ENH proof aim 3

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Nathan Dwarshuis 2021-09-09 13:33:54 -04:00
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@ -380,6 +380,7 @@
\newcommand{\inlinecode}{\texttt} \newcommand{\inlinecode}{\texttt}
\newcommand{\subcap}[2]{\subref{#1}) #2} \newcommand{\subcap}[2]{\subref{#1}) #2}
\newcommand{\sigkey}{Significance test key: *p<0.1; **p < 0.05; ***p<0.01} \newcommand{\sigkey}{Significance test key: *p<0.1; **p < 0.05; ***p<0.01}
\newcommand{\nVI}{NALM-6}
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
% ditto for environments % 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 cell expansion system and determine biologically-meaningful \glspl{cqa} and
\glspl{cpp} that could be used to optimize for highly-potent T cells. In \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 \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 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 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 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} mimics key components of the lymph nodes}
In this first aim, we demonstrated the process for manufacturing \glspl{dms}, 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 platform into a scalable manufacturing setting. We also demonstrated that the
\gls{dms} platform leads to higher overall expansion of T cells and higher \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 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{meyer15_immun}, and \gls{mab}-conjugated \gls{pdms}
beads\cite{Lambert2017} that respectively recapitulate the cellular membrane, beads\cite{Lambert2017} that respectively recapitulate the cellular membrane,
large interfacial contact area, 3D-structure, or soft surfaces T cells normally 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 experience \invivo{}. None of these have been shown to expand high quality T
quality T cells as outlined in \cref{sec:background_quality}. cells as outlined in \cref{sec:background_quality}.
\subsection{Microcarriers in Bioprocessing} \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} was added to tubes analogously to \gls{ptnl} and incubated for \SI{45}{\minute}
prior to analyzing on a \bd{} Accuri 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$\upalpha$ The anti-CD19-CD8-CD137-CD3$\upzeta$ \gls{car} sequence with the EF1$\upalpha$
promotor\cite{Milone2009} was synthesized (Aldevron) and subcloned into a 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 \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}. 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
% phenotype4446. 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} \chapter{AIM 3}\label{aim3}
\section{Introduction} \section{Introduction}
@ -4156,44 +4136,47 @@ lower-differentiated T cells with higher potency\cite{Ghassemi2018}.
\section{Methods} \section{Methods}
\subsection{CD19-CAR T Cell Generation}
\subsection{T Cell Culture} \subsection{T Cell Culture}
T cells were grown as described in \cref{sec:tcellculture}. 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} \subsection{\Invivo{} Therapeutic Efficacy in NSG Mice Model}
% METHOD describe how the luciferase cells were generated (eg the kwong lab) % METHOD describe how the luciferase cells were generated (eg the kwong lab)
% METHOD use actual product numbers for mice % METHOD use actual product numbers for mice
All mice in this study were male \gls{nsg} mice from Jackson Laboratories. At 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 day 0 (\SI{-7}{\day} relative to T cell injection), \num{1e6} firefly
luciferase-expressing \product{Nalm-6 cells}{ATCC}{CRL-3273} suspended in luciferase-expressing\footnote{luciferase transduction was performed and
ice-cold \gls{pbs} were injected via tail vein into each mouse. At day 7, saline verified by Ian Miller in the Kwong Lab at Georgia Tech} \product{\nVI{}
or \gls{car} T cells at the indicated doses from either bead or cells}{ATCC}{CRL-3273} suspended in ice-cold \gls{pbs} were injected via tail
\gls{dms}-expanded T cell cultures (for \SI{14}{\day}) were injected into each vein into each mouse. At day 7, saline or \gls{car} T cells at the indicated
mouse via tail vein. Tumor burden was quantified longitudinally via an doses from either bead or \gls{dms}-expanded T cell cultures (for \SI{14}{\day})
\gls{ivis} Spectrum (Perkin Elmer). Briefly, \SI{200}{\ug} luciferin at were injected into each mouse via tail vein. Tumor burden was quantified
\SI{15}{\mg\per\ml} in \gls{pbs} was injected intraperitoneally under isoflurane longitudinally via an \gls{ivis} Spectrum (Perkin Elmer). Briefly, \SI{200}{\ug}
anesthesia into each mouse and allowed to circulate for at least luciferin at \SI{15}{\mg\per\ml} in \gls{pbs} was injected intraperitoneally
\SI{10}{\minute} before imaging. Mice were anesthetized again and imaged using under isoflurane anesthesia into each mouse and allowed to circulate for at
the \gls{ivis}. Mice from each treatment group/dose were anesthetized, injected, least \SI{10}{\minute} before imaging. Mice were anesthetized again and imaged
and imaged together, and exposure time of the \gls{ivis} was limited to avoid using the \gls{ivis}. Mice from each treatment group/dose were anesthetized,
saturation based on the signal from the saline group. \gls{ivis} images were injected, and imaged together; exposure time of the \gls{ivis} was limited to
processed by normalizing them to common minimum and maximum photon counts and avoid saturation based on the signal from the saline group. \gls{ivis} images
total flux was estimated in terms of photons/second. Endpoint for each mouse was were scaled to common minimum and maximum photon counts. Endpoint for each mouse
determined by \gls{iacuc} euthanasia criteria (hunched back, paralysis, was determined by \gls{iacuc} euthanasia criteria (hunched back, paralysis,
blindness, lethargy, and weight loss). Mice were euthanized according to these blindness, lethargy, and weight loss). Mice were euthanized according to these
endpoint criteria using carbon dioxide asphyxiation. endpoint criteria using carbon dioxide asphyxiation.
\subsection{Statistics} \subsection{Statistics}
For the \invivo{} model, the survival curves were created and statistically Survival curves were created and statistically analyzed using GraphPad Prism
analyzed using GraphPad Prism using the Mantel-Cox test to assess significance using the Mantel-Cox test to assess significance between survival groups.
between survival groups.
\section{Results} \section{Results}
\subsection{DMSs Lead to Greater \invivo{} Anti-Tumor Activity}
\begin{figure*}[ht!] \begin{figure*}[ht!]
\begingroup \begingroup
@ -4212,7 +4195,6 @@ between survival groups.
\input{../tables/mouse_dose_car.tex} \input{../tables/mouse_dose_car.tex}
\end{table} \end{table}
\subsection{DMSs Lead to Greater \invivo{} Anti-Tumor Activity}
\begin{figure*}[ht!] \begin{figure*}[ht!]
\begingroup \begingroup
@ -4248,7 +4230,7 @@ between survival groups.
\caption[Mouse Dosing IVIS and Survival Results] \caption[Mouse Dosing IVIS and Survival Results]
{T cells expanded with \glspl{dms} confer greater anti-tumor potency \invivo{} {T cells expanded with \glspl{dms} confer greater anti-tumor potency \invivo{}
even at lower doses. 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} \gls{nsg} mice injected with varying doses of T cells}
\subcap{fig:mouse_dosing_ivis_plots}{Plots showing quantified photon counts \subcap{fig:mouse_dosing_ivis_plots}{Plots showing quantified photon counts
of the results from (\subref{fig:mouse_dosing_ivis_plots}).} 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 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 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} 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 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} 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 luciferase\cite{Fraietta2018}. After \SI{7}{\day} of tumor cell growth
(\cref{fig:mouse_dosing_overview}), we intravenously injected saline or three (\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} 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 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 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 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 fraction of \cdp{45RA} cells, which is present on lower-differentiated
\glspl{tscm} (\cref{fig:mouse_dosing_qc_mem}). However, the \pthp{} of \glspl{tn} and \glspl{tscm} (\cref{fig:mouse_dosing_qc_mem}). However, the
the final product was higher in \gls{dms} (\cref{fig:mouse_dosing_qc_cd4}). The \pthp{} of the final product was higher in \gls{dms}
\gls{dms} T cells also expanded more robustly than the beads (\cref{fig:mouse_dosing_qc_cd4}). The \gls{dms} T cells also expanded more
(\cref{fig:mouse_dosing_qc_growth}). robustly than the beads (\cref{fig:mouse_dosing_qc_growth}).
In the Nalm-6/\gls{nsg} xenograft model, we observed lower tumor burden and In the \nVI{}/\gls{nsg} xenograft model, bead and \gls{dms}-treated mice at all
significantly longer survival of bead and \gls{dms}-treated mice at all doses doses had lower tumor burden and significantly longer survival compared to the
compared to the saline groups (\cref{fig:mouse_dosing_ivis}). Importantly, at saline groups (\cref{fig:mouse_dosing_ivis}). Importantly, at each dose the
each dose we observed that the \gls{dms}-treated mice had much lower tumor \gls{dms}-treated mice had much lower tumor burden and significantly higher
burden and significantly higher survival than their bead-treated counterparts survival than their bead-treated counterparts
(\cref{fig:mouse_dosing_ivis_survival}). When factoring the percentage T cells (\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 in each dose that expressed the \gls{car}, survival of the low \gls{dms} dose
\gls{dms} dose (which had similar total \gls{car} T cells compared to the bead (which had similar total \gls{car} T cells compared to the bead medium dose and
medium dose and less than the bead high dose) is significantly higher than that less than the bead high dose) was significantly higher than that of both the
of both the bead medium dose and the bead high dose bead medium dose and the bead high dose
(\cref{fig:mouse_dosing_ivis_survival_comp}). Overall, the Kaplan-Meier survival (\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 \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 elsewhere\cite{Fraietta2018}. However, most of the mice euthanized from day 40
to day 46 (\cref{fig:mouse_dosing_ivis_survival_full}) where most of the mice through day 46 from \gls{dms} groups showed no or very small fragment of spleen
euthanized from day 40 through day 46 from \gls{dms} groups showed no or very which was due to \gls{gvhd} responses
small fragment of spleen which was due to \gls{gvhd} responses. Similar (\cref{fig:mouse_dosing_ivis_survival_full}). Similar \gls{gvhd} responses
\gls{gvhd} responses were reported earlier in \gls{nsg} mice where the mice \SIrange{40}{50}{\day} after injection have been reported by others in \gls{nsg}
injected with human \gls{pbmc} exhibited acute \gls{gvhd} between mice injected with human \gls{pbmc}\cite{Ali2012}. Both survival analyses (up to
\SIrange{40}{50}{\day} post intravenous injection\cite{Ali2012}. Notably, both day 40 in \cref{fig:mouse_dosing_ivis_survival} and up to day 46 in
survival analyses (up to day 40 in \cref{fig:mouse_dosing_ivis_survival} and up \cref{fig:mouse_dosing_ivis_survival_full}) confirmed that \gls{dms}-expanded
to day 46 in \cref{fig:mouse_dosing_ivis_survival_full}) confirmed that groups outperformed bead-expanded groups in terms of prolonging survival of
\gls{dms}-expanded groups outperformed bead-expanded groups in terms of \nVI{} tumor challenged \gls{nsg} mice.
prolonging survival of Nalm-6 tumor challenged \gls{nsg} mice.
Together, these data suggested that \glspl{dms} produce T cells that are not 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 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 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 are effective at lower doses. Given the \gls{qc} data of T cells prior to
cells prior to injecting into the mice, it seems that this advantage is either injection, it seems that this advantage for \gls{dms} groups was either due to
due to the higher \pthp{} or the overall fitness of the T cells given the higher higher \pthp{} or greater overall fitness (implied by higher fold change)
expansion in the case of \gls{dms}
(\cref{fig:mouse_dosing_qc_cd4,fig:mouse_dosing_qc_growth}). It was likely not (\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 due to memory phenotype given that this was actually slightly higher for the
case of beads (\cref{fig:mouse_dosing_qc_mem}). bead culture (\cref{fig:mouse_dosing_qc_mem}).
\subsection{Beads and DMSs Perform Similarly at Earlier Timepoints} \subsection{Beads and DMSs Perform Similarly at Earlier Timepoints}
We then asked how T cells harvested using either beads or \gls{dms} performed We then asked how T cells activated using beads or \gls{dms} performed when
when harvested at earlier timepoints\cite{Ghassemi2018}. We performed the same harvested at earlier timepoints\cite{Ghassemi2018}. We performed the same
experiments as described in \cref{fig:mouse_dosing_overview} with the 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 \SI{10}{\day}, or \SI{14}{\day} of expansion
(\cref{fig:mouse_timecourse_overview}). T cells were frozen after harvest, and (\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 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 the first experiment). All other characteristics of the experiment were the
same. same.
@ -4348,20 +4328,19 @@ same.
\label{fig:mouse_timecourse_overview} \label{fig:mouse_timecourse_overview}
\end{figure*} \end{figure*}
As was the case with the first \invivo{} experiment, T cells activated with As was the case with the first \invivo{} experiment, \gls{dms} cultures expanded
\glspl{dms} expanded much more efficiently compared to those expanded with beads much more efficiently than bead cultures
(\cref{fig:mouse_timecourse_qc_growth}). When we quantified the \ptcarp{} of T (\cref{fig:mouse_timecourse_qc_growth}). When we quantified the \ptcarp{} at
cells harvested at each timepoint, we noted that the bead group had much higher each timepoint, the bead group had much higher \ptcar{} expression at earlier
\ptcar{} expression at earlier timpoints compared to \gls{dms}, while they timpoints compared to \gls{dms}, while they equalized at later timepoints
equalized at later timepoints (\cref{fig:mouse_timecourse_qc_car}). In addition, (\cref{fig:mouse_timecourse_qc_car}). In addition, overall \ptcar{} expression
overall \ptcar{} expression decreased at later timepoints, indicating that decreased at later timepoints, indicating that transduced cells either grew
\gls{car} transduced T cells either grow slower or died faster compared to slower or died faster compared to untransduced cells. The \pthp{} was higher
untransduced cells. The \pthp{} of the harvested T cells was higher overall in overall in \gls{dms} groups but decreased with increasing timepoints
\gls{dms} expanded T cells but decreased with increasing timepoints (\cref{fig:mouse_timecourse_qc_cd4}). The \ptmemp{} was similar at day 6 between
(\cref{fig:mouse_timecourse_qc_cd4}). The \ptmemp{} was similar at day 6 bead and \gls{dms} groups but the \gls{dms} group had higher \ptmemp{} at day 14
between bead and \gls{dms} groups but the \gls{dms} group had higher \ptmemp{} despite the overall \ptmemp{} decreasing with time
at day 14 despite the overall \ptmemp{} decreasing with time as shown elsewhere (\cref{fig:mouse_timecourse_qc_mem}).
(\cref{fig:mouse_timecourse_qc_mem})\cite{Ghassemi2018}.
\begin{figure*}[ht!] \begin{figure*}[ht!]
\begingroup \begingroup
@ -4386,20 +4365,19 @@ at day 14 despite the overall \ptmemp{} decreasing with time as shown elsewhere
\label{fig:mouse_timecourse_qc} \label{fig:mouse_timecourse_qc}
\end{figure*} \end{figure*}
We analyzed the tumor burden using \gls{ivis} which showed that mice that Analyzing the tumor burden using \gls{ivis} showed that mice who received T
received T cells from any group performed better than those that received only cells from any group had less tumor than those that received only saline
saline (\cref{fig:mouse_timecourse_ivis}). Note that unlike the previous (\cref{fig:mouse_timecourse_ivis}). Unlike the previous experiment, most mice
experiment, many of the mice survived until day 40 at which point \gls{gvhd} survived until day 40 after which \gls{gvhd} began to take effect (upon
began to take effect (after euthanizing the mice at day 42, most had small or no euthanization at day 42, most had little or no spleen). When comparing bead and
spleen). When comparing bead and \gls{dms} groups, the \gls{dms} T cells still \gls{dms} groups, the \gls{dms} groups had lower tumor than the bead group, at
seemed superior to the bead group, at least initially (note that in this case least initially (note that in this experiment they had similar numbers of
they had similar numbers of \ptcar{} cells). At day 6, both \gls{dms} and bead \ptcar{} cells). For day 6 groups, both treatments seemed to eradicate the tumor
groups seemed to eradicate the tumor initially, after which it came back after initially, then it came back after \SI{21}{\day} for the beads and \SI{28}{\day}
day 21 for the bead and day 28 for the \gls{dms} group. The day 10 groups for \glspl{dms}. The day 10 groups performed somewhere in between, where they
performed somewhere in between, where they increased linearly unlike the day 6 increased linearly unlike the day 6 groups but not as quickly as the day 14
groups but not as quickly as the day 14 groups. In the case of the \gls{dms} day groups. In the case of the \gls{dms} day 10 group, a few mice actually had less
10 group, it also appeared like a few mice actually performed better than all tumor burden overall than all other groups.
other groups in regard to the final tumor burden.
\begin{figure*}[ht!] \begin{figure*}[ht!]
\begingroup \begingroup
@ -4433,85 +4411,76 @@ other groups in regard to the final tumor burden.
\endgroup \endgroup
\caption[Mouse Summary] \caption[Mouse Summary]
{Summary of cells injected into mice during for {Summary of T cells injected into mice for the
\subcap{fig:mouse_summary_1}{the first mouse experiment} and \subcap{fig:mouse_summary_1}{first} and \subcap{fig:mouse_summary_2}{second}
\subcap{fig:mouse_summary_2}{the second mouse experiment}. The y axis experiments. The y-axis maximum is set to the maximum cell number
maximum is set to the maximum number of cells injected between both injected between both experiments (\num{1.25e6}). NOTE: the \gls{car} was
experiments (\num{1.25e6}). Note that the \gls{car} was quantified using a quantified using a separate panel from the other markers. }
separate panel than the rest of the markers.
}
\label{fig:mouse_summary} \label{fig:mouse_summary}
\end{figure*} \end{figure*}
The total number of T cells injected for each \invivo{} experiment are shown in When we tested bead- and \gls{dms}-expanded \gls{car} T cells, the latter
\cref{fig:mouse_summary}. 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 When comparing total number of injected T cells with different phenotypes, the
\gls{dms} expanded \gls{car} T cells outperformed bead groups in prolonging number of \ptmem{} (both with and without CD45RA) cells was lower in the
survival of Nalm-6 tumor challenged (intravenously injected) \gls{nsg} mice. low-dose \gls{dms} group compared to the med-dose bead group (which had similar
\gls{dms} expanded CAR-T cells were very effective in clearing tumor cells as numbers of \gls{car} T cells) (\cref{fig:mouse_summary_1}). This could mean
early as \SI{7}{\day} post \gls{car} T injection even at low total T cell dose several things. First, the \ptmem{} phenotype may have nothing to do with the
compared to the bead groups where tumor burden was higher than \gls{dms} groups results seen here, at least in this model. While this may have been the case in
across all the total T cell doses tested here. More interestingly, when only our hands, this would contradict previous evidence suggesting that \gls{tn} and
\gls{car}-expressing T cell doses between bead and \gls{dms} groups were \gls{tcm} cells work better in almost the same model (the only difference being
compared, \gls{dms} group had significantly higher survival effects over similar Raji cells in place of \nVI{} cells, both of which express
or higher CAR expression T cell doses from bead group. All these results suggest CD19)\cite{Sommermeyer2015}. Second, the distribution of \gls{car} T cells
that the T cells in \gls{dms} groups (compared to bead group) resulted in highly across different subtypes of T cells was different between the \gls{dms} and
effective \gls{car} T cells that can efficiently kill tumor cells. 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 We can make a similar observation for the number of \pth{} T cells injected
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
(\cref{fig:mouse_summary_1}). In this case, either the \pth{} phenotype doesn't (\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 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 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 that favors the \gls{dms} group. Previous groups have shown that \pthp{} T cells
\gls{all} model, previous groups have shown that \pthp{} T cells are important are important for response (albeit for a glioblastoma model and not a B-cell
for response\cite{Wang2018}. \gls{all} model)\cite{Wang2018}.
When testing \gls{car} T cells at earlier timepoints relative to day 14 as used 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} in the first \invivo{} experiment, none of the \gls{car} treatments seemed to
treatments seemed to work as well as they did in the first experiment. However, work as well as they did in the first experiment. However, the total number of
the total number of \gls{car} T cells was generally much lower in this second \gls{car} T cells was generally much lower in this second experiment relative to
experiment relative to the first (\cref{fig:mouse_summary}). Only the day 6 the first (\cref{fig:mouse_summary}). Only the day 6 group had \gls{car} T cell
group had \gls{car} T cell numbers comparable to the weakest dose of bead cells numbers comparable to the weakest dose of bead cells given in the first
given in the first experiment, and these T cells were harvested at earlier experiment, and these T cells were harvested at earlier timepoints than the
timepoints than the first mouse experiment and thus may not be safely first mouse experiment and thus are not directly comparable. Furthermore, the
comparable. Furthermore, the \ptcarp{} decreased over time, which suggested that \ptcarp{} decreased over time, which suggested that the transduced T cells grew
the transduced T cells grew slower. This has been observed elsewhere and could slower. This has been observed elsewhere and could be due to tonic
be due to tonic signaling\cite{GomesSilva2017}. The lower overall \gls{car} signaling\cite{GomesSilva2017}. The lower overall \gls{car} doses may explain
doses may explain why at best, the tumor seemed to be in remission only why at best, the tumor seemed to be in remission only temporarily. Even so, the
temporarily. Even so, the \gls{dms} group seemed to perform better at day 6 as \gls{dms} group seemed to perform better at day 6 as it held off the tumor
it held off the tumor longer, and also slowed the tumor progression relative to longer, and also slowed the tumor progression relative to the bead group at day
the bead group at day 14 (\cref{fig:mouse_timecourse_ivis_plots}). 14 (\cref{fig:mouse_timecourse_ivis_plots}).
Taken together, these data suggest that the \gls{dms} platform produces T cells 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 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 mechanism, our data suggests that the responses are unsurprisingly influenced by
the \ptcarp{} of the final product. Followup experiments would need to be the \ptcarp{} of the final product. Followup experiments are needed to determine
performed to determine the precise phenotype responsible for these responses in the precise phenotype responsible for these results.
our hands.
\chapter{CONCLUSIONS AND FUTURE WORK}\label{conclusions} \chapter{CONCLUSIONS AND FUTURE WORK}\label{conclusions}
\section{Conclusions} \section{Conclusions}
This dissertation describes the development of a novel T cell expansion 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 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 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 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} \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 \gls{dms} coating process, we still see high variance across time and personnel
(\cref{fig:dms_coating}). This is less than ideal for translation. (\cref{fig:dms_coating}). This is less than ideal for translation.