Successful leukapheresis and CAR T cell manufacturing
The current recommended dose of tisagenlecleucel contains 0.2 to 5.0
x106 CAR-positive viable T cells per kg of body weight
for patients 50kg or less, or 0.1 to 2.5 x108CAR-positive viable T cells for patients more than 50kg40. In order to achieve this dose, an adequate
quantity of T cells must first be collected from the patient; therefore,
a minimum absolute lymphocyte count (ALC) of ~500
cells/uL and a CD3+ cell count of ~150 cells/uL is
recommended prior to starting apheresis 41. Factors
that would affect both ALC and CD3+ cell count include timing of
proximal cytotoxic therapy or progressive leukemic disease leading to
bone marrow replacement of cancer cells. Some patients are never able to
achieve these minimal peripheral blood parameters due to the nature of
their highly refractory B-ALL. For these patients, the prospect of an
allogeneic CAR T cell product, which remain in early clinical
investigation, is attractive 42.
Once an adequate quantity of T cells is collected, characteristics of
the leukapheresis product may directly affect the quality and/or
performance of the CAR T cell product. Predicting the performance of CAR
T cell products is quite difficult using in vitro testing, so at this
time, performance is best assessed after infusion into the patient using
the metrics of disease response, in vivo proliferation and CAR T cell
persistence. Expansion is a vital element to disease response. In the
ELIANA trial, expansion (measured as the geometric mean of the area
under the concentration-time curve in peripheral blood from time 0 to
day 28 as expressed in copies per microgram of DNA times days) was
315,000 in patients with a response and 301,000 in patients without a
response21. In addition, responders to
tisagenlecleucel have a shorter median time to maximum expansion of 11
days compared to 13 days in non-responding patients21,37. Much research has been dedicated to
understanding the mechanisms behind poor expansion and persistence of
the T cell product in order to maximize the anti-leukemic property of
this drug.
First, recent studies have demonstrated that T cell phenotype plays an
important role in predicting a CAR T cell product’s subsequent clinical
activity. The presence of naïve and early memory T cells with
significant proliferative potenital in the pre-manufactured product was
found to correlate with a biomarker of successful CAR performance in
pediatric B-ALL 43. Peripheral blood samples that
contained a higher percentage of naïve and stem central memory cells
directly correlates with T cell expansion potential in vivo44, and CAR T cell products that contain more central
memory T cells persist longer, which can mediate a more successful
clinical response 45,46. Interestingly, it has also
been recently shown that the distribution of T cell subsets in
peripheral blood samples varied across different pediatric cancers, thus
indicating that disease biology may further play a role in altering the
patients’ T cell developmental phenotype at collection44, which can inform collection practices as CAR T
cells are applied to other diseases.
Another factor that can contribute to differences in T cell fitness lies
in the previously exposed chemotherapy regimen. For example,
chemotherapy regimens containing clofarabine or doxorubicin has been
implicated in both quantitatively insufficient and poor-quality CAR T
cell products 44,47 (we strongly discourage use of
clofarabine prior to collection). Additionally, clinical data suggest
that prior treatment with cyclophosphamide and cytarabine selectively
reduces early lineage T cells that are associated with productive CAR T
cell expansion 43. Therefore, it is important to
understand how different chemotherapies affect T cells as it can have a
direct impact on the quality of T cells collected. Early collection of T
cells prior to intensive regimens of cytotoxic chemotherapy should be
considered in patients identified as having a high risk of relapse or
those with relapsed disease, which may improve the quality of the
apheresis product and, thus, the resultant manufactured CAR T cell
product.
Finally, differences in the CAR design and manufacturing processes may
also play an important role in predicting the clinical performance of
the final CAR T product. CAR T cell products that have shown efficacy in
clinical trials to this point, including tisagenlecleucel, are second
generation products 48-50. Tisagenlecleucel utilizes a
4-1BB based co-stimulatory domain and has been shown to persist in the
blood for a median duration of 168 days (range 20-167 days) compared to
CAR constructs using CD28 co-stimulatory domains, whose persistence is
approximately 1 to 2 months 21,47,51. There are
patients from the first CHOP studies with persistent CAR T cells for
5-10 years. This longer persistence is likely due to the reduced
propensity for T cell exhaustion induced by tonic CAR signaling when
co-stimulation is mediated by a 4-1BB domain 52. Data
thus far suggests that CAR persistence is an important factor in
achieving a durable remission in ALL without further anti-leukemia
therapy. This association is harder to discern in lymphoma patients
treated to date 53.