ADD a bunch of discussion stuff
This commit is contained in:
parent
9f990e5da0
commit
3b28dc5612
175
tex/thesis.tex
175
tex/thesis.tex
|
@ -1366,6 +1366,181 @@ the responses.
|
|||
|
||||
\section{discussion}
|
||||
|
||||
% TODO this is fluffy
|
||||
We have developed a T cell expansion system that recapitulates key features of
|
||||
the in vivo lymph node microenvironment using DMSs functionalized with
|
||||
activating mAbs. This strategy provided superior expansion with higher number of
|
||||
naïve/memory and CD4+ T cells compared to state-of-the-art microbead technology
|
||||
(Figure 2). Other groups have used biomaterials approaches to mimic the in vivo
|
||||
microenvironment13–15,17,34; however, to our knowledge this is the first system
|
||||
that specifically drives naïve/memory and CD4+ T cell formation in a scalable,
|
||||
potentially bioreactor-compatible manufacturing process.
|
||||
|
||||
Memory and naïve T cells have been shown to be important clinically. Compared to
|
||||
effectors, they have a higher proliferative capacity and are able to engraft for
|
||||
months; thus they are able to provide long-term immunity with smaller
|
||||
doses19,35. Indeed, less differentiated T cells have led to greater survival
|
||||
both in mouse tumor models and human patients20,36,37. Furthermore, clinical
|
||||
response rates have been positively correlated with T cell expansion, implying
|
||||
that highly-proliferative naïve and memory T cells are a significant
|
||||
contributor18,38. Circulating memory T cells have also been found in complete
|
||||
responders who received CAR T cell therapy39.
|
||||
|
||||
Similarly, CD4 T cells have been shown to play an important role in CAR T cell
|
||||
immunotherapy. It has been shown that CAR T doses with only CD4 or a mix of CD4
|
||||
and CD8 T cells confer greater tumor cytotoxicity than only CD8 T cells22,40.
|
||||
There are several possible reasons for these observations. First, CD4 T cells
|
||||
secrete proinflammatory cytokines upon stimulation which may have a synergistic
|
||||
effect on CD8 T cells. Second, CD4 T cells may be less prone to exhaustion and
|
||||
may more readily adopt a memory phenotype compared to CD8 T cells22. Third, CD8
|
||||
T cells may be more susceptible than CD4 T cells to dual stimulation via the CAR
|
||||
and endogenous T Cell Receptor (TCR), which could lead to overstimulation,
|
||||
exhaustion, and apoptosis23. Despite evidence for the importance of CD4 T cells,
|
||||
more work is required to determine the precise ratios of CD4 and CD8 T cell
|
||||
subsets to be included in CAR T cell therapy given a disease state.
|
||||
|
||||
% TODO this might be more appropriate for aim 2b where I actually talk about
|
||||
% the signaling and why this might matter
|
||||
There are several plausible explanations for the observed phenotypic differences
|
||||
between beads and DMSs. First, the DMSs are composed of a collagen derivative
|
||||
(gelatin); collagen has been shown to costimulate activated T cells via α1β1 and
|
||||
α2β1 integrins, leading to enhanced proliferation, increased IFNγ production,
|
||||
and upregulated CD25 (IL2Rα) surface expression8,10,11,41,42. Second, there is
|
||||
evidence that providing a larger contact area for T cell activation provides
|
||||
greater stimulation16,43; the DMSs have a rougher interface than the 5 µm
|
||||
magnetic beads, and thus could facilitate these larger contact areas. Third, the
|
||||
DMSs may allow the T cells to cluster more densely compared to beads, as
|
||||
evidenced by the large clusters on the outside of the DMSs (Figure 1f) as well
|
||||
as the significant fraction of DMSs found within their interiors (Supplemental
|
||||
Figure 2a and b). This may alter the local cytokine environment and trigger
|
||||
different signaling pathways. Particularly, IL15 and IL21 are secreted by T
|
||||
cells and known to drive memory phenotype44–46. We noted that the IL15 and IL21
|
||||
concentration was higher in a majority of samples when comparing beads and DMSs
|
||||
across multiple timepoints (Supplemental Figure 18) in addition to many other
|
||||
cytokines. IL15 and IL21 are added exogenously to T cell cultures to enhance
|
||||
memory frequency,45,47 and our data here suggest that the DMSs are better at
|
||||
naturally producing these cytokines and limiting this need. Furthermore, IL15
|
||||
unique signals in a trans manner in which IL15 is presented on IL15R to
|
||||
neighboring cells48. The higher cell density in the DMS cultures would lead to
|
||||
more of these trans interactions, and therefore upregulate the IL15 pathway and
|
||||
lead to more memory T cells.
|
||||
|
||||
% TODO this mentions the DOE which is in the next aim
|
||||
When analyzing all our experiments comprehensively using causal inference, we
|
||||
found that all three of our responses were significantly increased when
|
||||
controlling for covariates (Figure 3, Table 2). By extension, this implies that
|
||||
not only will DMSs lead to higher fold change overall, but also much higher fold
|
||||
change in absolute numbers of memory and CD4+ T cells. Furthermore, we found
|
||||
that using a Grex bioreactor is detrimental to fold change and memory percent
|
||||
while helping CD4+. Since there are multiple consequences to using a Grex
|
||||
compared to tissue-treated plates, we can only speculate as to why this might be
|
||||
the case. Firstly, when using a Grex we did not expand the surface area on which
|
||||
the cells were growing in a comparable way to that of polystyrene plates. In
|
||||
conjunction with our DOE data {Figure X} which shows that high DMS
|
||||
concentrations favor CD4+ and don’t favor memory fraction, one possible
|
||||
explanation is that the T cells spent longer times in highly activating
|
||||
conditions (since the beads and DMSs would have been at higher per-area
|
||||
concentrations in the Grex vs polystyrene plates). Furthermore, the simple fact
|
||||
that the T cells spent more time at high surface densities could simply mean
|
||||
that the T cells didn’t expands as much due to spacial constraints. This would
|
||||
all be despite the fact that Grex bioreactors are designed to lead to better T
|
||||
cell expansion due to their gas-permeable membranes and higher media-loading
|
||||
capacities. If anything, our data suggests we were using the bioreactor
|
||||
sub-optimally, and the hypothesized causes for why our T cells did not expand
|
||||
could be verified with additional experiments varying the starting cell density
|
||||
and/or using larger bioreactors.
|
||||
|
||||
A key question in the space of cell manufacturing is that of donor variability.
|
||||
To state this precisely, this is a second order interaction effect that
|
||||
represents the change in effect of treatment (eg bead vs DMS) given the donor.
|
||||
While our meta-analysis was relatively large compared to many published
|
||||
experiments usually seen for technologies at this developmental stage, we have a
|
||||
limited ability in answering this question. We can control for donor as a
|
||||
covariate, and indeed our models show that many of the donor characteristics are
|
||||
strongly associated with each response on average, but these are first order
|
||||
effects and represent the association of age, gender, demographic, etc given
|
||||
everything else in the model is held constant. Second order interactions require
|
||||
that our treatments be relatively balanced and random across each donor, which
|
||||
is a dubious assumption for our dataset. However, this can easily be solved by
|
||||
performing more experiments with these restrictions in mind, which will be a
|
||||
subject of our future work.
|
||||
|
||||
Furthermore, this dataset offers an interesting insight toward novel hypothesis
|
||||
that might be further investigated. One limitation of our dataset is that we
|
||||
were unable to investigate the effects of time using a method such as
|
||||
autoregression, and instead relied on aggregate measures such as the total
|
||||
amount of a reagent added over the course of the expansion. Further studies
|
||||
should be performed to investigate the temporal relationship between phenotype,
|
||||
cytokine concentrations, feed rates, and other measurements which may perturb
|
||||
cell cultures, as this will be the foundation of modern process control
|
||||
necessary to have a fully-automated manufacturing system.
|
||||
|
||||
In addition to larger numbers of potent T cells, other advantages of our DMS
|
||||
approach are that the DMSs are large enough to be filtered (approximately 300
|
||||
µm) using standard 40 µm cell filters or similar. If the remaining cells inside
|
||||
that DMSs are also desired, digestion with dispase or collagenase may be used.
|
||||
Collagenase D may be selective enough to dissolve the DMSs yet preserve surface
|
||||
markers which may be important to measure as critical quality attributes CQAs
|
||||
{Figure X}. Furthermore, our system should be compatible with
|
||||
large-scale static culture systems such as the G-Rex bioreactor or perfusion
|
||||
culture systems, which have been previously shown to work well for T cell
|
||||
expansion12,50,51. The microcarriers used to create the DMSs also have a
|
||||
regulatory history in human cell therapies that will aid in clinical
|
||||
translation.; they are already a component in an approved retinal pigment
|
||||
epithelial cell product for Parkinson’s patients, and are widely available in 30
|
||||
countries26.
|
||||
|
||||
It is important to note that all T cell cultures in this study were performed up
|
||||
to 14 days. Others have demonstrated that potent memory T cells may be obtained
|
||||
simply by culturing T cells as little as 5 days using traditional beads30. It is
|
||||
unknown if the naïve/memory phenotype of our DMS system could be further
|
||||
improved by reducing the culture time, but we can hypothesize that similar
|
||||
results would be observed given the lower number of doublings in a 5 day
|
||||
culture. We should also note that we investigated one subtype (\ptmem{}) in
|
||||
this study. Future work will focus on other memory subtypes such as tissue
|
||||
resident memory and stem memory T cells, as well as the impact of using the DMS
|
||||
system on the generation of these subtypes.
|
||||
|
||||
% TODO this sounds sketchy
|
||||
Another advantage is that the DMS system appears to induce a faster growth rate
|
||||
of T cells given the same IL2 concentration compared to beads (Supplemental
|
||||
Figure 8) along with retaining naïve and memory phenotype. This has benefits in
|
||||
multiple contexts. Firstly, some patients have small starting T cell populations
|
||||
(such as infants or those who are severely lymphodepleted), and thus require
|
||||
more population doublings to reach a usable dose. Our data suggests the time to
|
||||
reach this dose would be reduced, easing scheduling a reducing cost. Secondly,
|
||||
the allogeneic T cell model would greatly benefit from a system that could
|
||||
create large numbers of T cells with naïve and memory phenotype. In contrast to
|
||||
the autologous model which is currently used for Kymriah and Yescarta,
|
||||
allogeneic T cell therapy would reduce cost by spreading manufacturing expenses
|
||||
across many doses for multiple patients52. Since it is economically advantageous
|
||||
to grow as many T cells as possible in one batch in the allogeneic model
|
||||
(reduced start up and harvesting costs, fewer required cell donations), the DMSs
|
||||
offer an advantage over current technology.
|
||||
|
||||
% TODO this is already stated in the innovation section
|
||||
It should be noted that while we demonstrate a method providing superior
|
||||
performance compared to bead-based expansion, the cell manufacturing field would
|
||||
tremendously benefit from simply having an alternative to state-of-the-art
|
||||
methods. The patents for bead-based expansion are owned by few companies and
|
||||
licensed accordingly; having an alternative would provide more competition in
|
||||
the market, reducing costs and improving access for academic researchers and
|
||||
manufacturing companies.
|
||||
|
||||
% TODO this isn't relevent to this aim but should be said somewhere
|
||||
Finally, while we have demonstrated the DMS system in the context of CAR T
|
||||
cells, this method can theoretically be applied to any T cell immunotherapy
|
||||
which responds to anti-CD3/CD28 mAb and cytokine stimulation. These include
|
||||
tumor infiltrating lymphocytes (TILs), virus-specific T cells (VSTs), T cells
|
||||
engineered to express γδTCR (TEGs), γδ T cells, T cells with transduced-TCR, and
|
||||
CAR-TCR T cells53–58. Similar to CD19-CARs used in liquid tumors, these T cell
|
||||
immunotherapies would similarly benefit from the increased proliferative
|
||||
capacity, metabolic fitness, migration, and engraftment potential characteristic
|
||||
of naïve and memory phenotypes59–61. Indeed, since these T cell immunotherapies
|
||||
are activated and expanded with either soluble mAbs or bead-immobilized mAbs,
|
||||
our system will likely serve as a drop-in substitution to provide these
|
||||
benefits.
|
||||
|
||||
\chapter{aim 2}\label{aim2}
|
||||
|
||||
\section{introduction}
|
||||
|
|
Loading…
Reference in New Issue