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ENH proof aim 3

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@ -380,6 +380,7 @@
\newcommand{\inlinecode}{\texttt}
\newcommand{\subcap}[2]{\subref{#1}) #2}
\newcommand{\sigkey}{Significance test key: *p<0.1; **p < 0.05; ***p<0.01}
\newcommand{\nVI}{NALM-6}
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
% ditto for environments
@ -587,7 +588,7 @@ The goal of this dissertation was to develop a microcarrier-based \gls{dms} T
cell expansion system and determine biologically-meaningful \glspl{cqa} and
\glspl{cpp} that could be used to optimize for highly-potent T cells. In
\cref{aim1}, we developed and characterized the \gls{dms} system, including
quality control steps. We also demonstrated the feasibility of expanding
\gls{qc} steps. We also demonstrated the feasibility of expanding
high-quality T cells. In \cref{aim2a,aim2b}, we used \gls{doe} methodology to
optimize the \gls{dms} platform, and we developed a computational pipeline to
identify and model the effects of measurable \glspl{cqa} and \glspl{cpp} on the
@ -687,7 +688,7 @@ The specific aims of this dissertation are outlined in
mimics key components of the lymph nodes}
In this first aim, we demonstrated the process for manufacturing \glspl{dms},
including quality control steps that are necessary for translation of this
including \gls{qc} steps that are necessary for translation of this
platform into a scalable manufacturing setting. We also demonstrated that the
\gls{dms} platform leads to higher overall expansion of T cells and higher
overall fractions of potent memory and CD4+ subtypes desired for T cell
@ -1038,8 +1039,8 @@ Matrigel\cite{Rio2018} or 3d-printed lattices\cite{Delalat2017}, ellipsoid
beads\cite{meyer15_immun}, and \gls{mab}-conjugated \gls{pdms}
beads\cite{Lambert2017} that respectively recapitulate the cellular membrane,
large interfacial contact area, 3D-structure, or soft surfaces T cells normally
experience \textit{in vivo}. None of these have been shown to expand high
quality T cells as outlined in \cref{sec:background_quality}.
experience \invivo{}. None of these have been shown to expand high quality T
cells as outlined in \cref{sec:background_quality}.
\subsection{Microcarriers in Bioprocessing}
@ -1672,7 +1673,7 @@ to secondary controls (\gls{pe}-\gls{stp} with no \gls{ptnl}).
was added to tubes analogously to \gls{ptnl} and incubated for \SI{45}{\minute}
prior to analyzing on a \bd{} Accuri
\subsection{CAR Plasmid and Lentiviral Transduction}
\subsection{CAR Plasmid and Lentiviral Transduction}\label{sec:transduction}
The anti-CD19-CD8-CD137-CD3$\upzeta$ \gls{car} sequence with the EF1$\upalpha$
promotor\cite{Milone2009} was synthesized (Aldevron) and subcloned into a
@ -4115,27 +4116,6 @@ results on expansion and memory phenotype. Essentially this would turn the
\glspl{dms} into stromal cells that present \il{15}, as seen to be important in
the early work with \il{15} in mice\cite{Lodolce1998}.
% DISCUSSION not sure if this belongs here, although it might make sense to offer
% alternative explanations of why the DMSs "work" given this negative data
% Second, there is evidence that providing a larger
% contact area for T cell activation provides greater stimulation16,43; the DMSs
% have a rougher interface than the 5 µm magnetic beads, and thus could facilitate
% these larger contact areas. Third, the DMSs may allow the T cells to cluster
% more densely compared to beads, as evidenced by the large clusters on the
% outside of the DMSs (Figure 1f) as well as the significant fraction of DMSs
% found within their interiors (Supplemental Figure 2a and b). This may alter the
% local cytokine environment and trigger different signaling pathways.
% Particularly, IL15 and IL21 are secreted by T cells and known to drive memory
% phenotype44–46. We noted that the IL15 and IL21 concentration was higher in a
% majority of samples when comparing beads and DMSs across multiple timepoints
% (Supplemental Figure 18) in addition to many other cytokines. IL15 and IL21 are
% added exogenously to T cell cultures to enhance memory frequency,45,47 and our
% data here suggest that the DMSs are better at naturally producing these
% cytokines and limiting this need. Furthermore, IL15 unique signals in a trans
% manner in which IL15 is presented on IL15R to neighboring cells48. The higher
% cell density in the DMS cultures would lead to more of these trans interactions,
% and therefore upregulate the IL15 pathway and lead to more memory T cells.
\chapter{AIM 3}\label{aim3}
\section{Introduction}
@ -4156,44 +4136,47 @@ lower-differentiated T cells with higher potency\cite{Ghassemi2018}.
\section{Methods}
\subsection{CD19-CAR T Cell Generation}
\subsection{T Cell Culture}
T cells were grown as described in \cref{sec:tcellculture}.
\subsection{CD19-CAR T Cell Generation}
T cells were grown as described in \cref{sec:transduction}.
\subsection{\Invivo{} Therapeutic Efficacy in NSG Mice Model}
% METHOD describe how the luciferase cells were generated (eg the kwong lab)
% METHOD use actual product numbers for mice
All mice in this study were male \gls{nsg} mice from Jackson Laboratories. At
day 0 (\SI{-7}{\day} relative to T cell injection), 1e6 firefly
luciferase-expressing \product{Nalm-6 cells}{ATCC}{CRL-3273} suspended in
ice-cold \gls{pbs} were injected via tail vein into each mouse. At day 7, saline
or \gls{car} T cells at the indicated doses from either bead or
\gls{dms}-expanded T cell cultures (for \SI{14}{\day}) were injected into each
mouse via tail vein. Tumor burden was quantified longitudinally via an
\gls{ivis} Spectrum (Perkin Elmer). Briefly, \SI{200}{\ug} luciferin at
\SI{15}{\mg\per\ml} in \gls{pbs} was injected intraperitoneally under isoflurane
anesthesia into each mouse and allowed to circulate for at least
\SI{10}{\minute} before imaging. Mice were anesthetized again and imaged using
the \gls{ivis}. Mice from each treatment group/dose were anesthetized, injected,
and imaged together, and exposure time of the \gls{ivis} was limited to avoid
saturation based on the signal from the saline group. \gls{ivis} images were
processed by normalizing them to common minimum and maximum photon counts and
total flux was estimated in terms of photons/second. Endpoint for each mouse was
determined by \gls{iacuc} euthanasia criteria (hunched back, paralysis,
day 0 (\SI{-7}{\day} relative to T cell injection), \num{1e6} firefly
luciferase-expressing\footnote{luciferase transduction was performed and
verified by Ian Miller in the Kwong Lab at Georgia Tech} \product{\nVI{}
cells}{ATCC}{CRL-3273} suspended in ice-cold \gls{pbs} were injected via tail
vein into each mouse. At day 7, saline or \gls{car} T cells at the indicated
doses from either bead or \gls{dms}-expanded T cell cultures (for \SI{14}{\day})
were injected into each mouse via tail vein. Tumor burden was quantified
longitudinally via an \gls{ivis} Spectrum (Perkin Elmer). Briefly, \SI{200}{\ug}
luciferin at \SI{15}{\mg\per\ml} in \gls{pbs} was injected intraperitoneally
under isoflurane anesthesia into each mouse and allowed to circulate for at
least \SI{10}{\minute} before imaging. Mice were anesthetized again and imaged
using the \gls{ivis}. Mice from each treatment group/dose were anesthetized,
injected, and imaged together; exposure time of the \gls{ivis} was limited to
avoid saturation based on the signal from the saline group. \gls{ivis} images
were scaled to common minimum and maximum photon counts. Endpoint for each mouse
was determined by \gls{iacuc} euthanasia criteria (hunched back, paralysis,
blindness, lethargy, and weight loss). Mice were euthanized according to these
endpoint criteria using carbon dioxide asphyxiation.
\subsection{Statistics}
For the \invivo{} model, the survival curves were created and statistically
analyzed using GraphPad Prism using the Mantel-Cox test to assess significance
between survival groups.
Survival curves were created and statistically analyzed using GraphPad Prism
using the Mantel-Cox test to assess significance between survival groups.
\section{Results}
\subsection{DMSs Lead to Greater \invivo{} Anti-Tumor Activity}
\begin{figure*}[ht!]
\begingroup
@ -4212,7 +4195,6 @@ between survival groups.
\input{../tables/mouse_dose_car.tex}
\end{table}
\subsection{DMSs Lead to Greater \invivo{} Anti-Tumor Activity}
\begin{figure*}[ht!]
\begingroup
@ -4248,7 +4230,7 @@ between survival groups.
\caption[Mouse Dosing IVIS and Survival Results]
{T cells expanded with \glspl{dms} confer greater anti-tumor potency \invivo{}
even at lower doses.
\subcap{fig:mouse_dosing_ivis_images}{IVIS images of Nalm-6 tumor-bearing
\subcap{fig:mouse_dosing_ivis_images}{IVIS images of \nVI{} tumor-bearing
\gls{nsg} mice injected with varying doses of T cells}
\subcap{fig:mouse_dosing_ivis_plots}{Plots showing quantified photon counts
of the results from (\subref{fig:mouse_dosing_ivis_plots}).}
@ -4266,11 +4248,11 @@ between survival groups.
We asked if the higher memory/naive phenotype and more balanced CD4/CD8 ratio of
our \gls{dms}-expanded \gls{car} T cells would lead to better anti-tumor potency
in vivo compared to bead-expanded \gls{car} T cells. We also asked if this
\invivo{} compared to bead-expanded \gls{car} T cells. We also asked if this
superior anti-tumor potency would hold true at lower doses of \gls{car}
expressing T cells in the DMS group vs the bead group. To test this, we used a
human xenograft model of B cell \gls{all} by intravenously injecting \gls{nsg}
mice with \num{1e6} Nalm-6 tumor cells expression firefly
mice with \num{1e6} \nVI{} tumor cells expressing firefly
luciferase\cite{Fraietta2018}. After \SI{7}{\day} of tumor cell growth
(\cref{fig:mouse_dosing_overview}), we intravenously injected saline or three
doses (high, medium, and low) of \gls{car} T cells from either bead or \gls{dms}
@ -4280,57 +4262,55 @@ groups using the \gls{ptnl} assay (\cref{tab:mouse_dosing_results}).
Before injecting the T cells into the mice, we quantified their phenotype and
growth. We observed that for this expansion, the bead and \gls{dms} T cells
produced similar numbers of \ptmem{} T cells, and the beads even had a higher
fraction of CD45RA, which is present on lower-differentiated \glspl{tn} and
\glspl{tscm} (\cref{fig:mouse_dosing_qc_mem}). However, the \pthp{} of
the final product was higher in \gls{dms} (\cref{fig:mouse_dosing_qc_cd4}). The
\gls{dms} T cells also expanded more robustly than the beads
(\cref{fig:mouse_dosing_qc_growth}).
fraction of \cdp{45RA} cells, which is present on lower-differentiated
\glspl{tn} and \glspl{tscm} (\cref{fig:mouse_dosing_qc_mem}). However, the
\pthp{} of the final product was higher in \gls{dms}
(\cref{fig:mouse_dosing_qc_cd4}). The \gls{dms} T cells also expanded more
robustly than the beads (\cref{fig:mouse_dosing_qc_growth}).
In the Nalm-6/\gls{nsg} xenograft model, we observed lower tumor burden and
significantly longer survival of bead and \gls{dms}-treated mice at all doses
compared to the saline groups (\cref{fig:mouse_dosing_ivis}). Importantly, at
each dose we observed that the \gls{dms}-treated mice had much lower tumor
burden and significantly higher survival than their bead-treated counterparts
In the \nVI{}/\gls{nsg} xenograft model, bead and \gls{dms}-treated mice at all
doses had lower tumor burden and significantly longer survival compared to the
saline groups (\cref{fig:mouse_dosing_ivis}). Importantly, at each dose the
\gls{dms}-treated mice had much lower tumor burden and significantly higher
survival than their bead-treated counterparts
(\cref{fig:mouse_dosing_ivis_survival}). When factoring the percentage T cells
in each dose that expressed the \gls{car}, we note that survival of the low
\gls{dms} dose (which had similar total \gls{car} T cells compared to the bead
medium dose and less than the bead high dose) is significantly higher than that
of both the bead medium dose and the bead high dose
in each dose that expressed the \gls{car}, survival of the low \gls{dms} dose
(which had similar total \gls{car} T cells compared to the bead medium dose and
less than the bead high dose) was significantly higher than that of both the
bead medium dose and the bead high dose
(\cref{fig:mouse_dosing_ivis_survival_comp}). Overall, the Kaplan-Meier survival
of Nalm-6 tumor bearing \gls{nsg} mice shown in the
of \nVI{} tumor bearing \gls{nsg} mice shown in the
\cref{fig:mouse_dosing_ivis_survival} was up to day 40 as reported
elsewhere\cite{Fraietta2018}. However, we also included a Kaplan-Meier figure up
to day 46 (\cref{fig:mouse_dosing_ivis_survival_full}) where most of the mice
euthanized from day 40 through day 46 from \gls{dms} groups showed no or very
small fragment of spleen which was due to \gls{gvhd} responses. Similar
\gls{gvhd} responses were reported earlier in \gls{nsg} mice where the mice
injected with human \gls{pbmc} exhibited acute \gls{gvhd} between
\SIrange{40}{50}{\day} post intravenous injection\cite{Ali2012}. Notably, both
survival analyses (up to day 40 in \cref{fig:mouse_dosing_ivis_survival} and up
to day 46 in \cref{fig:mouse_dosing_ivis_survival_full}) confirmed that
\gls{dms}-expanded groups outperformed bead-expanded groups in terms of
prolonging survival of Nalm-6 tumor challenged \gls{nsg} mice.
elsewhere\cite{Fraietta2018}. However, most of the mice euthanized from day 40
through day 46 from \gls{dms} groups showed no or very small fragment of spleen
which was due to \gls{gvhd} responses
(\cref{fig:mouse_dosing_ivis_survival_full}). Similar \gls{gvhd} responses
\SIrange{40}{50}{\day} after injection have been reported by others in \gls{nsg}
mice injected with human \gls{pbmc}\cite{Ali2012}. Both survival analyses (up to
day 40 in \cref{fig:mouse_dosing_ivis_survival} and up to day 46 in
\cref{fig:mouse_dosing_ivis_survival_full}) confirmed that \gls{dms}-expanded
groups outperformed bead-expanded groups in terms of prolonging survival of
\nVI{} tumor challenged \gls{nsg} mice.
Together, these data suggested that \glspl{dms} produce T cells that are not
only more potent that bead-expanded T cells (even when accounting for
differences in \gls{car} expression) but also showed that \gls{dms} expanded T
cells are effective at lower doses. Given the quality control data of the T
cells prior to injecting into the mice, it seems that this advantage is either
due to the higher \pthp{} or the overall fitness of the T cells given the higher
expansion in the case of \gls{dms}
cells are effective at lower doses. Given the \gls{qc} data of T cells prior to
injection, it seems that this advantage for \gls{dms} groups was either due to
higher \pthp{} or greater overall fitness (implied by higher fold change)
(\cref{fig:mouse_dosing_qc_cd4,fig:mouse_dosing_qc_growth}). It was likely not
due to the memory phenotype given that it was actually slightly higher in the
case of beads (\cref{fig:mouse_dosing_qc_mem}).
due to memory phenotype given that this was actually slightly higher for the
bead culture (\cref{fig:mouse_dosing_qc_mem}).
\subsection{Beads and DMSs Perform Similarly at Earlier Timepoints}
We then asked how T cells harvested using either beads or \gls{dms} performed
when harvested at earlier timepoints\cite{Ghassemi2018}. We performed the same
We then asked how T cells activated using beads or \gls{dms} performed when
harvested at earlier timepoints\cite{Ghassemi2018}. We performed the same
experiments as described in \cref{fig:mouse_dosing_overview} with the
modification that T cells were only grown and harvested after \SI{6}{\day},
modification that T cells were only expanded and harvested after \SI{6}{\day},
\SI{10}{\day}, or \SI{14}{\day} of expansion
(\cref{fig:mouse_timecourse_overview}). T cells were frozen after harvest, and
all timepoints were thawed at the same time prior to injection. The dose of T
all timepoints were thawed simultaneously prior to injection. The dose of T
cells injected was \num{1.25e6} cells per mouse (the same as the high dose in
the first experiment). All other characteristics of the experiment were the
same.
@ -4348,20 +4328,19 @@ same.
\label{fig:mouse_timecourse_overview}
\end{figure*}
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
cells harvested at each timepoint, we noted that the bead group had much higher
\ptcar{} expression at earlier timpoints compared to \gls{dms}, while they
equalized at later timepoints (\cref{fig:mouse_timecourse_qc_car}). In addition,
overall \ptcar{} expression decreased at later timepoints, indicating that
\gls{car} transduced T cells either grow slower or died faster compared to
untransduced cells. The \pthp{} of the harvested T cells was higher overall in
\gls{dms} expanded T cells but decreased with increasing timepoints
(\cref{fig:mouse_timecourse_qc_cd4}). The \ptmemp{} was similar at day 6
between bead and \gls{dms} groups but the \gls{dms} group had higher \ptmemp{}
at day 14 despite the overall \ptmemp{} decreasing with time as shown elsewhere
(\cref{fig:mouse_timecourse_qc_mem})\cite{Ghassemi2018}.
As was the case with the first \invivo{} experiment, \gls{dms} cultures expanded
much more efficiently than bead cultures
(\cref{fig:mouse_timecourse_qc_growth}). When we quantified the \ptcarp{} at
each timepoint, the bead group had much higher \ptcar{} expression at earlier
timpoints compared to \gls{dms}, while they equalized at later timepoints
(\cref{fig:mouse_timecourse_qc_car}). In addition, overall \ptcar{} expression
decreased at later timepoints, indicating that transduced cells either grew
slower or died faster compared to untransduced cells. The \pthp{} was higher
overall in \gls{dms} groups but decreased with increasing timepoints
(\cref{fig:mouse_timecourse_qc_cd4}). The \ptmemp{} was similar at day 6 between
bead and \gls{dms} groups but the \gls{dms} group had higher \ptmemp{} at day 14
despite the overall \ptmemp{} decreasing with time
(\cref{fig:mouse_timecourse_qc_mem}).
\begin{figure*}[ht!]
\begingroup
@ -4386,20 +4365,19 @@ at day 14 despite the overall \ptmemp{} decreasing with time as shown elsewhere
\label{fig:mouse_timecourse_qc}
\end{figure*}
We analyzed the tumor burden using \gls{ivis} which showed that mice that
received T cells from any group performed better than those that received only
saline (\cref{fig:mouse_timecourse_ivis}). Note that unlike the previous
experiment, many of the mice survived until day 40 at which point \gls{gvhd}
began to take effect (after euthanizing the mice at day 42, most had small or no
spleen). When comparing bead and \gls{dms} groups, the \gls{dms} T cells still
seemed superior to the bead group, at least initially (note that in this case
they had similar numbers of \ptcar{} cells). At day 6, both \gls{dms} and bead
groups seemed to eradicate the tumor initially, after which it came back after
day 21 for the bead and day 28 for the \gls{dms} group. The day 10 groups
performed somewhere in between, where they increased linearly unlike the day 6
groups but not as quickly as the day 14 groups. In the case of the \gls{dms} day
10 group, it also appeared like a few mice actually performed better than all
other groups in regard to the final tumor burden.
Analyzing the tumor burden using \gls{ivis} showed that mice who received T
cells from any group had less tumor than those that received only saline
(\cref{fig:mouse_timecourse_ivis}). Unlike the previous experiment, most mice
survived until day 40 after which \gls{gvhd} began to take effect (upon
euthanization at day 42, most had little or no spleen). When comparing bead and
\gls{dms} groups, the \gls{dms} groups had lower tumor than the bead group, at
least initially (note that in this experiment they had similar numbers of
\ptcar{} cells). For day 6 groups, both treatments seemed to eradicate the tumor
initially, then it came back after \SI{21}{\day} for the beads and \SI{28}{\day}
for \glspl{dms}. The day 10 groups performed somewhere in between, where they
increased linearly unlike the day 6 groups but not as quickly as the day 14
groups. In the case of the \gls{dms} day 10 group, a few mice actually had less
tumor burden overall than all other groups.
\begin{figure*}[ht!]
\begingroup
@ -4433,85 +4411,76 @@ other groups in regard to the final tumor burden.
\endgroup
\caption[Mouse Summary]
{Summary of cells injected into mice during for
\subcap{fig:mouse_summary_1}{the first mouse experiment} and
\subcap{fig:mouse_summary_2}{the second mouse experiment}. The y axis
maximum is set to the maximum number of cells injected between both
experiments (\num{1.25e6}). Note that the \gls{car} was quantified using a
separate panel than the rest of the markers.
}
{Summary of T cells injected into mice for the
\subcap{fig:mouse_summary_1}{first} and \subcap{fig:mouse_summary_2}{second}
experiments. The y-axis maximum is set to the maximum cell number
injected between both experiments (\num{1.25e6}). NOTE: the \gls{car} was
quantified using a separate panel from the other markers. }
\label{fig:mouse_summary}
\end{figure*}
The total number of T cells injected for each \invivo{} experiment are shown in
\cref{fig:mouse_summary}.
When we tested bead- and \gls{dms}-expanded \gls{car} T cells, the latter
prolonged survival compared to the former in \nVI{} tumor challenged
(intravenously injected) \gls{nsg} mice. This held true when matching groups for
absolute \gls{car} dose. Furthermore, \gls{dms}-expanded \gls{car} T cells were
effective in clearing tumor cells as early as \SI{7}{\day} post T injection even
at low dose compared to the bead groups where tumor burden was higher than
\gls{dms} groups across all the total T cell doses tested here. These suggest
that \glspl{dms} (compared to beads) produced highly effective \gls{car} T cells
that can efficiently kill tumor cells.
When we tested bead and \gls{dms} expanded \gls{car} T cells, we found that the
\gls{dms} expanded \gls{car} T cells outperformed bead groups in prolonging
survival of Nalm-6 tumor challenged (intravenously injected) \gls{nsg} mice.
\gls{dms} expanded CAR-T cells were very effective in clearing tumor cells as
early as \SI{7}{\day} post \gls{car} T injection even at low total T cell dose
compared to the bead groups where tumor burden was higher than \gls{dms} groups
across all the total T cell doses tested here. More interestingly, when only
\gls{car}-expressing T cell doses between bead and \gls{dms} groups were
compared, \gls{dms} group had significantly higher survival effects over similar
or higher CAR expression T cell doses from bead group. All these results suggest
that the T cells in \gls{dms} groups (compared to bead group) resulted in highly
effective \gls{car} T cells that can efficiently kill tumor cells.
When comparing total number of injected T cells with different phenotypes, the
number of \ptmem{} (both with and without CD45RA) cells was lower in the
low-dose \gls{dms} group compared to the med-dose bead group (which had similar
numbers of \gls{car} T cells) (\cref{fig:mouse_summary_1}). This could mean
several things. First, the \ptmem{} phenotype may have nothing to do with the
results seen here, at least in this model. While this may have been the case in
our hands, this would contradict previous evidence suggesting that \gls{tn} and
\gls{tcm} cells work better in almost the same model (the only difference being
Raji cells in place of \nVI{} cells, both of which express
CD19)\cite{Sommermeyer2015}. Second, the distribution of \gls{car} T cells
across different subtypes of T cells was different between the \gls{dms} and
bead groups (with possibly higher correlation of \gls{car} expression and the
\ptmem{} phenotype). It is hard to assess this without strong assumptions as the
\gls{car} was quantified using a separate flow panel relative to the other
markers.
When comparing the total number of T cells of different phenotypes, we observed
that when comparing low-dose \gls{dms} group to the mid- bead groups (which had
similar numbers of \gls{car} T cells), the number of \ptmem{} (both with and
without CD45RA) T cells injected was much lower in the \gls{dms} group
(\cref{fig:mouse_summary_1}). This could mean several things. First, the
\ptmem{} phenotype may have nothing to do with the results seen here, at least
in this model. While this may have been the case in our hands, this would
contradict previous evidence suggesting that \gls{tn} and \gls{tcm} cells work
better in almost the same model (the only difference being Raji cells in place
of Nalm-6 cells, both of which express CD19)\cite{Sommermeyer2015}. Second, the
distribution of \gls{car} T cells across different subtypes of T cells was
different between the \gls{dms} and bead groups (with possibly higher
correlation of \gls{car} expression and the \ptmem{} phenotype). It is hard to
assess this without strong assumptions as the \gls{car} was quantified using a
separate flow panel relative to the other markers.
We can also make a similar observation for the number of \pth{} T cells injected
We can make a similar observation for the number of \pth{} T cells injected
(\cref{fig:mouse_summary_1}). In this case, either the \pth{} phenotype doesn't
matter in this model (or the \ptk{} population matters much more), or the
distribution of \gls{car} is different between CD4 and CD8 T cells in a manner
that favors the \gls{dms} group. While in a glioblastoma model and not a B-cell
\gls{all} model, previous groups have shown that \pthp{} T cells are important
for response\cite{Wang2018}.
that favors the \gls{dms} group. Previous groups have shown that \pthp{} T cells
are important for response (albeit for a glioblastoma model and not a B-cell
\gls{all} model)\cite{Wang2018}.
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,
the total number of \gls{car} T cells was generally much lower in this second
experiment relative to the first (\cref{fig:mouse_summary}). Only the day 6
group had \gls{car} T cell numbers comparable to the weakest dose of bead cells
given in the first experiment, and these T cells were harvested at earlier
timepoints than the first mouse experiment and thus may not be safely
comparable. Furthermore, the \ptcarp{} decreased over time, which suggested that
the transduced T cells grew slower. This has been observed elsewhere and could
be due to tonic signaling\cite{GomesSilva2017}. The lower overall \gls{car}
doses may explain why at best, the tumor seemed to be in remission only
temporarily. Even so, the \gls{dms} group seemed to perform better at day 6 as
it held off the tumor longer, and also slowed the tumor progression relative to
the bead group at day 14 (\cref{fig:mouse_timecourse_ivis_plots}).
in the first \invivo{} experiment, none of the \gls{car} treatments seemed to
work as well as they did in the first experiment. However, the total number of
\gls{car} T cells was generally much lower in this second experiment relative to
the first (\cref{fig:mouse_summary}). Only the day 6 group had \gls{car} T cell
numbers comparable to the weakest dose of bead cells given in the first
experiment, and these T cells were harvested at earlier timepoints than the
first mouse experiment and thus are not directly comparable. Furthermore, the
\ptcarp{} decreased over time, which suggested that the transduced T cells grew
slower. This has been observed elsewhere and could be due to tonic
signaling\cite{GomesSilva2017}. The lower overall \gls{car} doses may explain
why at best, the tumor seemed to be in remission only temporarily. Even so, the
\gls{dms} group seemed to perform better at day 6 as it held off the tumor
longer, and also slowed the tumor progression relative to the bead group at day
14 (\cref{fig:mouse_timecourse_ivis_plots}).
Taken together, these data suggest that the \gls{dms} platform produces T cells
that have an advantage \invivo{} over beads. While we may not know the exact
mechanism, our data suggests that the responses are unsurprisingly influenced by
the \ptcarp{} of the final product. Followup experiments would need to be
performed to determine the precise phenotype responsible for these responses in
our hands.
the \ptcarp{} of the final product. Followup experiments are needed to determine
the precise phenotype responsible for these results.
\chapter{CONCLUSIONS AND FUTURE WORK}\label{conclusions}
\section{Conclusions}
This dissertation describes the development of a novel T cell expansion
platform, including the fabrication, quality control, and biological validation
platform, including the fabrication, \gls{qc}, and biological validation
of its performance both \invitro{} and \invivo{}. Development of such a system
would be meaningful even if it only performed as well as current methods, as
adding another method to the arsenal of the growing T cell manufacturing
@ -4724,7 +4693,7 @@ function of surface density and the presentation method.
\subsection{Reducing Ligand Variance}
While we have robust quality control steps to quantify each step of the
While we have robust \gls{qc} steps to quantify each step of the
\gls{dms} coating process, we still see high variance across time and personnel
(\cref{fig:dms_coating}). This is less than ideal for translation.