From c5d80389e677b7bdf6b8d145734daeafeb92cf0f Mon Sep 17 00:00:00 2001
From: ndwarshuis <ndwar@yavin4.ch>
Date: Thu, 29 Jul 2021 19:05:37 -0400
Subject: [PATCH] ADD (some) future directions

---
 tex/thesis.tex | 62 +++++++++++++++++++++++++++++++++++++++++++++-----
 1 file changed, 56 insertions(+), 6 deletions(-)

diff --git a/tex/thesis.tex b/tex/thesis.tex
index 7330736..11a0a76 100644
--- a/tex/thesis.tex
+++ b/tex/thesis.tex
@@ -68,6 +68,7 @@
 \newacronym{pbs}{PBS}{phosphate buffered saline}
 \newacronym{bca}{BCA}{bicinchoninic acid assay}
 \newacronym{bsa}{BSA}{bovine serum albumin}
+\newacronym{hsa}{HSA}{human serum albumin}
 \newacronym{stp}{STP}{streptavidin}
 \newacronym{stppe}{STP-PE}{streptavidin-phycoerythrin}
 \newacronym{snb}{SNB}{sulfo-nhs-biotin}
@@ -3476,15 +3477,64 @@ In \cref{aim3} we determined that the \glspl{dms} expand T cells that also
 performed better than beads \invivo{}. In the first experiment we performed, the
 results were very clearly in favor of the \glspl{dms}. In the second experiment,
 even the \gls{dms} group failed to fully control the tumor burden, but this is
-not surprising given the low \ptcarp{} across all groups. Also, despite this, the
-\gls{dms} group appeared to control the tumor better on average for early, mid,
-and late T cell harvesting timepoints. It was not clear if this effect was due
-to increased \cdp{} or overall increased fitness of the \gls{dms}-expanded T
+not surprising given the low \ptcarp{} across all groups. Also, despite this,
+the \gls{dms} group appeared to control the tumor better on average for early,
+mid, and late T cell harvesting timepoints. It was not clear if this effect was
+due to increased \cdp{} or overall increased fitness of the \gls{dms}-expanded T
 cells given their higher expansion rate. The \ptmemp{} did not seem to be a
 factor given that it was nearly the same in the first experiment between
-\gls{dms} and bead groups despite the clear advantage seen in the \gls{dms} group.
+\gls{dms} and bead groups despite the clear advantage seen in the \gls{dms}
+group.
 
-\section{future work}
+\section{future directions}
+
+There are several important next steps to perform with this work:
+
+\subsection{Translation to GMP process}
+
+While this work was done with translatability and \gls{qc} in mind, an important
+feature that is missing from the process currently is the use of \gls{gmp}
+methods and materials. The microcarriers themselves are made from
+porcine-derived collagen, which itself is not \gls{gmp}-compliant due to its
+non-human animal origins. However, using any other source of collagen should
+work so long as the structure of the microcarriers remains relatively similar
+and it has lysine groups that can react with the \gls{snb} to attach \gls{stp}
+and \glspl{mab}. Obviously these would need to be tested and verified, but they
+should not be insurmountable. Furthermore, the \gls{mab} binding step requires
+\gls{bsa} to prevent adsorption to the non-polar polymer walls of the reaction
+tubes. A human carrier protein such as \gls{hsa} could be used in its place to
+eliminate the non-human animal origin material, but this could be much more
+expensive. Alternatively, the use of protein could be replaced altogether by a
+non-ionic detergent such as Tween-20 or Tween-80, which are already used for
+commercial \gls{mab} formulations for precisely this purpose {\#}. Validating
+the process with Tween would be the best next step to eliminate \gls{bsa} from
+the process. The \gls{stp} and \glspl{mab} in this process were not
+\gls{gmp}-grade; however, they are commonly used in clinical technology such as
+dynabeads and thus the research-grade proteins used here could be easily
+replaced. The \gls{snb} is a synthetic small molecule and thus does not have any
+animal-origin concerns.
+
+\subsection{Mechanistic investigation}
+
+% why do the dms work?
+% can we put anything on the dms to enhance their potency?
+
+\subsection{Assessing performance using unhealthy donors}
+
+All the work presented in this dissertation was performed using healthy donors.
+This was mostly due to the fact that it was much easier to obtain healthy donor
+cells and was much easier to control. However, it is indisputable that the most
+relevant test cases of the \gls{dms} will be for unhealthy patient T cells, at
+least in the case of autologous therapies. In particular, it will be interesting
+to see how the \gls{dms} performs when assessed head-to-head with bead-based
+expansion technology given that even in healthy donors, we observed the
+\gls{dms} platform to work where the beads failed
+(\cref{fig:dms_exp_fold_change}).
+
+\subsection{translation to bioreactors}
+
+% use dms in non-static bioreactors such as wave by first activating in a static
+% environment
 
 \onecolumn
 \clearpage