing and prepa- ration of the infusion varies between countries and health care systems. We propose that the decision as to who is responsible should be primarily based on competence, meaning that those individuals who normally thaw autol- ogous transplants are likely best qualified. On this basis, pharmacy, processing facility and clinical transplant staff are all acceptable candidates and bedside thawing is prefer- able.
Infusion of chimeric antigen receptor T cells
Before starting to thaw the CAR T-cell product, the patient should be assessed. Some factors to consider are shown in Table 6. A transfusion set is required for the administration of the cells. In general, a typical transfusion filter set with 50-200 μm pore size is used; this is, in fact, mandatory in some countries. Importantly, fluid infusion sets are not suitable because of the sub-micrometer bacte- rial filters. Transfusion sets with leukocyte-depletion fil- ters are also unacceptable. It should be noted that the manufacturers recommend the use of non-filtered tubing sets although our recommendations, and some local regu- latory requirements, deviate from this approach.
Pre-medication to prevent adverse reactions is reason- able with the important exception of corticosteroids
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