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ENH finish proofreading aim 1

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@ -2066,9 +2066,6 @@ MATLAB code and output for all the wash step calculations are given in
\label{fig:dms_expansion}
\end{figure*}
% DISCUSSION krish seems concerned about this isotype control figure, add some
% discussion saying that IL2 does not spontaneously activate T cells to appease
% him
We next sought to determine how our \glspl{dms} could expand T cells compared to
state-of-the-art methods used in industry. All bead expansions were performed as
per the manufacturer’s protocol, with the exception that the starting cell
@ -2082,7 +2079,9 @@ significantly greater expansion after \SI{12}{\day} of culture
(\cref{fig:dms_expansion_bead}). We also observed no T cell expansion using
\glspl{dms} coated with an isotype control mAb compared to \glspl{dms} coated
with \acd{3}/\acd{28} \glspl{mab} (\cref{fig:dms_expansion_isotype}), confirming
specificity of the expansion method.
specificity of the expansion method. Given that \il{2} does not lead to
expansion on its own, we know that the expansion of the T cells shown here is
due to the \acd{3} and \acd{28} \glspl{mab}\cite{Waysbort2013}.
\begin{figure*}[ht!]
\begingroup
@ -2577,11 +2576,7 @@ technology (\cref{fig:dms_exp}). Other groups have used biomaterials approaches
to mimic the \invivo{} microenvironment\cite{Cheung2018, Rio2018, Delalat2017,
Lambert2017, Matic2013}; 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. Given that the
isotype-control \glspl{mab} does not lead to expansion and that \il{2} does not
lead to expansion on its own (\cref{fig:dms_expansion_isotype}), we know that
the expansion of the T cells shown here is due to the \acd{3} and \acd{28}
\glspl{mab}\cite{Waysbort2013}.
potentially bioreactor-compatible manufacturing process.
Memory and naïve T cells have been shown to be important clinically. Compared to
\glspl{teff}, they have a higher proliferative capacity and are able to engraft
@ -2602,8 +2597,8 @@ 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 cells\cite{Wang2018}. 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
susceptible than CD4 T cells to dual stimulation via the \gls{car} and
endogenous \gls{tcr} , which could lead to overstimulation, exhaustion, and
apoptosis\cite{Yang2017}. 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.
@ -2611,32 +2606,32 @@ subsets to be included in CAR T cell therapy given a disease state.
% DISCUSSION 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.
controlling for covariates (\cref{fig:metaanalysis_fx,tab:ci_controlled}). By
extension, this implies that not only will \glspl{dms} 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. 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) which has been shown to skew toward \gls{teff}
populations\cite{Lozza2008}. 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
represents the change in effect of treatment (eg bead vs \gls{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
@ -2646,7 +2641,7 @@ 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.
subject of 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
@ -2658,73 +2653,51 @@ 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 expansion\cite{Forget2014,
Gerdemann2011, Jin2012}. 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
countries\cite{purcellmain}.
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
beads\cite{Ghassemi2018}. 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.
% 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
% beads\cite{Ghassemi2018}. 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.
% DISCUSSION 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 patients\cite{Harrison2019}. 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.
% 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 patients\cite{Harrison2019}. 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.
% DISCUSSION this is already stated in the innovation section
It should be noted that while we demonstrate a method providing superior
The \gls{dms} system could be used as a drop in replacement for beads in many of
current allogeneic therapies. Indeed, given its higher potential for expansion
(\cref{fig:dms_exp,tab:ci_controlled}, it may work in cases where the beads fail
(although this would need to be tested by gathering data with many unhealthy
donors). However, in the autologous setting patients only need a fixed dose, and
thus any expansion beyond the indicated dose would be wasted. Given this, it
will be interesting to apply this technology in an allogeneic paradigm where
this increased expansion potential would be well utilized.
Finally, we should note that while we demonstrated 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
tremendously benefit from simply having an alternative to state-of-the-art bead
based expansion. 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.
% DISCUSSION 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
\glspl{til}, virus-specific T cells (VSTs), T cells engineered to express
$\upgamma\updelta$TCR (TEGs), $\upgamma\updelta$ T cells, T cells with
transduced-TCR, and CAR-TCR T cells\cite{Cho2015, Straetemans2018, Robbins2011,
Brimnes2012, Baldan2015, Walseng2017}. 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 phenotypes\cite{Blanc2018, Lalor2016,
Rosato2019}. 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 2a}\label{aim2a}
\section{introduction}
@ -4368,11 +4341,25 @@ targeted specification. These \gls{qc} steps all rely on common, relatively
cost-effective assays such as the \gls{haba} assay, \gls{bca} assay, and
\glspl{elisa}, thus other labs and commercial entities should be able to perform
them. The microcarriers themselves are an off-the-shelf product available from
reputable vendors, further enhancing translatability. On average, we
demonstrated that the \gls{dms} outperforms state-of-the-art bead-based T cell
expansion technology in terms of total fold expansion, \ptmemp{}, and \pthp{} by
\SI{143}{\percent}, \SI{2.5}{\percent}, and \SI{9.8}{\percent} controlling for
donor, operator, and a variety of process conditions.
reputable vendors, and they have a regulatory history in human cell therapies
that will aid in clinical translation\cite{purcellmain}. Both these will help
in translatability. On average, we demonstrated that the \gls{dms} outperforms
state-of-the-art bead-based T cell expansion technology in terms of total fold
expansion, \ptmemp{}, and \pthp{} by \SI{143}{\percent}, \SI{2.5}{\percent}, and
\SI{9.8}{\percent} controlling for donor, operator, and a variety of process
conditions.
In addition to larger numbers of potent T cells, other advantages of our
\gls{dms} approach are that the \glspl{dms} are large enough to be filtered
(approximately \SI{300}{\um}) using standard \SI{40}{\um} cell filters or
similar. If the remaining cells inside that \glspl{dms} are also desired,
digestion with dispase or collagenase may be used. Collagenase D may be
selective enough to dissolve the \gls{dms} yet preserve surface markers which
may be important to measure as critical quality attributes \glspl{cqa}
(\cref{fig:collagenase_fx}). 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
expansion\cite{Forget2014, Gerdemann2011, Jin2012}.
In \cref{aim2a}, we developed a modeling pipeline that can be used by commercial
entities as the scale up this process to identify \glspl{cqa} and \gls{cpp}.
@ -4428,6 +4415,21 @@ factor given that it was nearly the same in the first experiment between
\gls{dms} and bead groups despite the clear advantage seen in the \gls{dms}
group.
Finally, while we have demonstrated the \gls{dms} system in the context of
\gls{car} T cells, this method can theoretically be applied to any T cell
immunotherapy which responds to \acd{3}/\acd{28} \gls{mab} and cytokine
stimulation. These include \glspl{til}, virus-specific T cells, T cells
engineered to express $\upgamma\updelta$ \glspl{tcr}, $\upgamma\updelta$ T
cells, T cells with transduced-\gls{tcr}, and \gls{car}-\gls{tcr} T
cells\cite{Cho2015, Straetemans2018, Robbins2011, Brimnes2012, Baldan2015,
Walseng2017}. Similar to \glspl{car} against CD19 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 phenotypes\cite{Blanc2018, Lalor2016, Rosato2019}. Indeed,
since these T cell immunotherapies are activated and expanded with either
soluble \glspl{mab} or bead-immobilized \glspl{mab}, our system will likely
serve as a drop-in substitution to provide these benefits.
\section{future directions}
There are several important next steps to perform with this work, many of which