REF update todos and comments for myself
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@ -415,8 +415,6 @@ better mimic these \invivo{} expansion conditions of T cells, can significantly
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improve the quality and quantity of manufactured T cells and provide better
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control on the resulting T cell phenotype.
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% TODO mention the Cloudz stuff that's in my presentation
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A variety of solutions have been proposed to make the T cell expansion process
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more physiological. One strategy is to use modified feeder cell cultures to
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provide activation signals similar to those of \glspl{dc}\cite{Forget2014}.
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@ -499,8 +497,6 @@ The specific aims of this dissertation are outlined in
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\subsection*{aim 1: develop and optimize a novel T cell expansion process that
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mimics key components of the lymph nodes}
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% TODO this might be easier to break apart in separate aims
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In this first aim, we demonstrated the process for manufacturing \glspl{dms},
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including quality control steps that are necessary for translation of this
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platform into a scalable manufacturing setting. We also demonstrate that the
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@ -540,6 +536,8 @@ present our final conclusions in Chapter~\ref{conclusions}.
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\chapter{background and significance}\label{background}
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\section*{background}
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% TODO mention cloudz stuff
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% TODO break this apart into mfg tech and T cell phenotypes/quality
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% TODO consider adding a separate section on microcarriers and their use in
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% bioprocess
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@ -1284,7 +1282,6 @@ to the microcarrier suspension (which itself is in \gls{pbs}) this result
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indicated that hydrolysis is not of concern when adding \gls{snb} within
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minutes.
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% TODO use the water vs pbs curve here
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\begin{figure*}[ht!]
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\begingroup
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@ -1369,7 +1366,7 @@ the \gls{mab} reaction should proceed in {\#}{mab curve}.
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\subsection{DMSs can efficiently expand T cells compared to beads}
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% TODO add other subfigures here
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% RESULT add other subfigures here
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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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@ -1384,7 +1381,7 @@ also observed no T cell expansion using \glspl{dms} coated with an isotype
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control mAb compared to \glspl{dms} coated with \acd{3}/\acd{28} \glspl{mab}
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{Figure X}, confirming specificity of the expansion method.
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% TODO make sure the day on these is correct
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% FIGURE make sure the day on these is correct
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\begin{figure*}[ht!]
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\begingroup
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@ -1431,7 +1428,7 @@ control mAb compared to \glspl{dms} coated with \acd{3}/\acd{28} \glspl{mab}
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\input{../tables/inside_fraction_regression.tex}
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\end{table}
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% TODO state the CI of what are inside the carriers
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% RESULT state the CI of what are inside the carriers
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We also asked how many cells were inside the \glspl{dms} vs. free-floating in
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suspension and/or loosely attached to the surface. We qualitatively verified the
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presence of cells inside the \glspl{dms} using a \gls{mtt} stain to opaquely
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@ -1468,8 +1465,7 @@ cells after \SI{14}{\day} were on the interior surface of the \glspl{dms}
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\label{fig:dms_flowchart}
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\end{figure*}
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% TODO double check the timing of this experiment (it might not be day 14)
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% FIGURE double check the timing of this experiment (it might not be day 14)
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\begin{figure*}[ht!]
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\begingroup
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@ -1542,9 +1538,9 @@ observing the CD4+ and CD8+ fractions of the naïve/memory subset (CD62L+CCR7+)
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\label{fig:dms_exp}
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\end{figure*}
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% TODO add a paragraph for this figure
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% RESULT add a paragraph for this figure
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% TODO this figure has weird proportions
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% FIGURE this figure has weird proportions
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\begin{figure*}[ht!]
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\begingroup
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@ -1633,9 +1629,7 @@ cells (albeit in this case at day 7 and with an undefined \gls{moi})
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we also found that the number of \ptcar{} T cells was greater for \gls{dms} than
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for bead (\cref{fig:car_bcma_total}).
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% TODO the right half if bigger than the left half
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% TODO add memory stuff to this since I have it (it wasn't the right size so I
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% haven't included it yet)?
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% FIGURE the right half if bigger than the left half
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\begin{figure*}[ht!]
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\begingroup
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@ -1653,7 +1647,6 @@ for bead (\cref{fig:car_bcma_total}).
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\label{fig:car_bcma}
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\end{figure*}
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\subsection{DMSs efficiently expand T cells in Grex bioreactors}
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% RESULT update this in light of the grex data
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@ -1666,7 +1659,7 @@ with past results, \glspl{dms}-expanded T cells had higher \pthp{} compared to
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beads, but only had slightly higher \ptmemp{} compared to beads
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(\cref{fig:grex_mem,fig:grex_cd4}).
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% TODO is this discussion stuff?
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% DISCUSSION is this discussion stuff?
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These discrepancies might be explained in light of our other data as follows.
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The Grex bioreactor has higher media capacity relative to its surface area, and
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we did not move the T cells to a larger bioreactor as they grew in contrast with
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@ -1717,8 +1710,6 @@ demonstrates that \gls{dms} could lead to more robust activation and fitness.
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\label{fig:grex_luminex}
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\end{figure*}
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% FIGURE grex + car (maybe, IDK if I actually have this data)
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\subsection{DMSs do not leave antibodies attached to cell product}
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We asked if \glspl{mab} from the \glspl{dms} detached from the \gls{dms} surface
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@ -1760,7 +1751,7 @@ include those experiments where the surface density of the CD3 and CD28
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\glspl{dms}). This ultimately resulted in a dataset with 162 runs spanning 15
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experiments between early 2017 and early 2021.
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% TODO add some correlation analysis to this
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% FIGURE add some correlation analysis to this
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Since the aim of the analysis was to perform causal inference, we determined 6
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possible treatment variables which we controlled when designing the experiments
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@ -1807,7 +1798,7 @@ significant in all cases except for the \dpthp{}; however, we also observe that
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relatively little of the variability is explained by these simple models ($R^2$
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between 0.17 and 0.44).
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% TODO add the regression diagnostics to this
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% RESULT add the regression diagnostics to this
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We then included all covariates and unbalanced treatment parameters and
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performed linear regression again
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(\cref{tab:ci_controlled,fig:metaanalysis_fx}). We observe that after
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@ -1830,7 +1821,7 @@ using a Grex entails changing the cell surface and feeding strategy for the T
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cells, and any one of these ‘mediating variables’ might actually be the cause of
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the responses.
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% TODO these tables have extra crap in them that I don't need to show
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% TABLE these tables have extra crap in them that I don't need to show
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\begin{table}[!h] \centering
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\caption{Causal Inference on treatment variables only}
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\label{tab:ci_treat}
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@ -1868,7 +1859,7 @@ the responses.
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\section{discussion}
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% TODO this is fluffy
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% DISCUSSION this is fluffy
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We have developed a T cell expansion system that recapitulates key features of
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the in vivo lymph node microenvironment using DMSs functionalized with
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activating mAbs. This strategy provided superior expansion with higher number of
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@ -1904,7 +1895,7 @@ 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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% TODO this mentions the DOE which is in the next aim
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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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@ -1980,7 +1971,7 @@ 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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% TODO this sounds sketchy
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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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@ -1997,7 +1988,7 @@ 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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% TODO this is already stated in the innovation section
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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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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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@ -2006,7 +1997,7 @@ 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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manufacturing companies.
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% TODO this isn't relevent to this aim but should be said somewhere
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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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@ -2177,7 +2168,6 @@ suggested that some of these unknown features belonged to the same molecules
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(not shown). Additional multidimensional NMR experiments will be required to
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determine their identity.
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% TODO add footnote saying what I did in this
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\subsection{machine learning and statistical analysis}
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Linear regression analysis of the \glspl{doe} was performed as described in
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@ -2308,9 +2298,7 @@ between T cells. Since \gls{il2} is secreted by activated T cells themselves,
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T cells in the \gls{dms} system may need less or no \gls{il2} if this hypothesis
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were true.
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% TODO this plots proportions look dumb
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% TODO explain what the NLS lines are in b
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% TODO plot the differences in lower IL2 concentrations to better show this
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% FIGURE this plots proportions look dumb
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\begin{figure*}[ht!]
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\begingroup
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@ -2334,7 +2322,7 @@ were true.
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\label{fig:il2_mod}
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\end{figure*}
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% TODO the nls stuff is a bit iffy
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% RESULT the nls stuff is a bit iffy
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We varied the concentration of \gls{il2} from \SIrange{0}{100}{\IU\per\ml} and
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expanded T cells as described in \cref{sec:tcellculture}. T cells grown with
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either method expanded robustly as \gls{il2} concentration was increased
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@ -2361,7 +2349,7 @@ at \SI{10}{\IU\per\ml} throughout the remainder of this aim.
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% different
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\subsection{DOE shows optimal conditions for expanded potent T cells}
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% TODO not all of these were actually use, explain why by either adding columns
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% RESULT not all of these were actually used, explain why by either adding columns
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% or marking with an asterisk
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\begin{table}[!h] \centering
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\caption{DOE Runs}
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@ -2392,7 +2380,7 @@ the range of \SIrange{10}{30}{\IU\per\ml}, \pdms{} in the range of
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\SIrange{500}{2500}{\dms\per\ml}, and \pmab{} in the range of
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\SIrange{60}{100}{\percent}.
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% TODO explain why not all runs were used
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% RESULT explain why not all runs were used
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After performing the first \gls{doe} we augmented the original design matrix
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with an \gls{adoe} which was built with three goals in mind. Firstly we wished
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to validate the first \gls{doe} by assessing the strength and responses of each
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@ -2475,7 +2463,7 @@ confidence to the location of this second order feature. The remainder of the
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responses showed mostly linear relationships in all parameter cases
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(\cref{fig:doe_responses_cd4,fig:doe_responses_mem4,fig:doe_responses_ratio}).
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% TODO it seems arbitrary that I went straight to a third order model, the real
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% RESULT it seems arbitrary that I went straight to a third order model, the real
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% reason is because it seemed weird that a second order model didn't find
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% anything to be significant
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We performed linear regression on the three input parameters as well as a binary
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@ -2501,7 +2489,7 @@ that our data might be underpowered for a model this complex. Further
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experiments beyond what was performed here may be needed to fully describe this
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response.
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% TODO combine these tables into one
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% TABLE combine these tables into one
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We performed linear regression on the other three responses, all of which
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performed much better than the \ptmem{} response as expected given the much
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lower apparent complexity in the response plots
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@ -2562,7 +2550,7 @@ Symbolic Regression (SR), was implemented to molecularly characterize TN+TCM
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cells and to extract predictive features of quality early on their expansion
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process (Fig.1d-e).
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% TODO this table looks like crap, break it up into smaller tables
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% TABLE this table looks like crap, break it up into smaller tables
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\begin{table}[!h] \centering
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\caption{Results for data-driven modeling}
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\label{tab:mod_results}
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@ -2656,6 +2644,8 @@ IL13 and IL15 were found predictive in combination with these using SR
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% optimization of process features
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% TODO this sounds like total fluff
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% DISCUSSION integrate figures
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CPPs modeling and understanding are critical to new product development and in
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cell therapy development, it can have life-saving implications. The challenges
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for effective modeling grow with the increasing complexity of processes due to
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@ -2805,7 +2795,7 @@ interest using \glspl{mab}.
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\subsection{DMSs temporal modulation}
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% TODO The concentration for the surface marker cleavage experiment was much
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% METHOD The concentration for the surface marker cleavage experiment was much
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% higher, if that matters
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\glspl{dms} were digested in active T cell cultures via addition of sterile
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\product{\gls{colb}}{Sigma}{11088807001} or
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@ -2866,7 +2856,7 @@ To block soluble \gls{il15}, we supplemented analogously with
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\subsection{adding or removing DMSs alters expansion and phenotype}
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% TODO state what collagenase actually targets
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% RESULT state what collagenase actually targets
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We hypothesized that adding or removing \gls{dms} in the middle of an active
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culture would alter the activation signal and hence the growth trajectory and
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phenotype of T cells. While adding \glspl{dms} was simple, the easiest way to
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@ -2882,7 +2872,7 @@ the \gls{cold} group were similar to that of the buffer group, while the
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enzymatic cleavage (\cref{fig:collagenase_fx}). Based on this result, we used
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\gls{cold} moving forward.
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% TODO this figure is tall and skinny like me
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% FIGURE this figure is tall and skinny like me
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\begin{figure*}[ht!]
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\begingroup
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@ -3015,10 +3005,6 @@ domains present on the \gls{dms} group could be creating pro-survival and
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pro-expansion signals to the T cells through \gls{a2b1} and \gls{a2b2}, causing
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them to grow better in the \gls{dms} system.
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% TODO perhaps these figs should be combined
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% TODO actually make the captions for these
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% TODO add some background into why integrins are important and the proposed mechanism
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% TODO add an experimental timeline to these showing when I added the mabs
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\begin{figure*}[ht!]
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\begingroup
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@ -3103,9 +3089,7 @@ is not due to signaling through \gls{a2b1} or \gls{a2b2}.
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\subsection{blocking IL15 signaling does not alter expansion or phenotype}
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% BACKGROUND why is IL15 important?
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% TODO cite the luminex data
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% RESULT cite the luminex data
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\gls{il15} is a cytokine responsible for memory T cell survival and maintenance.
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Furthermore, we observed in other experiments that it is secreted to a much
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greater extend in \gls{dms} compared to bead cultures. One of our driving
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@ -3115,7 +3099,6 @@ many conditions, we hypothesized that \gls{il15} may be responsible for this,
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and further that the unique \textit{cis/trans} activity of \gls{il15} may be
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more active in the \gls{dms} system due to higher cell density.
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% TODO add some background into why IL15 is important and the proposed mechanism
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\begin{figure*}[ht!]
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\begingroup
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@ -3139,8 +3122,8 @@ more active in the \gls{dms} system due to higher cell density.
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\label{fig:il15_1}
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\end{figure*}
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% TODO how did I determine how much to add?
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% TODO just gate these as normal because this looks sketchy
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% RESULT how did I determine how much to add?
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% FIGURE just gate these as normal because this looks sketchy
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We first tested this hypothesis by blocking \gls{il15r} with either a specific
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\gls{mab} or an \gls{igg} isotype control. We observed no difference in the
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expansion rate of blocked or unblocked cells (this experiment also had
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@ -3180,7 +3163,7 @@ We next tried blocking soluble \gls{il15} itself using either a \gls{mab} or an
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change, viability, or marker histograms between any of these markers, showing
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that blocking \gls{il15} led to no difference in growth or phenotype.
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% TODO this can probably be worded more specifically in terms of the cis/trans
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% RESULT this can probably be worded more specifically in terms of the cis/trans
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% action of IL15
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In summary, this data did not support the hypothesis that the \gls{dms} platform
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gains its advantages via the \gls{il15} pathway.
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@ -3203,7 +3186,7 @@ are included in the \ptmem{} phenotype). Taken together, these imply that
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temporally or spatially altering the \gls{dms} concentration, and thus the
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activation signal, has similar effects.
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% TODO this sounds like background?
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% BACKGROUND this sounds like background?
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% There are several plausible explanations for the observed phenotypic differences
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% between beads and DMSs. First, the DMSs are composed of a collagen derivative
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% (gelatin); collagen has been shown to costimulate activated T cells via
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@ -3247,7 +3230,7 @@ came back negative, we would be fairly confident that the \gls{a2b1} and
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We also failed to uphold our hypothesis that the \gls{dms} system gains its
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advantage via \gls{il15} signaling.
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% TODO not sure if this belongs here, although it might make sense to offer
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% DISCUSSION not sure if this belongs here, although it might make sense to offer
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% alternative explanations of why the DMSs "work" given this negative data
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% Second, there is evidence that providing a larger
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% contact area for T cell activation provides greater stimulation16,43; the DMSs
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@ -3296,7 +3279,7 @@ potency\cite{Ghassemi2018}.
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\subsection{\invivo{} therapeutic efficacy in NSG mice model}
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% TODO use actual product numbers for mice
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% METHOD use actual product numbers for mice
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All mice in this study were male \gls{nsg} mice from Jackson Laboratories. At
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day 0 (-7 day relative to T cell injection), 1e6 firefly luciferase-expressing
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\product{Nalm-6 cells}{ATCC}{CRL-3273} suspended in ice-cold PBS were injected
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@ -3403,7 +3386,7 @@ case of beads (\cref{fig:mouse_dosing_qc_mem}).
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\input{../tables/mouse_dose_car.tex}
|
||||
\end{table}
|
||||
|
||||
% TODO put growth first in this figure
|
||||
% FIGURE put growth first in this figure
|
||||
\begin{figure*}[ht!]
|
||||
\begingroup
|
||||
|
||||
|
|
@ -3424,7 +3407,7 @@ case of beads (\cref{fig:mouse_dosing_qc_mem}).
|
|||
\label{fig:mouse_dosing_qc}
|
||||
\end{figure*}
|
||||
|
||||
% TODO explain what statistical test was used here
|
||||
% FIGURE explain what statistical test was used here
|
||||
\begin{figure*}[ht!]
|
||||
\begingroup
|
||||
|
||||
|
|
@ -3479,7 +3462,7 @@ the same.
|
|||
\label{fig:mouse_timecourse_overview}
|
||||
\end{figure*}
|
||||
|
||||
% TODO find literature saying that CAR T cells grow slower
|
||||
% RESULT find literature saying that CAR T cells grow slower
|
||||
As was the case with the first \invivo{} experiment, T cells activated with
|
||||
\glspl{dms} expanded much more efficiently compared to those expanded with beads
|
||||
(\cref{fig:mouse_timecourse_qc_growth}). When we quantified the \ptcarp{} of T
|
||||
|
|
@ -3591,7 +3574,7 @@ results suggest that the higher proportion of memory T cells in DMS groups
|
|||
efficiently kill tumor cells as recently reported in
|
||||
literature\cite{Fraietta2018, Sommermeyer2015}.
|
||||
|
||||
% TODO try and find literature explaining what the ideal ratio is
|
||||
% DISCUSSION try and find literature explaining what the ideal ratio is
|
||||
When testing \gls{car} T cells at earlier timepoints relative to day 14 as used
|
||||
in the first \invivo{} experiment, we noted that none of the \gls{car}
|
||||
treatments seemed to work as well as they did in the first experiment. However,
|
||||
|
|
|
|||
Loading…
Reference in New Issue