ENH finish proofreading aim 1
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tex/thesis.tex
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tex/thesis.tex
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@ -2066,9 +2066,6 @@ MATLAB code and output for all the wash step calculations are given in
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\label{fig:dms_expansion}
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\end{figure*}
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% DISCUSSION krish seems concerned about this isotype control figure, add some
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% discussion saying that IL2 does not spontaneously activate T cells to appease
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% him
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We next sought to determine how our \glspl{dms} could expand T cells compared to
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state-of-the-art methods used in industry. All bead expansions were performed as
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per the manufacturer’s protocol, with the exception that the starting cell
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@ -2082,7 +2079,9 @@ significantly greater expansion after \SI{12}{\day} of culture
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(\cref{fig:dms_expansion_bead}). We also observed no T cell expansion using
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\glspl{dms} coated with an isotype control mAb compared to \glspl{dms} coated
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with \acd{3}/\acd{28} \glspl{mab} (\cref{fig:dms_expansion_isotype}), confirming
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specificity of the expansion method.
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specificity of the expansion method. Given that \il{2} does not lead to
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expansion on its own, we know that the expansion of the T cells shown here is
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due to the \acd{3} and \acd{28} \glspl{mab}\cite{Waysbort2013}.
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\begin{figure*}[ht!]
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\begingroup
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@ -2577,11 +2576,7 @@ technology (\cref{fig:dms_exp}). Other groups have used biomaterials approaches
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to mimic the \invivo{} microenvironment\cite{Cheung2018, Rio2018, Delalat2017,
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Lambert2017, Matic2013}; however, to our knowledge this is the first system
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that specifically drives naïve/memory and CD4+ T cell formation in a scalable,
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potentially bioreactor-compatible manufacturing process. Given that the
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isotype-control \glspl{mab} does not lead to expansion and that \il{2} does not
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lead to expansion on its own (\cref{fig:dms_expansion_isotype}), we know that
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the expansion of the T cells shown here is due to the \acd{3} and \acd{28}
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\glspl{mab}\cite{Waysbort2013}.
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potentially bioreactor-compatible manufacturing process.
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Memory and naïve T cells have been shown to be important clinically. Compared to
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\glspl{teff}, they have a higher proliferative capacity and are able to engraft
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@ -2602,8 +2597,8 @@ these observations. First, CD4 T cells secrete proinflammatory cytokines upon
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stimulation which may have a synergistic effect on CD8 T cells. Second, CD4 T
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cells may be less prone to exhaustion and may more readily adopt a memory
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phenotype compared to CD8 T cells\cite{Wang2018}. Third, CD8 T cells may be more
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susceptible than CD4 T cells to dual stimulation via the CAR and endogenous T
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Cell Receptor (TCR), which could lead to overstimulation, exhaustion, and
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susceptible than CD4 T cells to dual stimulation via the \gls{car} and
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endogenous \gls{tcr} , which could lead to overstimulation, exhaustion, and
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apoptosis\cite{Yang2017}. Despite evidence for the importance of CD4 T cells,
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more work is required to determine the precise ratios of CD4 and CD8 T cell
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subsets to be included in CAR T cell therapy given a disease state.
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@ -2611,32 +2606,32 @@ subsets to be included in CAR T cell therapy given a disease state.
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% DISCUSSION this mentions the DOE which is in the next aim
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When analyzing all our experiments comprehensively using causal inference, we
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found that all three of our responses were significantly increased when
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controlling for covariates (Figure 3, Table 2). By extension, this implies that
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not only will DMSs lead to higher fold change overall, but also much higher fold
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change in absolute numbers of memory and CD4+ T cells. Furthermore, we found
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that using a Grex bioreactor is detrimental to fold change and memory percent
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while helping CD4+. Since there are multiple consequences to using a Grex
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compared to tissue-treated plates, we can only speculate as to why this might be
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the case. Firstly, when using a Grex we did not expand the surface area on which
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the cells were growing in a comparable way to that of polystyrene plates. In
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conjunction with our DOE data {Figure X} which shows that high DMS
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concentrations favor CD4+ and don’t favor memory fraction, one possible
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explanation is that the T cells spent longer times in highly activating
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conditions (since the beads and DMSs would have been at higher per-area
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concentrations in the Grex vs polystyrene plates). Furthermore, the simple fact
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that the T cells spent more time at high surface densities could simply mean
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that the T cells didn’t expands as much due to spacial constraints. This would
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all be despite the fact that Grex bioreactors are designed to lead to better T
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cell expansion due to their gas-permeable membranes and higher media-loading
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capacities. If anything, our data suggests we were using the bioreactor
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sub-optimally, and the hypothesized causes for why our T cells did not expand
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could be verified with additional experiments varying the starting cell density
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and/or using larger bioreactors.
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controlling for covariates (\cref{fig:metaanalysis_fx,tab:ci_controlled}). By
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extension, this implies that not only will \glspl{dms} lead to higher fold
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change overall, but also much higher fold change in absolute numbers of memory
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and CD4+ T cells. Furthermore, we found that using a Grex bioreactor is
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detrimental to fold change and memory percent while helping CD4+. Since there
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are multiple consequences to using a Grex compared to tissue-treated plates, we
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can only speculate as to why this might be the case. Firstly, when using a Grex
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we did not expand the surface area on which the cells were growing in a
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comparable way to that of polystyrene plates. One possible explanation is that
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the T cells spent longer times in highly activating conditions (since the beads
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and DMSs would have been at higher per-area concentrations in the Grex vs
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polystyrene plates) which has been shown to skew toward \gls{teff}
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populations\cite{Lozza2008}. Furthermore, the simple fact that the T cells spent
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more time at high surface densities could simply mean that the T cells didn’t
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expands as much due to spacial constraints. This would all be despite the fact
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that Grex bioreactors are designed to lead to better T cell expansion due to
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their gas-permeable membranes and higher media-loading capacities. If anything,
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our data suggests we were using the bioreactor sub-optimally, and the
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hypothesized causes for why our T cells did not expand could be verified with
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additional experiments varying the starting cell density and/or using larger
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bioreactors.
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A key question in the space of cell manufacturing is that of donor variability.
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To state this precisely, this is a second order interaction effect that
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represents the change in effect of treatment (eg bead vs DMS) given the donor.
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While our meta-analysis was relatively large compared to many published
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represents the change in effect of treatment (eg bead vs \gls{dms}) given the
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donor. While our meta-analysis was relatively large compared to many published
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experiments usually seen for technologies at this developmental stage, we have a
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limited ability in answering this question. We can control for donor as a
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covariate, and indeed our models show that many of the donor characteristics are
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@ -2646,7 +2641,7 @@ everything else in the model is held constant. Second order interactions require
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that our treatments be relatively balanced and random across each donor, which
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is a dubious assumption for our dataset. However, this can easily be solved by
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performing more experiments with these restrictions in mind, which will be a
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subject of our future work.
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subject of future work.
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Furthermore, this dataset offers an interesting insight toward novel hypothesis
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that might be further investigated. One limitation of our dataset is that we
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@ -2658,73 +2653,51 @@ cytokine concentrations, feed rates, and other measurements which may perturb
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cell cultures, as this will be the foundation of modern process control
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necessary to have a fully-automated manufacturing system.
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In addition to larger numbers of potent T cells, other advantages of our DMS
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approach are that the DMSs are large enough to be filtered (approximately 300
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µm) using standard 40 µm cell filters or similar. If the remaining cells inside
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that DMSs are also desired, digestion with dispase or collagenase may be used.
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Collagenase D may be selective enough to dissolve the DMSs yet preserve surface
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markers which may be important to measure as critical quality attributes CQAs
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{Figure X}. Furthermore, our system should be compatible with large-scale static
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culture systems such as the G-Rex bioreactor or perfusion culture systems, which
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have been previously shown to work well for T cell expansion\cite{Forget2014,
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Gerdemann2011, Jin2012}. The microcarriers used to create the DMSs also have a
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regulatory history in human cell therapies that will aid in clinical
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translation.; they are already a component in an approved retinal pigment
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epithelial cell product for Parkinson’s patients, and are widely available in 30
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countries\cite{purcellmain}.
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It is important to note that all T cell cultures in this study were performed up
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to 14 days. Others have demonstrated that potent memory T cells may be obtained
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simply by culturing T cells as little as 5 days using traditional
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beads\cite{Ghassemi2018}. It is unknown if the naïve/memory phenotype of our DMS
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system could be further improved by reducing the culture time, but we can
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hypothesize that similar results would be observed given the lower number of
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doublings in a 5 day culture. We should also note that we investigated one
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subtype (\ptmem{}) in this study. Future work will focus on other memory
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subtypes such as tissue resident memory and stem memory T cells, as well as the
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impact of using the DMS system on the generation of these subtypes.
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% It is important to note that all T cell cultures in this study were performed up
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% to 14 days. Others have demonstrated that potent memory T cells may be obtained
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% simply by culturing T cells as little as 5 days using traditional
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% beads\cite{Ghassemi2018}. It is unknown if the naïve/memory phenotype of our DMS
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% system could be further improved by reducing the culture time, but we can
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% hypothesize that similar results would be observed given the lower number of
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% doublings in a 5 day culture. We should also note that we investigated one
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% subtype (\ptmem{}) in this study. Future work will focus on other memory
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% subtypes such as tissue resident memory and stem memory T cells, as well as the
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% impact of using the DMS system on the generation of these subtypes.
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% DISCUSSION this sounds sketchy
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Another advantage is that the DMS system appears to induce a faster growth rate
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of T cells given the same IL2 concentration compared to beads (Supplemental
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Figure 8) along with retaining naïve and memory phenotype. This has benefits in
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multiple contexts. Firstly, some patients have small starting T cell populations
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(such as infants or those who are severely lymphodepleted), and thus require
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more population doublings to reach a usable dose. Our data suggests the time to
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reach this dose would be reduced, easing scheduling a reducing cost. Secondly,
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the allogeneic T cell model would greatly benefit from a system that could
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create large numbers of T cells with naïve and memory phenotype. In contrast to
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the autologous model which is currently used for Kymriah and Yescarta,
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allogeneic T cell therapy would reduce cost by spreading manufacturing expenses
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across many doses for multiple patients\cite{Harrison2019}. Since it is
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economically advantageous to grow as many T cells as possible in one batch in
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the allogeneic model (reduced start up and harvesting costs, fewer required cell
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donations), the DMSs offer an advantage over current technology.
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% Another advantage is that the DMS system appears to induce a faster growth rate
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% of T cells given the same IL2 concentration compared to beads (Supplemental
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% Figure 8) along with retaining naïve and memory phenotype. This has benefits in
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% multiple contexts. Firstly, some patients have small starting T cell populations
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% (such as infants or those who are severely lymphodepleted), and thus require
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% more population doublings to reach a usable dose. Our data suggests the time to
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% reach this dose would be reduced, easing scheduling a reducing cost. Secondly,
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% the allogeneic T cell model would greatly benefit from a system that could
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% create large numbers of T cells with naïve and memory phenotype. In contrast to
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% the autologous model which is currently used for Kymriah and Yescarta,
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% allogeneic T cell therapy would reduce cost by spreading manufacturing expenses
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% across many doses for multiple patients\cite{Harrison2019}. Since it is
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% economically advantageous to grow as many T cells as possible in one batch in
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% the allogeneic model (reduced start up and harvesting costs, fewer required cell
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% donations), the DMSs offer an advantage over current technology.
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% DISCUSSION this is already stated in the innovation section
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It should be noted that while we demonstrate a method providing superior
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The \gls{dms} system could be used as a drop in replacement for beads in many of
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current allogeneic therapies. Indeed, given its higher potential for expansion
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(\cref{fig:dms_exp,tab:ci_controlled}, it may work in cases where the beads fail
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(although this would need to be tested by gathering data with many unhealthy
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donors). However, in the autologous setting patients only need a fixed dose, and
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thus any expansion beyond the indicated dose would be wasted. Given this, it
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will be interesting to apply this technology in an allogeneic paradigm where
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this increased expansion potential would be well utilized.
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Finally, we should note that while we demonstrated a method providing superior
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performance compared to bead-based expansion, the cell manufacturing field would
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tremendously benefit from simply having an alternative to state-of-the-art
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methods. The patents for bead-based expansion are owned by few companies and
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licensed accordingly; having an alternative would provide more competition in
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the market, reducing costs and improving access for academic researchers and
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tremendously benefit from simply having an alternative to state-of-the-art bead
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based expansion. The patents for bead-based expansion are owned by few companies
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and licensed accordingly; having an alternative would provide more competition
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in the market, reducing costs and improving access for academic researchers and
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manufacturing companies.
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% DISCUSSION this isn't relevent to this aim but should be said somewhere
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Finally, while we have demonstrated the DMS system in the context of CAR T
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cells, this method can theoretically be applied to any T cell immunotherapy
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which responds to anti-CD3/CD28 mAb and cytokine stimulation. These include
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\glspl{til}, virus-specific T cells (VSTs), T cells engineered to express
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$\upgamma\updelta$TCR (TEGs), $\upgamma\updelta$ T cells, T cells with
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transduced-TCR, and CAR-TCR T cells\cite{Cho2015, Straetemans2018, Robbins2011,
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Brimnes2012, Baldan2015, Walseng2017}. Similar to CD19-CARs used in liquid
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tumors, these T cell immunotherapies would similarly benefit from the increased
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proliferative capacity, metabolic fitness, migration, and engraftment potential
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characteristic of naïve and memory phenotypes\cite{Blanc2018, Lalor2016,
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Rosato2019}. Indeed, since these T cell immunotherapies are activated and
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expanded with either soluble mAbs or bead-immobilized mAbs, our system will
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likely serve as a drop-in substitution to provide these benefits.
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\chapter{aim 2a}\label{aim2a}
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\section{introduction}
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@ -4368,11 +4341,25 @@ targeted specification. These \gls{qc} steps all rely on common, relatively
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cost-effective assays such as the \gls{haba} assay, \gls{bca} assay, and
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\glspl{elisa}, thus other labs and commercial entities should be able to perform
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them. The microcarriers themselves are an off-the-shelf product available from
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reputable vendors, further enhancing translatability. On average, we
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demonstrated that the \gls{dms} outperforms state-of-the-art bead-based T cell
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expansion technology in terms of total fold expansion, \ptmemp{}, and \pthp{} by
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\SI{143}{\percent}, \SI{2.5}{\percent}, and \SI{9.8}{\percent} controlling for
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donor, operator, and a variety of process conditions.
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reputable vendors, and they have a regulatory history in human cell therapies
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that will aid in clinical translation\cite{purcellmain}. Both these will help
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in translatability. On average, we demonstrated that the \gls{dms} outperforms
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state-of-the-art bead-based T cell expansion technology in terms of total fold
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expansion, \ptmemp{}, and \pthp{} by \SI{143}{\percent}, \SI{2.5}{\percent}, and
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\SI{9.8}{\percent} controlling for donor, operator, and a variety of process
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conditions.
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In addition to larger numbers of potent T cells, other advantages of our
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\gls{dms} approach are that the \glspl{dms} are large enough to be filtered
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(approximately \SI{300}{\um}) using standard \SI{40}{\um} cell filters or
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similar. If the remaining cells inside that \glspl{dms} are also desired,
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digestion with dispase or collagenase may be used. Collagenase D may be
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selective enough to dissolve the \gls{dms} yet preserve surface markers which
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may be important to measure as critical quality attributes \glspl{cqa}
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(\cref{fig:collagenase_fx}). Furthermore, our system should be compatible with
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large-scale static culture systems such as the G-Rex bioreactor or perfusion
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culture systems, which have been previously shown to work well for T cell
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expansion\cite{Forget2014, Gerdemann2011, Jin2012}.
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In \cref{aim2a}, we developed a modeling pipeline that can be used by commercial
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entities as the scale up this process to identify \glspl{cqa} and \gls{cpp}.
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@ -4428,6 +4415,21 @@ factor given that it was nearly the same in the first experiment between
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\gls{dms} and bead groups despite the clear advantage seen in the \gls{dms}
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group.
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Finally, while we have demonstrated the \gls{dms} system in the context of
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\gls{car} T cells, this method can theoretically be applied to any T cell
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immunotherapy which responds to \acd{3}/\acd{28} \gls{mab} and cytokine
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stimulation. These include \glspl{til}, virus-specific T cells, T cells
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engineered to express $\upgamma\updelta$ \glspl{tcr}, $\upgamma\updelta$ T
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cells, T cells with transduced-\gls{tcr}, and \gls{car}-\gls{tcr} T
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cells\cite{Cho2015, Straetemans2018, Robbins2011, Brimnes2012, Baldan2015,
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Walseng2017}. Similar to \glspl{car} against CD19 used in liquid tumors, these
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T cell immunotherapies would similarly benefit from the increased proliferative
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capacity, metabolic fitness, migration, and engraftment potential characteristic
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of naïve and memory phenotypes\cite{Blanc2018, Lalor2016, Rosato2019}. Indeed,
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since these T cell immunotherapies are activated and expanded with either
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soluble \glspl{mab} or bead-immobilized \glspl{mab}, our system will likely
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serve as a drop-in substitution to provide these benefits.
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\section{future directions}
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There are several important next steps to perform with this work, many of which
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