Discussion
The SNBTS currently provides allogeneic third-party EBV-specific T cells
for patients with relapsed/refractory post-transplant
lymphoproliferative disease (PTLD). As of November 2020 more than 100
patients with relapsed or refractory PTLD have been treated from the
current bank under a Specials license, with a mean overall survival rate
of over 40% at three years post treatment. Patients in this cohort with
PTLD arising after solid organ transplant had better outcomes, with
survival of over 60% at three years post-treatment, and with minimal
adoptive cell therapy-related side-effects [5].
The current bank of EBV-specific T cells was manufactured from
2007-2014, and changes to GMP standards since this period have driven a
requirement to optimize and refine the current manufacturing processes
[20]. More recent methods for generation utilise cytokine release
assays to capture virus-specific T cells, though this requires a
different expansion process. LCL-based stimulation and expansion
protocols are still in regular use for development of anti-cancer
therapies [34-36], and therefore there is a need to identify optimal
methods for production and analysis.
In this study we demonstrated that optimization of the standard
autologous LCL-based method of EBV T cell manufacturing to a fully
GMP-compliant closed-process process is feasible without compromise in
quality of final cell product. The modifications to protocol, reagents
and culture process were assessed principally using flow cytometry,
which provides a rapid and quantitative method for analysis. Robust,
validated flow cytometric assays are a cornerstone of effective
reproducible cell therapy manufacture [21]. The use of flow
cytometric analysis and functional profiling of EBV-specific T cells
through cytokine expression in this study resulted in improved
characterization of both start material and final product, and effective
assessment of in-process culture optima, which has been used for
analysis of other T cell therapeutics including a SARS-CoV-2 T cell
product for COVID-19 treatment [37].
Intracellular cytokine staining for IL-2, TNF-α and
IFN-γ provides a reliable method for
discriminating the differentiation state of T cells [19, 22]. The
combination of multi-parameter cytokine secretion-based phenotyping with
t-SNE analysis forms a powerful tool for dissecting functional
subpopulations within the CD8+ cytotoxic T cell compartment, and was
used as the basis for analysis of improvements and refinements in
manufacturing of the current SNBTS EBV-specific T cell therapy used for
treatment of PTLD [20].
Using the combined surface marker and intracellular cytokine flow
cytometric phenotyping approach we were able to identify that multiple
rounds of LCL stimulation were unnecessary, and that extending
stimulation may increase the level of anergy or loss of function in the
T cells, as identified by a loss of absolute IFN-γ secretion and
increased expression of CD57, a marker of terminal effector
differentiation [23]. However, the reduction in stimulation round to
maximize functional responses needs to be balanced with the requirement
for high yields of cells for treatment of multiple patients from a
single manufacturing run.
Adoptive T cell therapy relies on large-scale expansion of functional T
cells to manufacture clinically relevant numbers for patient infusion,
conventionally through use of standard culture flasks or gas-permeable
bags. The introduction of large volume, high gas exchange culture
vessels (G-Rex flask, Wilson Wolf) has significantly improved the rate
and extent of T cell expansion capacity [24]. The G-Rex flasks are
GMP-compliant and are scalable up to 1L flasks which are qualified as an
FDA Class 1 medical device allowing full closed process manufacture.
This closed process manufacture involves suitable sealed flasks,
transfer bags, heat sealed tubing and the GatheRex cell harvester pump
(Wilson Wolf) to ensure sterility in the clinical product. We identified
that cell yields could also be improved by using G-Rex flasks for
culture with no significant changes in phenotype. A minor change in T
cell composition was identified in the G-Rex cultures, with increase in
the percentage of CD8 cells. This consistency of final product phenotype
was also retained when all reagents were converted to fully
GMP-compliant standards. GMP-compliant medium and cytokines with no
exogenous xenoproteins ensured that the modified process complied with
current regulatory requirements. T cells generated with GMP compliant
reagents and flasks suitable for closed process culture had a
significant increase in retention of the TCM compartment. This has
advantages for persistence of the cell therapy once administered to a
patient [25,26].
A principal concern with the current LCL-based stimulation process is
that high LCL (and therefore viral antigen load) ratio to T cells
combined with multiple rounds could drive T cell exhaustion [27,28]
and the reduced T cell: LCL ratio process outlined here quantified
whether this resulted in functional differences. The LCL process appears
robust, as reduced intensity stimulation over three rounds did not
significantly affect the phenotype of the T cells at end-point, although
the reduced ratio exhibited a significantly enhanced CD8+ cell secretion
of IFN-γ and TNF-α. The only modulation of culture processes that was
not undertaken was to replace or supplement IL-2 with other gamma-chain
specific T cell growth factors. However, other studies have concluded
that changing the cytokine-mediated expansion method from IL-2 to other
cytokines such as IL-7, IL-15, or IL-21 has no significant effect on the
overall phenotype or function of T cells for therapy [29]. The
increased production of IFN-γ and TNF-α in response to stimulation in
the reduced intensity LCL stimulation may suggest products made using
this protocol could have increased effector functions against
viral-infected cells following patient engraftment.
A key feature of this work was to identify a robust panel of surface and
intracellular markers which could effectively classify the T cell
differentiation status and development from initial material through to
final product. Our approach supplies clear data for this, and
demonstrates the utility of this approach for T cell therapies [37].
In addition, the use of t-SNE dimensionality reduction was very
effective at condensing multiple parameters into a single image which
could be used to identify the status of the material at any stage of
manufacture. These images are both illustrative and quantitative and
could therefore be used as part of a standardised product release
process. This cytometric phenotype and analysis approach is sufficiently
adaptable and inclusive that it would suitable for phenotypic and
functional assay of other cell therapies including virus-specific and
genetically-modified T cell therapies [30-33,37].