From fa716f9d78dc0e028a85c1a2aaa56a681111775d Mon Sep 17 00:00:00 2001 From: ndwarshuis Date: Wed, 4 Aug 2021 15:28:47 -0400 Subject: [PATCH] ENH finish proofreading aim 1 --- tex/thesis.tex | 204 +++++++++++++++++++++++++------------------------ 1 file changed, 103 insertions(+), 101 deletions(-) diff --git a/tex/thesis.tex b/tex/thesis.tex index 3e6a3a7..661df46 100644 --- a/tex/thesis.tex +++ b/tex/thesis.tex @@ -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